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Sample records for genome amplification wga

  1. Molecular analysis of single oocyst of Eimeria by whole genome amplification (WGA) based nested PCR.

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    Wang, Yunzhou; Tao, Geru; Cui, Yujuan; Lv, Qiyao; Xie, Li; Li, Yuan; Suo, Xun; Qin, Yinghe; Xiao, Lihua; Liu, Xianyong

    2014-09-01

    PCR-based molecular tools are widely used for the identification and characterization of protozoa. Here we report the molecular analysis of Eimeria species using combined methods of whole genome amplification (WGA) and nested PCR. Single oocyst of Eimeria stiedai or Eimeriamedia was directly used for random amplification of the genomic DNA with either primer extension preamplification (PEP) or multiple displacement amplification (MDA), and then the WGA product was used as template in nested PCR with species-specific primers for ITS-1, 18S rDNA and 23S rDNA of E. stiedai and E. media. WGA-based PCR was successful for the amplification of these genes from single oocyst. For the species identification of single oocyst isolated from mixed E. stiedai or E. media, the results from WGA-based PCR were exactly in accordance with those from morphological identification, suggesting the availability of this method in molecular analysis of eimerian parasites at the single oocyst level. WGA-based PCR method can also be applied for the identification and genetic characterization of other protists. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. A quantitative comparison of single-cell whole genome amplification methods.

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    Charles F A de Bourcy

    Full Text Available Single-cell sequencing is emerging as an important tool for studies of genomic heterogeneity. Whole genome amplification (WGA is a key step in single-cell sequencing workflows and a multitude of methods have been introduced. Here, we compare three state-of-the-art methods on both bulk and single-cell samples of E. coli DNA: Multiple Displacement Amplification (MDA, Multiple Annealing and Looping Based Amplification Cycles (MALBAC, and the PicoPLEX single-cell WGA kit (NEB-WGA. We considered the effects of reaction gain on coverage uniformity, error rates and the level of background contamination. We compared the suitability of the different WGA methods for the detection of copy-number variations, for the detection of single-nucleotide polymorphisms and for de-novo genome assembly. No single method performed best across all criteria and significant differences in characteristics were observed; the choice of which amplifier to use will depend strongly on the details of the type of question being asked in any given experiment.

  3. New perspectives on microbial community distortion after whole-genome amplification

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    Whole-genome amplification (WGA) has become an important tool to explore the genomic information of microorganisms in an environmental sample with limited biomass, however potential selective biases during the amplification processes are poorly understood. Here, we describe the e...

  4. Single-Cell Whole-Genome Amplification and Sequencing: Methodology and Applications.

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    Huang, Lei; Ma, Fei; Chapman, Alec; Lu, Sijia; Xie, Xiaoliang Sunney

    2015-01-01

    We present a survey of single-cell whole-genome amplification (WGA) methods, including degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), multiple displacement amplification (MDA), and multiple annealing and looping-based amplification cycles (MALBAC). The key parameters to characterize the performance of these methods are defined, including genome coverage, uniformity, reproducibility, unmappable rates, chimera rates, allele dropout rates, false positive rates for calling single-nucleotide variations, and ability to call copy-number variations. Using these parameters, we compare five commercial WGA kits by performing deep sequencing of multiple single cells. We also discuss several major applications of single-cell genomics, including studies of whole-genome de novo mutation rates, the early evolution of cancer genomes, circulating tumor cells (CTCs), meiotic recombination of germ cells, preimplantation genetic diagnosis (PGD), and preimplantation genomic screening (PGS) for in vitro-fertilized embryos.

  5. Monodisperse Picoliter Droplets for Low-Bias and Contamination-Free Reactions in Single-Cell Whole Genome Amplification.

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    Yohei Nishikawa

    Full Text Available Whole genome amplification (WGA is essential for obtaining genome sequences from single bacterial cells because the quantity of template DNA contained in a single cell is very low. Multiple displacement amplification (MDA, using Phi29 DNA polymerase and random primers, is the most widely used method for single-cell WGA. However, single-cell MDA usually results in uneven genome coverage because of amplification bias, background amplification of contaminating DNA, and formation of chimeras by linking of non-contiguous chromosomal regions. Here, we present a novel MDA method, termed droplet MDA, that minimizes amplification bias and amplification of contaminants by using picoliter-sized droplets for compartmentalized WGA reactions. Extracted DNA fragments from a lysed cell in MDA mixture are divided into 105 droplets (67 pL within minutes via flow through simple microfluidic channels. Compartmentalized genome fragments can be individually amplified in these droplets without the risk of encounter with reagent-borne or environmental contaminants. Following quality assessment of WGA products from single Escherichia coli cells, we showed that droplet MDA minimized unexpected amplification and improved the percentage of genome recovery from 59% to 89%. Our results demonstrate that microfluidic-generated droplets show potential as an efficient tool for effective amplification of low-input DNA for single-cell genomics and greatly reduce the cost and labor investment required for determination of nearly complete genome sequences of uncultured bacteria from environmental samples.

  6. Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance

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    Haque Kashif A

    2005-09-01

    Full Text Available Abstract Background Whole genome amplification (WGA promises to eliminate practical molecular genetic analysis limitations associated with genomic DNA (gDNA quantity. We evaluated the performance of multiple displacement amplification (MDA WGA using gDNA extracted from lymphoblastoid cell lines (N = 27 with a range of starting gDNA input of 1–200 ng into the WGA reaction. Yield and composition analysis of whole genome amplified DNA (wgaDNA was performed using three DNA quantification methods (OD, PicoGreen® and RT-PCR. Two panels of N = 15 STR (using the AmpFlSTR® Identifiler® panel and N = 49 SNP (TaqMan® genotyping assays were performed on each gDNA and wgaDNA sample in duplicate. gDNA and wgaDNA masses of 1, 4 and 20 ng were used in the SNP assays to evaluate the effects of DNA mass on SNP genotyping assay performance. A total of N = 6,880 STR and N = 56,448 SNP genotype attempts provided adequate power to detect differences in STR and SNP genotyping performance between gDNA and wgaDNA, and among wgaDNA produced from a range of gDNA templates inputs. Results The proportion of double-stranded wgaDNA and human-specific PCR amplifiable wgaDNA increased with increased gDNA input into the WGA reaction. Increased amounts of gDNA input into the WGA reaction improved wgaDNA genotyping performance. Genotype completion or genotype concordance rates of wgaDNA produced from all gDNA input levels were observed to be reduced compared to gDNA, although the reduction was not always statistically significant. Reduced wgaDNA genotyping performance was primarily due to the increased variance of allelic amplification, resulting in loss of heterozygosity or increased undetermined genotypes. MDA WGA produces wgaDNA from no template control samples; such samples exhibited substantial false-positive genotyping rates. Conclusion The amount of gDNA input into the MDA WGA reaction is a critical determinant of genotyping performance of wgaDNA. At least 10 ng of

  7. Systematic evaluation of bias in microbial community profiles induced by whole genome amplification.

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    Direito, Susana O L; Zaura, Egija; Little, Miranda; Ehrenfreund, Pascale; Röling, Wilfred F M

    2014-03-01

    Whole genome amplification methods facilitate the detection and characterization of microbial communities in low biomass environments. We examined the extent to which the actual community structure is reliably revealed and factors contributing to bias. One widely used [multiple displacement amplification (MDA)] and one new primer-free method [primase-based whole genome amplification (pWGA)] were compared using a polymerase chain reaction (PCR)-based method as control. Pyrosequencing of an environmental sample and principal component analysis revealed that MDA impacted community profiles more strongly than pWGA and indicated that this related to species GC content, although an influence of DNA integrity could not be excluded. Subsequently, biases by species GC content, DNA integrity and fragment size were separately analysed using defined mixtures of DNA from various species. We found significantly less amplification of species with the highest GC content for MDA-based templates and, to a lesser extent, for pWGA. DNA fragmentation also interfered severely: species with more fragmented DNA were less amplified with MDA and pWGA. pWGA was unable to amplify low molecular weight DNA (microbial communities in low-biomass environments and for currently planned astrobiological missions to Mars. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

  8. Development of a fluorescence-activated cell sorting method coupled with whole genome amplification to analyze minority and trace Dehalococcoides genomes in microbial communities.

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    Lee, Patrick K H; Men, Yujie; Wang, Shanquan; He, Jianzhong; Alvarez-Cohen, Lisa

    2015-02-03

    Dehalococcoides mccartyi are functionally important bacteria that catalyze the reductive dechlorination of chlorinated ethenes. However, these anaerobic bacteria are fastidious to isolate, making downstream genomic characterization challenging. In order to facilitate genomic analysis, a fluorescence-activated cell sorting (FACS) method was developed in this study to separate D. mccartyi cells from a microbial community, and the DNA of the isolated cells was processed by whole genome amplification (WGA) and hybridized onto a D. mccartyi microarray for comparative genomics against four sequenced strains. First, FACS was successfully applied to a D. mccartyi isolate as positive control, and then microarray results verified that WGA from 10(6) cells or ∼1 ng of genomic DNA yielded high-quality coverage detecting nearly all genes across the genome. As expected, some inter- and intrasample variability in WGA was observed, but these biases were minimized by performing multiple parallel amplifications. Subsequent application of the FACS and WGA protocols to two enrichment cultures containing ∼10% and ∼1% D. mccartyi cells successfully enabled genomic analysis. As proof of concept, this study demonstrates that coupling FACS with WGA and microarrays is a promising tool to expedite genomic characterization of target strains in environmental communities where the relative concentrations are low.

  9. Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics.

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    Hosokawa, Masahito; Nishikawa, Yohei; Kogawa, Masato; Takeyama, Haruko

    2017-07-12

    Massively parallel single-cell genome sequencing is required to further understand genetic diversities in complex biological systems. Whole genome amplification (WGA) is the first step for single-cell sequencing, but its throughput and accuracy are insufficient in conventional reaction platforms. Here, we introduce single droplet multiple displacement amplification (sd-MDA), a method that enables massively parallel amplification of single cell genomes while maintaining sequence accuracy and specificity. Tens of thousands of single cells are compartmentalized in millions of picoliter droplets and then subjected to lysis and WGA by passive droplet fusion in microfluidic channels. Because single cells are isolated in compartments, their genomes are amplified to saturation without contamination. This enables the high-throughput acquisition of contamination-free and cell specific sequence reads from single cells (21,000 single-cells/h), resulting in enhancement of the sequence data quality compared to conventional methods. This method allowed WGA of both single bacterial cells and human cancer cells. The obtained sequencing coverage rivals those of conventional techniques with superior sequence quality. In addition, we also demonstrate de novo assembly of uncultured soil bacteria and obtain draft genomes from single cell sequencing. This sd-MDA is promising for flexible and scalable use in single-cell sequencing.

  10. Small sample whole-genome amplification

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    Hara, Christine; Nguyen, Christine; Wheeler, Elizabeth; Sorensen, Karen; Arroyo, Erin; Vrankovich, Greg; Christian, Allen

    2005-11-01

    Many challenges arise when trying to amplify and analyze human samples collected in the field due to limitations in sample quantity, and contamination of the starting material. Tests such as DNA fingerprinting and mitochondrial typing require a certain sample size and are carried out in large volume reactions; in cases where insufficient sample is present whole genome amplification (WGA) can be used. WGA allows very small quantities of DNA to be amplified in a way that enables subsequent DNA-based tests to be performed. A limiting step to WGA is sample preparation. To minimize the necessary sample size, we have developed two modifications of WGA: the first allows for an increase in amplified product from small, nanoscale, purified samples with the use of carrier DNA while the second is a single-step method for cleaning and amplifying samples all in one column. Conventional DNA cleanup involves binding the DNA to silica, washing away impurities, and then releasing the DNA for subsequent testing. We have eliminated losses associated with incomplete sample release, thereby decreasing the required amount of starting template for DNA testing. Both techniques address the limitations of sample size by providing ample copies of genomic samples. Carrier DNA, included in our WGA reactions, can be used when amplifying samples with the standard purification method, or can be used in conjunction with our single-step DNA purification technique to potentially further decrease the amount of starting sample necessary for future forensic DNA-based assays.

  11. Comparison of whole genome amplification techniques for human single cell exome sequencing.

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    Borgström, Erik; Paterlini, Marta; Mold, Jeff E; Frisen, Jonas; Lundeberg, Joakim

    2017-01-01

    Whole genome amplification (WGA) is currently a prerequisite for single cell whole genome or exome sequencing. Depending on the method used the rate of artifact formation, allelic dropout and sequence coverage over the genome may differ significantly. The largest difference between the evaluated protocols was observed when analyzing the target coverage and read depth distribution. These differences also had impact on the downstream variant calling. Conclusively, the products from the AMPLI1 and MALBAC kits were shown to be most similar to the bulk samples and are therefore recommended for WGA of single cells. In this study four commercial kits for WGA (AMPLI1, MALBAC, Repli-G and PicoPlex) were used to amplify human single cells. The WGA products were exome sequenced together with non-amplified bulk samples from the same source. The resulting data was evaluated in terms of genomic coverage, allelic dropout and SNP calling.

  12. Current Developments in Prokaryotic Single Cell Whole Genome Amplification

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    Goudeau, Danielle; Nath, Nandita; Ciobanu, Doina; Cheng, Jan-Fang; Malmstrom, Rex

    2014-03-14

    Our approach to prokaryotic single-cell Whole Genome Amplification at the JGI continues to evolve. To increase both the quality and number of single-cell genomes produced, we explore all aspects of the process from cell sorting to sequencing. For example, we now utilize specialized reagents, acoustic liquid handling, and reduced reaction volumes eliminate non-target DNA contamination in WGA reactions. More specifically, we use a cleaner commercial WGA kit from Qiagen that employs a UV decontamination procedure initially developed at the JGI, and we use the Labcyte Echo for tip-less liquid transfer to set up 2uL reactions. Acoustic liquid handling also dramatically reduces reagent costs. In addition, we are exploring new cell lysis methods including treatment with Proteinase K, lysozyme, and other detergents, in order to complement standard alkaline lysis and allow for more efficient disruption of a wider range of cells. Incomplete lysis represents a major hurdle for WGA on some environmental samples, especially rhizosphere, peatland, and other soils. Finding effective lysis strategies that are also compatible with WGA is challenging, and we are currently assessing the impact of various strategies on genome recovery.

  13. Whole genome amplification in preimplantation genetic diagnosis*

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    Zheng, Ying-ming; Wang, Ning; Li, Lei; Jin, Fan

    2011-01-01

    Preimplantation genetic diagnosis (PGD) refers to a procedure for genetically analyzing embryos prior to implantation, improving the chance of conception for patients at high risk of transmitting specific inherited disorders. This method has been widely used for a large number of genetic disorders since the first successful application in the early 1990s. Polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) are the two main methods in PGD, but there are some inevitable shortcomings limiting the scope of genetic diagnosis. Fortunately, different whole genome amplification (WGA) techniques have been developed to overcome these problems. Sufficient DNA can be amplified and multiple tasks which need abundant DNA can be performed. Moreover, WGA products can be analyzed as a template for multi-loci and multi-gene during the subsequent DNA analysis. In this review, we will focus on the currently available WGA techniques and their applications, as well as the new technical trends from WGA products. PMID:21194180

  14. Review:Whole genome amplification in preimplantation genetic diagnosis

    Institute of Scientific and Technical Information of China (English)

    Ying-ming ZHENG; Ning WANG; Lei LI; Fan JIN

    2011-01-01

    Preimplantation genetic diagnosis(PGD)refers to a procedure for genetically analyzing embryos prior to implantation,improving the chance of conception for patients at high risk of transmitting specific inherited disorders.This method has been widely used for a large number of genetic disorders since the first successful application in the early 1990s.Polymerase chain reaction(PCR)and fluorescent in situ hybridization(FISH)are the two main methods in PGD,but there are some inevitable shortcomings limiting the scope of genetic diagnosis.Fortunately,different whole genome amplification(WGA)techniques have been developed to overcome these problems.Sufficient DNA can be amplified and multiple tasks which need abundant DNA can be performed.Moreover,WGA products can be analyzed as a template for multi-loci and multi-gene during the subsequent DNA analysis.In this review,we will focus on the currently available WGA techniques and their applications,as well as the new technical trends from WGA products.

  15. The effect of whole genome amplification on samples originating from more than one donor

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    Thacker, C.R.; Balogh, M.K.; Børsting, Claus

    2006-01-01

    In this study, the GenomiPhi(TM) DNA Amplification Kit (Amersham Biosciences) was used to investigate the potential of whole genome amplification (WGA) when considering samples originating from more than one donor. DNA was extracted from blood samples, quantified and normalised before being mixed...

  16. Whole community genome amplification (WCGA) leads to compositional bias in methane oxidizing communities as assessed by pmoA based microarray analyses and QPCR

    NARCIS (Netherlands)

    Bodelier, P.L.E.; Kamst, M.; Meima-Franke, M.; Stralis-Pavese, N.; Bodrossy, L.

    2009-01-01

    Whole-genome amplification (WGA) using multiple displacement amplification (MDA) has recently been introduced to the field of environmental microbiology. The amplification of single-cell genomes or whole-community metagenomes decreases the minimum amount of DNA needed for subsequent molecular

  17. Highly efficient PCR assay to discriminate allelic DNA methylation status using whole genome amplification

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    Ito Takashi

    2011-06-01

    Full Text Available Abstract Background We previously developed a simple method termed HpaII-McrBC PCR (HM-PCR to discriminate allelic methylation status of the genomic sites of interest, and successfully applied it to a comprehensive analysis of CpG islands (CGIs on human chromosome 21q. However, HM-PCR requires 200 ng of genomic DNA to examine one target site, thereby precluding its application to such samples that are limited in quantity. Findings We developed HpaII-McrBC whole-genome-amplification PCR (HM-WGA-PCR that uses whole-genome-amplified DNA as the template. HM-WGA-PCR uses only 1/100th the genomic template material required for HM-PCR. Indeed, we successfully analyzed 147 CGIs by HM-WGA-PCR using only ~300 ng of DNA, whereas previous HM-PCR study had required ~30 μg. Furthermore, we confirmed that allelic methylation status revealed by HM-WGA-PCR is identical to that by HM-PCR in every case of the 147 CGIs tested, proving high consistency between the two methods. Conclusions HM-WGA-PCR would serve as a reliable alternative to HM-PCR in the analysis of allelic methylation status when the quantity of DNA available is limited.

  18. Single Cell HLA Matching Feasibility by Whole Genomic Amplification and Nested PCR

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    Xiao-hong Li; Fang-yin Meng

    2004-01-01

    @@ PCR based single-cell DNA analysis has been widely used in forensic science, preimplantation genetic diagnosis and so on. However, the original sample cannot be efficiently retrieved following single cell PCR, consequently the amount of information gained is limited. HLA system is too sophisticated that it is very hard to complete HLA typing by single cell. A Taq polymerase-based method using random primers to amplify whole genome termed as whole genome amplification (WGA) has demonstrated to be a useful method in increasing the copies of minimum sample. We establish a technique in this study to amplify HLA-A and HLA-B loci at same time in a single cell using WGA.

  19. Whole genome amplification of Chelex-extracted DNA from a single mite: a method for studying genetics of the predatory mite Phytoseiulus persimilis.

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    Konakandla, Bhanu; Park, Yoonseong; Margolies, David

    2006-01-01

    We developed and optimized a method using Chelex DNA extraction followed by whole genome amplification (WGA) to overcome problems conducting molecular genetic studies due to the limited amount of DNA obtainable from individual small organisms such as predatory mites. The DNA from a single mite, Phytoseiulus persimilis Athias-Henrot (Acari: Phytoseiidae), isolated in Chelex suspension was subjected to WGA. More than 1000-fold amplification of the DNA was achieved using as little as 0.03 ng genomic DNA template. The DNA obtained by the WGA was used for polymerase chain reaction followed by direct sequencing. From WGA DNA, nuclear DNA intergenic spacers ITS1 and ITS2 and a mitochondrial DNA 12S marker were tested in three different geographical populations of the predatory mite: California, the Netherlands, and Sicily. We found a total of four different alleles of the 12S in the Sicilian population, but no polymorphism was identified in the ITS marker. The combination of Chelex DNA extraction and WGA is thus shown to be a simple and robust technique for examining molecular markers for multiple loci by using individual mites. We conclude that the methods, Chelex extraction of DNA followed by WGA, provide a large quantity of DNA template that can be used for multiple PCR reactions useful for genetic studies requiring the genotypes of individual mites.

  20. Inability of 'Whole Genome Amplification' to Improve Success Rates for the Biomolecular Detection of Tuberculosis in Archaeological Samples.

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    Jannine Forst

    Full Text Available We assessed the ability of whole genome amplification (WGA to improve the efficiency of downstream polymerase chain reactions (PCRs directed at ancient DNA (aDNA of members of the Mycobacterium tuberculosis complex (MTBC. Using extracts from a variety of bones and a tooth from human skeletons with or without lesions indicative of tuberculosis, from multiple time periods, we obtained inconsistent results. We conclude that WGA does not provide any advantage in studies of MTBC aDNA. The sporadic nature of our results are probably due to the fact that WGA is itself a PCR-based procedure which, although designed to deal with fragmented DNA, might be inefficient with the low concentration of templates in an aDNA extract. As such, WGA is subject to similar, if not the same, restrictions as PCR when applied to aDNA.

  1. A simple method for encapsulating single cells in alginate microspheres allows for direct PCR and whole genome amplification.

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    Saharnaz Bigdeli

    Full Text Available Microdroplets are an effective platform for segregating individual cells and amplifying DNA. However, a key challenge is to recover the contents of individual droplets for downstream analysis. This paper offers a method for embedding cells in alginate microspheres and performing multiple serial operations on the isolated cells. Rhodobacter sphaeroides cells were diluted in alginate polymer and sprayed into microdroplets using a fingertip aerosol sprayer. The encapsulated cells were lysed and subjected either to conventional PCR, or whole genome amplification using either multiple displacement amplification (MDA or a two-step PCR protocol. Microscopic examination after PCR showed that the lumen of the occupied microspheres contained fluorescently stained DNA product, but multiple displacement amplification with phi29 produced only a small number of polymerase colonies. The 2-step WGA protocol was successful in generating fluorescent material, and quantitative PCR from DNA extracted from aliquots of microspheres suggested that the copy number inside the microspheres was amplified up to 3 orders of magnitude. Microspheres containing fluorescent material were sorted by a dilution series and screened with a fluorescent plate reader to identify single microspheres. The DNA was extracted from individual isolates, re-amplified with full-length sequencing adapters, and then a single isolate was sequenced using the Illumina MiSeq platform. After filtering the reads, the only sequences that collectively matched a genome in the NCBI nucleotide database belonged to R. sphaeroides. This demonstrated that sequencing-ready DNA could be generated from the contents of a single microsphere without culturing. However, the 2-step WGA strategy showed limitations in terms of low genome coverage and an uneven frequency distribution of reads across the genome. This paper offers a simple method for embedding cells in alginate microspheres and performing PCR on isolated

  2. Selective WGA uptake in the hippocampus from the locus coeruleus of DBH-WGA transgenic mice

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    Susan G eWalling

    2012-05-01

    Full Text Available We generated transgenic mice in which a transsynaptic tracer, wheat germ agglutinin (WGA, was specifically expressed in the locus coeruleus neurons under the control of the dopamine-β-hydroxylase gene promoter. WGA protein was produced in more than 95% of the tyrosine hydroxylase-positive locus coeruleus neurons sampled. Transynaptic transfer of WGA was most evident in CA3 neurons of the hippocampus, but appeared absent in CA1 neurons. Faint but significant WGA immunoreactivity was observed surrounding the nuclei of dentate granule cells. Putative hilar mossy cells, identified by the presence of calretinin in the ventral hippocampus, appeared uniformly positive for transynaptically transferred WGA protein. GAD67-positive interneurons in the hilar and CA3 regions tended to be WGA-positive, although a subset of them did not show WGA co-localization. The same mixed WGA uptake profile was apparent when examining co-localization with parvalbumin. The selective uptake of WGA by dentate granule cells, mossy cells and CA3 pyramidal neurons is consistent with evidence for a large proportion of conventional synapses adjacent to locus coeruleus axonal varicosities in these regions. The lack of WGA uptake in the CA1 region and its relatively sparse innervation by dopamine-β-hydroxylase-positive fibers suggest that a majority of the tyrosine hydroxylase-positive classical synapses revealed by electron microscopy in that region may be producing dopamine. The overall pattern of WGA uptake in these transgenic mice suggests a selective role for the granule cell-mossy cell-CA3 network in processing novelty or the salient environmental contingency changes signaled by locus coeruleus activity.

  3. Improved genome recovery and integrated cell-size analyses of individual uncultured microbial cells and viral particles.

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    Stepanauskas, Ramunas; Fergusson, Elizabeth A; Brown, Joseph; Poulton, Nicole J; Tupper, Ben; Labonté, Jessica M; Becraft, Eric D; Brown, Julia M; Pachiadaki, Maria G; Povilaitis, Tadas; Thompson, Brian P; Mascena, Corianna J; Bellows, Wendy K; Lubys, Arvydas

    2017-07-20

    Microbial single-cell genomics can be used to provide insights into the metabolic potential, interactions, and evolution of uncultured microorganisms. Here we present WGA-X, a method based on multiple displacement amplification of DNA that utilizes a thermostable mutant of the phi29 polymerase. WGA-X enhances genome recovery from individual microbial cells and viral particles while maintaining ease of use and scalability. The greatest improvements are observed when amplifying high G+C content templates, such as those belonging to the predominant bacteria in agricultural soils. By integrating WGA-X with calibrated index-cell sorting and high-throughput genomic sequencing, we are able to analyze genomic sequences and cell sizes of hundreds of individual, uncultured bacteria, archaea, protists, and viral particles, obtained directly from marine and soil samples, in a single experiment. This approach may find diverse applications in microbiology and in biomedical and forensic studies of humans and other multicellular organisms.Single-cell genomics can be used to study uncultured microorganisms. Here, Stepanauskas et al. present a method combining improved multiple displacement amplification and FACS, to obtain genomic sequences and cell size information from uncultivated microbial cells and viral particles in environmental samples.

  4. Whole Genome Amplification of Day 3 or Day 5 Human Embryos Biopsies Provides a Suitable DNA Template for PCR-Based Techniques for Genotyping, a Complement of Preimplantation Genetic Testing

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    Elizabeth Schaeffer

    2017-01-01

    Full Text Available Our objective was to determine if whole genome amplification (WGA provides suitable DNA for qPCR-based genotyping for human embryos. Single blastomeres (Day 3 or trophoblastic cells (Day 5 were isolated from 342 embryos for WGA. Comparative Genomic Hybridization determined embryo sex as well as Trisomy 18 or Trisomy 21. To determine the embryo’s sex, qPCR melting curve analysis for SRY and DYS14 was used. Logistic regression indicated a 4.4%, 57.1%, or 98.8% probability of a male embryo when neither gene, SRY only, or both genes were detected, respectively (accuracy = 94.1%, kappa = 0.882, and p<0.001. Fluorescent Capillary Electrophoresis for the amelogenin genes (AMEL was also used to determine sex. AMELY peak’s height was higher and this peak’s presence was highly predictive of male embryos (AUC = 0.93, accuracy = 81.7%, kappa = 0.974, and p<0.001. Trisomy 18 and Trisomy 21 were determined using the threshold cycle difference for RPL17 and TTC3, respectively, which were significantly lower in the corresponding embryos. The Ct difference for TTC3 specifically determined Trisomy 21 (AUC = 0.89 and RPL17 for Trisomy 18 (AUC = 0.94. Here, WGA provides adequate DNA for PCR-based techniques for preimplantation genotyping.

  5. Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing

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    Hou, Yong; Wu, Kui; Shi, Xulian

    2015-01-01

    methods, focusing particularly on variations detection. Low-coverage whole-genome sequencing revealed that DOP-PCR had the highest duplication ratio, but an even read distribution and the best reproducibility and accuracy for detection of copy-number variations (CNVs). However, MDA had significantly...... performance using SCRS amplified by different WGA methods. It will guide researchers to determine which WGA method is best suited to individual experimental needs at single-cell level....

  6. [Investigation of RNA viral genome amplification by multiple displacement amplification technique].

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    Pang, Zheng; Li, Jian-Dong; Li, Chuan; Liang, Mi-Fang; Li, De-Xin

    2013-06-01

    In order to facilitate the detection of newly emerging or rare viral infectious diseases, a negative-strand RNA virus-severe fever with thrombocytopenia syndrome bunyavirus, and a positive-strand RNA virus-dengue virus, were used to investigate RNA viral genome unspecific amplification by multiple displacement amplification technique from clinical samples. Series of 10-fold diluted purified viral RNA were utilized as analog samples with different pathogen loads, after a series of reactions were sequentially processed, single-strand cDNA, double-strand cDNA, double-strand cDNA treated with ligation without or with supplemental RNA were generated, then a Phi29 DNA polymerase depended isothermal amplification was employed, and finally the target gene copies were detected by real time PCR assays to evaluate the amplification efficiencies of various methods. The results showed that multiple displacement amplification effects of single-strand or double-strand cDNA templates were limited, while the fold increases of double-strand cDNA templates treated with ligation could be up to 6 X 10(3), even 2 X 10(5) when supplemental RNA existed, and better results were obtained when viral RNA loads were lower. A RNA viral genome amplification system using multiple displacement amplification technique was established in this study and effective amplification of RNA viral genome with low load was achieved, which could provide a tool to synthesize adequate viral genome for multiplex pathogens detection.

  7. Novel approach for deriving genome wide SNP analysis data from archived blood spots

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    2012-01-01

    Background The ability to transport and store DNA at room temperature in low volumes has the advantage of optimising cost, time and storage space. Blood spots on adapted filter papers are popular for this, with FTA (Flinders Technology Associates) Whatman™TM technology being one of the most recent. Plant material, plasmids, viral particles, bacteria and animal blood have been stored and transported successfully using this technology, however the method of porcine DNA extraction from FTA Whatman™TM cards is a relatively new approach, allowing nucleic acids to be ready for downstream applications such as PCR, whole genome amplification, sequencing and subsequent application to single nucleotide polymorphism microarrays has hitherto been under-explored. Findings DNA was extracted from FTA Whatman™TM cards (following adaptations of the manufacturer’s instructions), whole genome amplified and subsequently analysed to validate the integrity of the DNA for downstream SNP analysis. DNA was successfully extracted from 288/288 samples and amplified by WGA. Allele dropout post WGA, was observed in less than 2% of samples and there was no clear evidence of amplification bias nor contamination. Acceptable call rates on porcine SNP chips were also achieved using DNA extracted and amplified in this way. Conclusions DNA extracted from FTA Whatman cards is of a high enough quality and quantity following whole genomic amplification to perform meaningful SNP chip studies. PMID:22974252

  8. Whole genome amplification - Review of applications and advances

    Energy Technology Data Exchange (ETDEWEB)

    Hawkins, Trevor L.; Detter, J.C.; Richardson, Paul

    2001-11-15

    The concept of Whole Genome Amplification is something that has arisen in the past few years as modifications to the polymerase chain reaction (PCR) have been adapted to replicate regions of genomes which are of biological interest. The applications here are many--forensics, embryonic disease diagnosis, bio terrorism genome detection, ''imoralization'' of clinical samples, microbial diversity, and genotyping. The key question is if DNA can be replicated a genome at a time without bias or non random distribution of the target. Several papers published in the last year and currently in preparation may lead to the conclusion that whole genome amplification may indeed be possible and therefore open up a new avenue to molecular biology.

  9. Improving enrichment of circulating fetal DNA for genetic testing: size fractionation followed by whole gene amplification.

    Science.gov (United States)

    Jorgez, Carolina J; Bischoff, Farideh Z

    2009-01-01

    Among the pitfalls of using cell-free fetal DNA in plasma for prenatal diagnosis is quality of the recovered DNA fragments and concomitant presence of maternal DNA (>95%). Our objective is to provide alternative methods for achieving enrichment and high-quality fetal DNA from plasma. Cell-free DNA from 31 pregnant women and 18 controls (10 males and 8 females) were size separated using agarose gel electrophoresis. DNA fragments of 100-300, 500-700 and 1,500-2,000 bp were excised and extracted, followed by whole genome amplification (WGA) of recovered fragments. Levels of beta-globin and DYS1 were measured. Distribution of beta-globin size fragments was similar among pregnant women and controls. Among control male cases, distribution of size fragments was the same for both beta-globin and DYS1. Among maternal cases confirmed to be male, the smallest size fragment (100-300 bp) accounted for nearly 50% (39.76 +/- 17.55%) of the recovered DYS1-DNA (fetal) and only 10% (10.40 +/- 6.49%) of beta-globin (total) DNA. After WGA of plasma fragments from pregnant women, DYS1 sequence amplification was best observed when using the 100-300 bp fragments as template. Combination of electrophoresis for size separation and WGA led to enriched fetal DNA from plasma. This novel combination of strategies is more likely to permit universal clinical applications of cell-free fetal DNA. Copyright 2009 S. Karger AG, Basel.

  10. Inspecting Targeted Deep Sequencing of Whole Genome Amplified DNA Versus Fresh DNA for Somatic Mutation Detection: A Genetic Study in Myelodysplastic Syndrome Patients.

    Science.gov (United States)

    Palomo, Laura; Fuster-Tormo, Francisco; Alvira, Daniel; Ademà, Vera; Armengol, María Pilar; Gómez-Marzo, Paula; de Haro, Nuri; Mallo, Mar; Xicoy, Blanca; Zamora, Lurdes; Solé, Francesc

    2017-08-01

    Whole genome amplification (WGA) has become an invaluable method for preserving limited samples of precious stock material and has been used during the past years as an alternative tool to increase the amount of DNA before library preparation for next-generation sequencing. Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell disorders characterized by presenting somatic mutations in several myeloid-related genes. In this work, targeted deep sequencing has been performed on four paired fresh DNA and WGA DNA samples from bone marrow of MDS patients, to assess the feasibility of using WGA DNA for detecting somatic mutations. The results of this study highlighted that, in general, the sequencing and alignment statistics of fresh DNA and WGA DNA samples were similar. However, after variant calling and when considering variants detected at all frequencies, there was a high level of discordance between fresh DNA and WGA DNA (overall, a higher number of variants was detected in WGA DNA). After proper filtering, a total of three somatic mutations were detected in the cohort. All somatic mutations detected in fresh DNA were also identified in WGA DNA and validated by whole exome sequencing.

  11. Assessment of whole genome amplification-induced bias through high-throughput, massively parallel whole genome sequencing

    Directory of Open Access Journals (Sweden)

    Plant Ramona N

    2006-08-01

    Full Text Available Abstract Background Whole genome amplification is an increasingly common technique through which minute amounts of DNA can be multiplied to generate quantities suitable for genetic testing and analysis. Questions of amplification-induced error and template bias generated by these methods have previously been addressed through either small scale (SNPs or large scale (CGH array, FISH methodologies. Here we utilized whole genome sequencing to assess amplification-induced bias in both coding and non-coding regions of two bacterial genomes. Halobacterium species NRC-1 DNA and Campylobacter jejuni were amplified by several common, commercially available protocols: multiple displacement amplification, primer extension pre-amplification and degenerate oligonucleotide primed PCR. The amplification-induced bias of each method was assessed by sequencing both genomes in their entirety using the 454 Sequencing System technology and comparing the results with those obtained from unamplified controls. Results All amplification methodologies induced statistically significant bias relative to the unamplified control. For the Halobacterium species NRC-1 genome, assessed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 119 times greater than those from unamplified material, 164.0 times greater for Repli-G, 165.0 times greater for PEP-PCR and 252.0 times greater than the unamplified controls for DOP-PCR. For Campylobacter jejuni, also analyzed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 15 times greater than those from unamplified material, 19.8 times greater for Repli-G, 61.8 times greater for PEP-PCR and 220.5 times greater than the unamplified controls for DOP-PCR. Conclusion Of the amplification methodologies examined in this paper, the multiple displacement amplification products generated the least bias, and produced significantly higher yields of amplified DNA.

  12. Genome position and gene amplification

    Czech Academy of Sciences Publication Activity Database

    Jirsová, Pavla; Snijders, A.M.; Kwek, S.; Roydasgupta, R.; Fridlyand, J.; Tokuyasu, T.; Pinkel, D.; Albertson, D. G.

    2007-01-01

    Roč. 8, č. 6 (2007), r120 ISSN 1474-760X Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : gene amplification * array comparative genomic hybridization * oncogene Subject RIV: BO - Biophysics Impact factor: 6.589, year: 2007

  13. Generation of recombinant pestiviruses using a full genome amplification strategy

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Reimann, Ilona; Uttenthal, Åse

    Aim Complete genome amplification of viral RNA provides a new tool for generation of modified pestiviruses. We have recently reported a full genome amplification strategy for direct recovery of infectious pestivirus (Rasmussen et al., 2008). This comprised rescue of BDV strain “Gifhorn” from a full......-length RT-PCR amplicon demonstrating that long RT-PCR can be used for direct generation of an infectious pestivirus. The strategy is not limited to amplification of BDV “Gifhorn”, but can be further utilized for amplification of a diverse selection of pestivirus strains and for the generation of modified...... was reverse transcribed to cDNA at 50C for 90 minutes using SuperScript III reverse transcriptase (Invitrogen). Full-length PCR amplification was performed using primers specific for the extreme 5’- and 3’-ends of the viral genomes. A T7 promoter was incorporated in the 5’-primers for direct in vitro...

  14. Nanoliter reactors improve multiple displacement amplification of genomes from single cells.

    Directory of Open Access Journals (Sweden)

    Yann Marcy

    2007-09-01

    Full Text Available Since only a small fraction of environmental bacteria are amenable to laboratory culture, there is great interest in genomic sequencing directly from single cells. Sufficient DNA for sequencing can be obtained from one cell by the Multiple Displacement Amplification (MDA method, thereby eliminating the need to develop culture methods. Here we used a microfluidic device to isolate individual Escherichia coli and amplify genomic DNA by MDA in 60-nl reactions. Our results confirm a report that reduced MDA reaction volume lowers nonspecific synthesis that can result from contaminant DNA templates and unfavourable interaction between primers. The quality of the genome amplification was assessed by qPCR and compared favourably to single-cell amplifications performed in standard 50-microl volumes. Amplification bias was greatly reduced in nanoliter volumes, thereby providing a more even representation of all sequences. Single-cell amplicons from both microliter and nanoliter volumes provided high-quality sequence data by high-throughput pyrosequencing, thereby demonstrating a straightforward route to sequencing genomes from single cells.

  15. Environmental whole-genome amplification to access microbial populations in contaminated sediments

    Energy Technology Data Exchange (ETDEWEB)

    Abulencia, Carl B [Diversa Corporation; Wyborski, Denise L. [Diversa Corporation; Garcia, Joseph A. [Diversa Corporation; Podar, Mircea [ORNL; Chen, Wenqiong [Diversa Corporation; Chang, Sherman H. [Diversa Corporation; Chang, Hwai W. [Diversa Corporation; Watson, David B [ORNL; Brodie, Eoin L. [Lawrence Berkeley National Laboratory (LBNL); Hazen, Terry [Lawrence Berkeley National Laboratory (LBNL); Keller, Martin [ORNL

    2006-05-01

    Low-biomass samples from nitrate and heavy metal contaminated soils yield DNA amounts that have limited use for direct, native analysis and screening. Multiple displacement amplification (MDA) using {phi}29 DNA polymerase was used to amplify whole genomes from environmental, contaminated, subsurface sediments. By first amplifying the genomic DNA (gDNA), biodiversity analysis and gDNA library construction of microbes found in contaminated soils were made possible. The MDA method was validated by analyzing amplified genome coverage from approximately five Escherichia coli cells, resulting in 99.2% genome coverage. The method was further validated by confirming overall representative species coverage and also an amplification bias when amplifying from a mix of eight known bacterial strains. We extracted DNA from samples with extremely low cell densities from a U.S. Department of Energy contaminated site. After amplification, small-subunit rRNA analysis revealed relatively even distribution of species across several major phyla. Clone libraries were constructed from the amplified gDNA, and a small subset of clones was used for shotgun sequencing. BLAST analysis of the library clone sequences showed that 64.9% of the sequences had significant similarities to known proteins, and 'clusters of orthologous groups' (COG) analysis revealed that more than half of the sequences from each library contained sequence similarity to known proteins. The libraries can be readily screened for native genes or any target of interest. Whole-genome amplification of metagenomic DNA from very minute microbial sources, while introducing an amplification bias, will allow access to genomic information that was not previously accessible.

  16. Environmental Whole-Genome Amplification to Access Microbial Diversity in Contaminated Sediments

    Energy Technology Data Exchange (ETDEWEB)

    Abulencia, C.B.; Wyborski, D.L.; Garcia, J.; Podar, M.; Chen, W.; Chang, S.H.; Chang, H.W.; Watson, D.; Brodie,E.I.; Hazen, T.C.; Keller, M.

    2005-12-10

    Low-biomass samples from nitrate and heavy metal contaminated soils yield DNA amounts that have limited use for direct, native analysis and screening. Multiple displacement amplification (MDA) using ?29 DNA polymerase was used to amplify whole genomes from environmental, contaminated, subsurface sediments. By first amplifying the genomic DNA (gDNA), biodiversity analysis and gDNA library construction of microbes found in contaminated soils were made possible. The MDA method was validated by analyzing amplified genome coverage from approximately five Escherichia coli cells, resulting in 99.2 percent genome coverage. The method was further validated by confirming overall representative species coverage and also an amplification bias when amplifying from a mix of eight known bacterial strains. We extracted DNA from samples with extremely low cell densities from a U.S. Department of Energy contaminated site. After amplification, small subunit rRNA analysis revealed relatively even distribution of species across several major phyla. Clone libraries were constructed from the amplified gDNA, and a small subset of clones was used for shotgun sequencing. BLAST analysis of the library clone sequences showed that 64.9 percent of the sequences had significant similarities to known proteins, and ''clusters of orthologous groups'' (COG) analysis revealed that more than half of the sequences from each library contained sequence similarity to known proteins. The libraries can be readily screened for native genes or any target of interest. Whole-genome amplification of metagenomic DNA from very minute microbial sources, while introducing an amplification bias, will allow access to genomic information that was not previously accessible.

  17. Selective whole genome amplification for resequencing target microbial species from complex natural samples.

    Science.gov (United States)

    Leichty, Aaron R; Brisson, Dustin

    2014-10-01

    Population genomic analyses have demonstrated power to address major questions in evolutionary and molecular microbiology. Collecting populations of genomes is hindered in many microbial species by the absence of a cost effective and practical method to collect ample quantities of sufficiently pure genomic DNA for next-generation sequencing. Here we present a simple method to amplify genomes of a target microbial species present in a complex, natural sample. The selective whole genome amplification (SWGA) technique amplifies target genomes using nucleotide sequence motifs that are common in the target microbe genome, but rare in the background genomes, to prime the highly processive phi29 polymerase. SWGA thus selectively amplifies the target genome from samples in which it originally represented a minor fraction of the total DNA. The post-SWGA samples are enriched in target genomic DNA, which are ideal for population resequencing. We demonstrate the efficacy of SWGA using both laboratory-prepared mixtures of cultured microbes as well as a natural host-microbe association. Targeted amplification of Borrelia burgdorferi mixed with Escherichia coli at genome ratios of 1:2000 resulted in >10(5)-fold amplification of the target genomes with genomic extracts from Wolbachia pipientis-infected Drosophila melanogaster resulted in up to 70% of high-throughput resequencing reads mapping to the W. pipientis genome. By contrast, 2-9% of sequencing reads were derived from W. pipientis without prior amplification. The SWGA technique results in high sequencing coverage at a fraction of the sequencing effort, thus allowing population genomic studies at affordable costs. Copyright © 2014 by the Genetics Society of America.

  18. Generation of recombinant pestiviruses using a full-genome amplification strategy

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Reimann, I.; Uttenthal, Åse

    2010-01-01

    -Gifhorn genome was generated by long RTPCR and then RNA transcripts derived from this amplicon were used to rescue infectious virus. Here, we have now used this full-genome amplification strategy for efficient and robust amplification of three additional pestivirus strains: the vaccine strain C and the virulent...... Paderborn strain of Classical swine fever virus plus the CP7 strain of Bovine viral diarrhoea virus. The amplicons were cloned directly into a stable single-copy bacterial artificial chromosome generating full-length pestivirus DNAs from which infectious RNA transcripts could be also derived....

  19. An evaluation of multiple annealing and looping based genome amplification using a synthetic bacterial community

    KAUST Repository

    Wang, Yong

    2016-02-23

    The low biomass in environmental samples is a major challenge for microbial metagenomic studies. The amplification of a genomic DNA was frequently applied to meeting the minimum requirement of the DNA for a high-throughput next-generation-sequencing technology. Using a synthetic bacterial community, the amplification efficiency of the Multiple Annealing and Looping Based Amplification Cycles (MALBAC) kit that is originally developed to amplify the single-cell genomic DNA of mammalian organisms is examined. The DNA template of 10 pg in each reaction of the MALBAC amplification may generate enough DNA for Illumina sequencing. Using 10 pg and 100 pg templates for each reaction set, the MALBAC kit shows a stable and homogeneous amplification as indicated by the highly consistent coverage of the reads from the two amplified samples on the contigs assembled by the original unamplified sample. Although GenomePlex whole genome amplification kit allows one to generate enough DNA using 100 pg of template in each reaction, the minority of the mixed bacterial species is not linearly amplified. For both of the kits, the GC-rich regions of the genomic DNA are not efficiently amplified as suggested by the low coverage of the contigs with the high GC content. The high efficiency of the MALBAC kit is supported for the amplification of environmental microbial DNA samples, and the concerns on its application are also raised to bacterial species with the high GC content.

  20. Atividades biológicas das lectinas PHA, WGA, jacalina e artocarpina Biological activities of PHA, WGA, jacalin and artocarpin lectins

    Directory of Open Access Journals (Sweden)

    Ana Maria Sell

    2000-05-01

    Full Text Available As lectinas são (glicoproteínas que se ligam a açúcares. O interesse destas moléculas na investigação científica se deve às diversas atividades biológicas a elas atribuídas. Além da identificação de grupos sangüíneos, da caracterização de microorganismos e da estimulação mitogênica de células imunes, as lectinas estão envolvidas nos processos de reconhecimento e das interações celulares. No presente trabalho, revisamos algumas atividades biológicas das lectinas PHA (fitohemaglutinina de Phaseolus vulgaris, WGA (aglutinina de germe de trigo, jacalina e artocarpina (lectinas de Artocarpus integrifolia. PHA, jacalina e artocarpina são mitogênicas para os linfócitos, estimulam a produção de citocinas endógenas, a atração e a ativação de leucócitos. WGA é uma lectina não-mitogênica, no entanto, modula a resposta de defesa estimulando a liberação de superóxido pelos neutrófilos. O entendimento das atividades biológicas permitirá o uso destas moléculas como uma ferramenta útil no diagnóstico e tratamento de muitas doenças.Lectins are ubiquitous (glycoproteins, which exhibit a specific and reversibly carbohydrate binding activity. Because of their structural complexity and variability, they can participate in many physiological functions. Besides the blood groups identification, microorganism characterization and mitogenic immune cell proliferation, they are involved in the cell recognition and cell-cell interaction. In the present work we revised some biological activities of the PHA (Phaseolus vulgaris phytohemagglutinin, WGA (wheat germ agglutinin, jacalin and artocarpin (Artocarpus integrifolia lectins. PHA, jacalin and artocarpin may stimulate lymphocyte proliferation, attract and activate mononuclear cells and stimulate endogenous citokines production. WGA is a nonmitogenic lectin, however it may stimulate neutrophil superoxide production. The understanding of the lectin biological activities may

  1. Clinical Characteristics and Outcome of Patients with Neuroblastoma Presenting Genomic Amplification of Loci Other than MYCN

    Science.gov (United States)

    Guimier, Anne; Ferrand, Sandrine; Pierron, Gaëlle; Couturier, Jérôme; Janoueix-Lerosey, Isabelle; Combaret, Valérie; Mosseri, Véronique; Thebaud, Estelle; Gambart, Marion; Plantaz, Dominique; Marabelle, Aurélien; Coze, Carole; Rialland, Xavier; Fasola, Sylvie; Lapouble, Eve; Fréneaux, Paul; Peuchmaur, Michel; Michon, Jean; Delattre, Olivier; Schleiermacher, Gudrun

    2014-01-01

    Background Somatically acquired genomic alterations with MYCN amplification (MNA) are key features of neuroblastoma (NB), the most common extra-cranial malignant tumour of childhood. Little is known about the frequency, clinical characteristics and outcome of NBs harbouring genomic amplification(s) distinct from MYCN. Methods Genomic profiles of 1100 NBs from French centres studied by array-CGH were re-examined specifically to identify regional amplifications. Patients were included if amplifications distinct from the MYCN locus were seen. A subset of NBs treated at Institut Curie and harbouring MNA as determined by array-CGH without other amplification was also studied. Clinical and histology data were retrospectively collected. Results In total, 56 patients were included and categorised into 3 groups. Group 1 (n = 8) presented regional amplification(s) without MNA. Locus 12q13-14 was a recurrent amplified region (4/8 cases). This group was heterogeneous in terms of INSS stages, primary localisations and histology, with atypical clinical features. Group 2 (n = 26) had MNA as well as other regional amplifications. These patients shared clinical features of those of a group of NBs MYCN amplified (Group 3, n = 22). Overall survival for group 1 was better than that of groups 2 and 3 (5 year OS: 87.5%±11% vs 34.9%±7%, log-rank p<0.05). Conclusion NBs harbouring regional amplification(s) without MNA are rare and seem to show atypical features in clinical presentation and genomic profile. Further high resolution genetic explorations are justified in this heterogeneous group, especially when considering these alterations as predictive markers for targeted therapy. PMID:25013904

  2. Clinical characteristics and outcome of patients with neuroblastoma presenting genomic amplification of loci other than MYCN.

    Directory of Open Access Journals (Sweden)

    Anne Guimier

    Full Text Available Somatically acquired genomic alterations with MYCN amplification (MNA are key features of neuroblastoma (NB, the most common extra-cranial malignant tumour of childhood. Little is known about the frequency, clinical characteristics and outcome of NBs harbouring genomic amplification(s distinct from MYCN.Genomic profiles of 1100 NBs from French centres studied by array-CGH were re-examined specifically to identify regional amplifications. Patients were included if amplifications distinct from the MYCN locus were seen. A subset of NBs treated at Institut Curie and harbouring MNA as determined by array-CGH without other amplification was also studied. Clinical and histology data were retrospectively collected.In total, 56 patients were included and categorised into 3 groups. Group 1 (n = 8 presented regional amplification(s without MNA. Locus 12q13-14 was a recurrent amplified region (4/8 cases. This group was heterogeneous in terms of INSS stages, primary localisations and histology, with atypical clinical features. Group 2 (n = 26 had MNA as well as other regional amplifications. These patients shared clinical features of those of a group of NBs MYCN amplified (Group 3, n = 22. Overall survival for group 1 was better than that of groups 2 and 3 (5 year OS: 87.5%±11% vs 34.9%±7%, log-rank p<0.05.NBs harbouring regional amplification(s without MNA are rare and seem to show atypical features in clinical presentation and genomic profile. Further high resolution genetic explorations are justified in this heterogeneous group, especially when considering these alterations as predictive markers for targeted therapy.

  3. WGA-Alexa transsynaptic labeling in the phrenic motor system of adult rats: Intrapleural injection versus intradiaphragmatic injection.

    Science.gov (United States)

    Buttry, Janelle L; Goshgarian, Harry G

    2015-02-15

    Intrapleural injection of CTB-Alexa 488, a retrograde tracer, provides an alternative labeling technique to the surgically invasive laparotomy required for intradiaphragmatic injection. However, CTB-Alexa 488 is incapable of crossing synapses restricting the tracer to the phrenic nuclei and the intercostal motor nuclei in the spinal cord. Intrapleural injection of WGA-Alexa 488, a transsynaptic tracer, provides a method to label the respiratory motor pathway in both the spinal cord and medulla. Intradiaphragmatic injection of WGA-Alexa 594 and vagal nerve injections of True blue were used to confirm the phrenic nuclei and to differentiate between the rVRG and the NA in the medulla. Following intrapleural injection, WGA-Alexa 488 was retrogradely transported to the phrenic nuclei and to the intercostal motor nuclei. Subsequently WGA-Alexa 488 was transsynaptically transported from the phrenic motoneurons to the pre-motor neurons in the rVRG that provide the descending drive to the phrenic neurons during inspiration. In addition WGA-Alexa 488 was identified in select cells of the NA confirmed by a dual label of both WGA-Alexa 488 and True blue. WGA-Alexa 488 demonstrates retrograde transsynaptic labeling following intrapleural injection whereas the previous method of injecting CTB-Alexa 488 only demonstrates retrograde labeling. Intrapleural injection of WGA-Alexa fluor conjugates is an effective method to transsynaptically label the phrenic motor system providing an alternative for the invasive laparotomy required for intradiaphragmatic injections. Furthermore, the study provides the first anatomical evidence of a direct synaptic relationship between rVRG and select NA cells. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Digital Droplet Multiple Displacement Amplification (ddMDA for Whole Genome Sequencing of Limited DNA Samples.

    Directory of Open Access Journals (Sweden)

    Minsoung Rhee

    Full Text Available Multiple displacement amplification (MDA is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently, the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an E. coli genome per droplet, ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology.

  5. 多重置换扩增在植入前遗传学诊断中的应用及展望%Applications and prospect of multiple displacement amplification in preimplantation genetic diagnosis

    Institute of Scientific and Technical Information of China (English)

    张印峰; 罗海宁; 黎小佩; 张云山

    2012-01-01

    多重置换扩增是一种新兴的全基因组扩增技术,能对单个细胞进行全基因扩增,产生大量的优质DNA,具有高扩增效率和高保真性等特点.多重置换扩增联合常规PCR已被成功用于植入前遗传学诊断,进一步扩展了后者的应用范围.%Multiple displacement amplification (MDA) is a new technology for whole genome amplification (WGA),which can generate large amount of high-quality DNA and features high amplification efficiency and fidelity.MDA combined with conventional PCR techniques has been successfully applied for preimplantation genetic diagnosis,which has broaden latter's clinical applications.

  6. Genomic Amplification of an Endogenous Retrovirus in Zebrafish T-Cell Malignancies

    Directory of Open Access Journals (Sweden)

    J. Kimble Frazer

    2012-01-01

    Full Text Available Genomic instability plays a crucial role in oncogenesis. Somatically acquired mutations can disable some genes and inappropriately activate others. In addition, chromosomal rearrangements can amplify, delete, or even fuse genes, altering their functions and contributing to malignant phenotypes. Using array comparative genomic hybridization (aCGH, a technique to detect numeric variations between different DNA samples, we examined genomes from zebrafish (Danio rerio T-cell leukemias of three cancer-prone lines. In all malignancies tested, we identified recurring amplifications of a zebrafish endogenous retrovirus. This retrovirus, ZFERV, was first identified due to high expression of proviral transcripts in thymic tissue from larval and adult fish. We confirmed ZFERV amplifications by quantitative PCR analyses of DNA from wild-type fish tissue and normal and malignant D. rerio T cells. We also quantified ZFERV RNA expression and found that normal and neoplastic T cells both produce retrovirally encoded transcripts, but most cancers show dramatically increased transcription. In aggregate, these data imply that ZFERV amplification and transcription may be related to T-cell leukemogenesis. Based on these data and ZFERV’s phylogenetic relation to viruses of the murine-leukemia-related virus class of gammaretroviridae, we posit that ZFERV may be oncogenic via an insertional mutagenesis mechanism.

  7. Development of a multiplex polymerase chain reaction-sequence-specific primer method for NKG2D and NKG2F single-nucleotide polymorphism typing using isothermal multiple displacement amplification products.

    Science.gov (United States)

    Kaewmanee, M; Phoksawat, W; Romphruk, A; Romphruk, A V; Jumnainsong, A; Leelayuwat, C

    2013-06-01

    Natural killer group 2 member D (NKG2D) on immune effector cells recognizes multiple stress-inducible ligands. NKG2D single-nucleotide polymorphism (SNP) haplotypes were related to the levels of cytotoxic activity of peripheral blood mononuclear cells. Indeed, these polymorphisms were also located in NKG2F. Isothermal multiple displacement amplification (IMDA) is used for whole genome amplification (WGA) that can amplify very small genomic DNA templates into microgram with whole genome coverage. This is particularly useful in the cases of limited amount of valuable DNA samples requiring multi-locus genotyping. In this study, we evaluated the quality and applicability of IMDA to genetic studies in terms of sensitivity, efficiency of IMDA re-amplification and stability of IMDA products. The smallest amount of DNA to be effectively amplified by IMDA was 200 pg yielding final DNA of approximately 16 µg within 1.5 h. IMDA could be re-amplified only once (second round of amplification), and could be kept for 5 months at 4°C and more than a year at -20°C without loosing genome coverage. The amplified products were used successfully to setup a multiplex polymerase chain reaction-sequence-specific primer for SNP typing of the NKG2D/F genes. The NKG2D/F multiplex polymerase chain reaction (PCR) contained six PCR mixtures for detecting 10 selected SNPs, including 8 NKG2D/F SNP haplotypes and 2 additional NKG2D coding SNPs. This typing procedure will be applicable in both clinical and research laboratories. Thus, our data provide useful information and limitations for utilization of genome-wide amplification using IMDA and its application for multiplex NKG2D/F typing. © 2013 John Wiley & Sons Ltd.

  8. Amplification of HER2 is a marker for global genomic instability

    International Nuclear Information System (INIS)

    Ellsworth, Rachel E; Ellsworth, Darrell L; Patney, Heather L; Deyarmin, Brenda; Love, Brad; Hooke, Jeffrey A; Shriver, Craig D

    2008-01-01

    Genomic alterations of the proto-oncogene c-erbB-2 (HER-2/neu) are associated with aggressive behavior and poor prognosis in patients with breast cancer. The variable clinical outcomes seen in patients with similar HER2 status, given similar treatments, suggests that the effects of amplification of HER2 can be influenced by other genetic changes. To assess the broader genomic implications of structural changes at the HER2 locus, we investigated relationships between genomic instability and HER2 status in patients with invasive breast cancer. HER2 status was determined using the PathVysion ® assay. DNA was extracted after laser microdissection from the 181 paraffin-embedded HER2 amplified (n = 39) or HER2 negative (n = 142) tumor specimens with sufficient tumor available to perform molecular analysis. Allelic imbalance (AI) was assessed using a panel of microsatellite markers representing 26 chromosomal regions commonly altered in breast cancer. Student t-tests and partial correlations were used to investigate relationships between genomic instability and HER2 status. The frequency of AI was significantly higher (P < 0.005) in HER2 amplified (27%) compared to HER2 negative tumors (19%). Samples with HER2 amplification showed significantly higher levels of AI (P < 0.05) at chromosomes 11q23, 16q22-q24 and 18q21. Partial correlations including ER status and tumor grade supported associations between HER2 status and alterations at 11q13.1, 16q22-q24 and 18q21. The poor prognosis associated with HER2 amplification may be attributed to global genomic instability as cells with high frequencies of chromosomal alterations have been associated with increased cellular proliferation and aggressive behavior. In addition, high levels of DNA damage may render tumor cells refractory to treatment. In addition, specific alterations at chromosomes 11q13, 16q22-q24, and 18q21, all of which have been associated with aggressive tumor behavior, may serve as genetic modifiers to HER2

  9. Amplification of HER2 is a marker for global genomic instability

    Directory of Open Access Journals (Sweden)

    Love Brad

    2008-10-01

    Full Text Available Abstract Background Genomic alterations of the proto-oncogene c-erbB-2 (HER-2/neu are associated with aggressive behavior and poor prognosis in patients with breast cancer. The variable clinical outcomes seen in patients with similar HER2 status, given similar treatments, suggests that the effects of amplification of HER2 can be influenced by other genetic changes. To assess the broader genomic implications of structural changes at the HER2 locus, we investigated relationships between genomic instability and HER2 status in patients with invasive breast cancer. Methods HER2 status was determined using the PathVysion® assay. DNA was extracted after laser microdissection from the 181 paraffin-embedded HER2 amplified (n = 39 or HER2 negative (n = 142 tumor specimens with sufficient tumor available to perform molecular analysis. Allelic imbalance (AI was assessed using a panel of microsatellite markers representing 26 chromosomal regions commonly altered in breast cancer. Student t-tests and partial correlations were used to investigate relationships between genomic instability and HER2 status. Results The frequency of AI was significantly higher (P P Conclusion The poor prognosis associated with HER2 amplification may be attributed to global genomic instability as cells with high frequencies of chromosomal alterations have been associated with increased cellular proliferation and aggressive behavior. In addition, high levels of DNA damage may render tumor cells refractory to treatment. In addition, specific alterations at chromosomes 11q13, 16q22-q24, and 18q21, all of which have been associated with aggressive tumor behavior, may serve as genetic modifiers to HER2 amplification. These data not only improve our understanding of HER in breast pathogenesis but may allow more accurate risk profiles and better treatment options to be developed.

  10. Improved acid tolerance of Lactobacillus pentosus by error-prone whole genome amplification.

    Science.gov (United States)

    Ye, Lidan; Zhao, Hua; Li, Zhi; Wu, Jin Chuan

    2013-05-01

    Acid tolerance of Lactobacillus pentosus ATCC 8041 was improved by error-prone amplification of its genomic DNA using random primers and Taq DNA polymerase. The resulting amplification products were transferred into wild-type L. pentosus by electroporation and the transformants were screened for growth on low-pH agar plates. After only one round of mutation, one mutant (MT3) was identified that was able to completely consume 20 g/L of glucose to produce lactic acid at a yield of 95% in 1L MRS medium at pH 3.8 within 36 h, whereas no growth or lactic acid production was observed for the wild-type strain under the same conditions. The acid tolerance of mutant MT3 remained genetically stable for at least 25 subcultures. Therefore, the error-prone whole genome amplification technique is a very powerful tool for improving phenotypes of this lactic acid bacterium and may also be applicable for other microorganisms. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. An evaluation of multiple annealing and looping based genome amplification using a synthetic bacterial community

    KAUST Repository

    Wang, Yong; Gao, Zhaoming; Xu, Ying; Li, Guangyu; He, Lisheng; Qian, Peiyuan

    2016-01-01

    -generation-sequencing technology. Using a synthetic bacterial community, the amplification efficiency of the Multiple Annealing and Looping Based Amplification Cycles (MALBAC) kit that is originally developed to amplify the single-cell genomic DNA of mammalian organisms

  12. Chromosomal Localization of DNA Amplifications in Neuroblastoma Tumors Using cDNA Microarray Comparative Genomic Hybridization

    Directory of Open Access Journals (Sweden)

    Ben Beheshti

    2003-01-01

    Full Text Available Conventional comparative genomic hybridization (CGH profiling of neuroblastomas has identified many genomic aberrations, although the limited resolution has precluded a precise localization of sequences of interest within amplicons. To map high copy number genomic gains in clinically matched stage IV neuroblastomas, CGH analysis using a 19,200-feature cDNA microarray was used. A dedicated (freely available algorithm was developed for rapid in silico determination of chromosomal localizations of microarray cDNA targets, and for generation of an ideogram-type profile of copy number changes. Using these methodologies, novel gene amplifications undetectable by chromosome CGH were identified, and larger MYCN amplicon sizes (in one tumor up to 6 Mb than those previously reported in neuroblastoma were identified. The genes HPCAL1, LPIN1/KIAA0188, NAG, and NSE1/LOC151354 were found to be coamplified with MYCN. To determine whether stage IV primary tumors could be further subclassified based on their genomic copy number profiles, hierarchical clustering was performed. Cluster analysis of microarray CGH data identified three groups: 1 no amplifications evident, 2 a small MYCN amplicon as the only detectable imbalance, and 3 a large MYCN amplicon with additional gene amplifications. Application of CGH to cDNA microarray targets will help to determine both the variation of amplicon size and help better define amplification-dependent and independent pathways of progression in neuroblastoma.

  13. Identification of nine genomic regions of amplification in urothelial carcinoma, correlation with stage, and potential prognostic and therapeutic value.

    Directory of Open Access Journals (Sweden)

    Yvonne Chekaluk

    Full Text Available We performed a genome wide analysis of 164 urothelial carcinoma samples and 27 bladder cancer cell lines to identify copy number changes associated with disease characteristics, and examined the association of amplification events with stage and grade of disease. Multiplex inversion probe (MIP analysis, a recently developed genomic technique, was used to study 80 urothelial carcinomas to identify mutations and copy number changes. Selected amplification events were then analyzed in a validation cohort of 84 bladder cancers by multiplex ligation-dependent probe assay (MLPA. In the MIP analysis, 44 regions of significant copy number change were identified using GISTIC. Nine gene-containing regions of amplification were selected for validation in the second cohort by MLPA. Amplification events at these 9 genomic regions were found to correlate strongly with stage, being seen in only 2 of 23 (9% Ta grade 1 or 1-2 cancers, in contrast to 31 of 61 (51% Ta grade 3 and T2 grade 2 cancers, p<0.001. These observations suggest that analysis of genomic amplification of these 9 regions might help distinguish non-invasive from invasive urothelial carcinoma, although further study is required. Both MIP and MLPA methods perform well on formalin-fixed paraffin-embedded DNA, enhancing their potential clinical use. Furthermore several of the amplified genes identified here (ERBB2, MDM2, CCND1 are potential therapeutic targets.

  14. Sequencing Single Cell Microbial Genomes with Microfluidic Amplifications Tools (MICW - Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

    Energy Technology Data Exchange (ETDEWEB)

    Quake, Steve

    2011-10-12

    Stanford University's Steve Quake on "Sequencing Single Cell Microbial Genomes with Microfluidic Amplification Tools" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

  15. A subset of herpes simplex virus replication genes induces DNA amplification within the host cell genome

    Energy Technology Data Exchange (ETDEWEB)

    Heilbronn, R.; zur Hausen, H. (Deutsches Krebsforschungszentrum, Heidelberg (West Germany))

    1989-09-01

    Herpes simplex virus (HSV) induces DNA amplification of target genes within the host cell chromosome. To characterize the HSV genes that mediate the amplification effect, combinations of cloned DNA fragments covering the entire HSV genome were transiently transfected into simian virus 40 (SV40)-transformed hamster cells. This led to amplification of the integrated SV40 DNA sequences to a degree comparable to that observed after transfection of intact virion DNA. Transfection of combinations of subclones and of human cytomegalovirus immediate-early promoter-driven expression constructs for individual open reading frames led to the identification of sic HSV genes which together were necessary and sufficient for the induction of DNA amplification: UL30 (DNA polymerase), UL29 (major DNA-binding protein), UL5, UL8, UL42, and UL52. All of these genes encode proteins necessary for HSV DNA replication. However, an additional gene coding for an HSV origin-binding protein (UL9) was required for origin-dependent HSV DNA replication but was dispensable for SV40 DNA amplification. The results show that a subset of HSV replication genes is sufficient for the induction of DNA amplification. This opens the possibility that HSV expresses functions sufficient for DNA amplification but separate from those responsible for lytic viral growth. HSV infection may thereby induce DNA amplification within the host cell genome without killing the host by lytic viral growth. This may lead to persistence of a cell with a new genetic phenotype, which would have implications for the pathogenicity of the virus in vivo.

  16. The pattern and extent of retrograde transsynaptic transport of WGA-Alexa 488 in the phrenic motor system is dependent upon the site of application.

    Science.gov (United States)

    Goshgarian, Harry G; Buttry, Janelle L

    2014-01-30

    The first aim of the study was to determine if WGA-Alexa 488 would undergo retrograde transsynaptic transport in the phrenic motor system as we have shown with WGA-HRP in a previous study. The advantage of using WGA-Alexa 488 is that labeled neurons could be isolated and analyzed for intracellular molecular mechanisms without exposing tissue sections to chemicals for histochemical staining. The second aim of the study was to investigate the pattern and extent of labeling that occurs when WGA-Alexa 488 is applied to the cervical phrenic nerve as compared to intradiaphragmatic injection. After injecting the hemidiaphragm ipsilateral to a C2 spinal cord hemisection, WGA-Alexa 488 presumably diffused to the contralateral hemidiaphragm and labeled the phrenic nuclei bilaterally. In all animals with hemidiaphragmatic injection, the rostral ventral respiratory group (rVRG) was also labeled bilaterally in the medulla. Thus, injection of WGA-Alexa 488 into the diaphragm results in retrograde transsynaptic transport in the phrenic motor system. After applying WGA-Alexa 488 to the ipsilateral intact cervical phrenic nerve in both C2 hemisected rats and rats with a sham hemisection, only ipsilateral phrenic neurons were labeled; there was no labeling of the rVRG or any other center in the medulla. These results suggest that WGA-Alexa 488 must be applied in the vicinity of the phrenic myoneural junction where there is a high concentration of WGA receptors in order for transsynaptic transport to occur. The present study provides investigators with a new tool to study plasticity in the respiratory system after spinal cord injury. Copyright © 2013. Published by Elsevier B.V.

  17. Systematic evaluation of bias in microbial community profiles induced by whole genome amplification

    NARCIS (Netherlands)

    Direito, S.O.L.; Zaura, E.; Little, M.; Ehrenfreund, P.; Röling, W.F.M.

    2014-01-01

    Whole genome amplification methods facilitate the detection and characterization of microbial communities in low biomass environments. We examined the extent to which the actual community structure is reliably revealed and factors contributing to bias. One widely used [multiple displacement

  18. Systematic evaluation of bias in microbial community profiles induced by whole genome amplification.

    NARCIS (Netherlands)

    Direito, S.; Zaura, E.; Little, M.; Ehrenfreund, P.; Roling, W.F.M.

    2014-01-01

    Whole genome amplification methods facilitate the detection and characterization of microbial communities in low biomass environments. We examined the extent to which the actual community structure is reliably revealed and factors contributing to bias. One widely used [multiple displacement

  19. Specific single-cell isolation and genomic amplification of uncultured microorganisms

    DEFF Research Database (Denmark)

    Kvist, Thomas; Ahring, Birgitte Kiær; Lasken, R.S.

    2007-01-01

    We in this study describe a new method for genomic studies of individual uncultured prokaryotic organisms, which was used for the isolation and partial genome sequencing of a soil archaeon. The diversity of Archaea in a soil sample was mapped by generating a clone library using group-specific pri......We in this study describe a new method for genomic studies of individual uncultured prokaryotic organisms, which was used for the isolation and partial genome sequencing of a soil archaeon. The diversity of Archaea in a soil sample was mapped by generating a clone library using group......-specific primers in combination with a terminal restriction fragment length polymorphism profile. Intact cells were extracted from the environmental sample, and fluorescent in situ hybridization probing with Cy3-labeled probes designed from the clone library was subsequently used to detect the organisms...... of interest. Single cells with a bright fluorescent signal were isolated using a micromanipulator and the genome of the single isolated cells served as a template for multiple displacement amplification (MDA) using the Phi29 DNA polymerase. The generated MDA product was afterwards used for 16S rRNA gene...

  20. Trypanosomosis in The Gambia: prevalence in working horses and donkeys detected by whole genome amplification and PCR, and evidence for interactions between trypanosome species

    Directory of Open Access Journals (Sweden)

    Jallow Jibril

    2008-02-01

    Full Text Available Abstract Background The Gambia has an increasing population of equidae largely used for agriculture and transportation. A review of cases at The Gambian Horse and Donkey Trust (GHDT indicated that a common reason for presentation is a poorly defined medical condition often attributed to trypanosomosis. There are few reports describing the prevalence or the range of clinical signs associated with infection with different species of trypanosomes in horses and donkeys, but given the importance of these animals, the role of trypanosomosis requires investigation. Results In total 241 animals from the Central River Division in The Gambia (183 horses and 58 donkeys were screened using Whole Genome Amplification (WGA followed by trypanosome species identification using polymerase chain reaction (PCR. The results indicated overall trypanosome prevalence of 91%; with an infection rate of 31% for Trypanosoma congolense Savannah, 87% for Trypanosoma vivax and 18% for Trypanosoma brucei sp. Multiple species were present in 43% of infections. Microscopy had a good specificity (100% and positive predictive value (100% for trypanosome detection, but the sensitivity (20% and negative predictive value (10.5% were low relative to PCR-based diagnosis. Infection with T congolense showed the greatest negative effect on packed cell volume (PCV, while infection with T. brucei sp also had a significant, although lesser, negative effect on PCV. In addition, cases positive by microscopy were associated with significantly lower PCV. However, concurrent infection with T. vivax appeared to cause less effect on PCV, compared to animals infected with T. congolense alone. Conclusion The prevalence of Trypanosomosis was high in both horses and donkeys. Infection with T. congolense appeared to have the greatest clinical significance, while T. vivax infection may be of limited clinical significance in this population. Indeed, there is evidence of T. vivax co-infection ameliorating

  1. Quantification of trace-level DNA by real-time whole genome amplification.

    Science.gov (United States)

    Kang, Min-Jung; Yu, Hannah; Kim, Sook-Kyung; Park, Sang-Ryoul; Yang, Inchul

    2011-01-01

    Quantification of trace amounts of DNA is a challenge in analytical applications where the concentration of a target DNA is very low or only limited amounts of samples are available for analysis. PCR-based methods including real-time PCR are highly sensitive and widely used for quantification of low-level DNA samples. However, ordinary PCR methods require at least one copy of a specific gene sequence for amplification and may not work for a sub-genomic amount of DNA. We suggest a real-time whole genome amplification method adopting the degenerate oligonucleotide primed PCR (DOP-PCR) for quantification of sub-genomic amounts of DNA. This approach enabled quantification of sub-picogram amounts of DNA independently of their sequences. When the method was applied to the human placental DNA of which amount was accurately determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES), an accurate and stable quantification capability for DNA samples ranging from 80 fg to 8 ng was obtained. In blind tests of laboratory-prepared DNA samples, measurement accuracies of 7.4%, -2.1%, and -13.9% with analytical precisions around 15% were achieved for 400-pg, 4-pg, and 400-fg DNA samples, respectively. A similar quantification capability was also observed for other DNA species from calf, E. coli, and lambda phage. Therefore, when provided with an appropriate standard DNA, the suggested real-time DOP-PCR method can be used as a universal method for quantification of trace amounts of DNA.

  2. Novel degenerate PCR method for whole genome amplification applied to Peru Margin (ODP Leg 201 subsurface samples

    Directory of Open Access Journals (Sweden)

    Amanda eMartino

    2012-01-01

    Full Text Available A degenerate PCR-based method of whole-genome amplification, designed to work fluidly with 454 sequencing technology, was developed and tested for use on deep marine subsurface DNA samples. The method, which we have called Random Amplification Metagenomic PCR (RAMP, involves the use of specific primers from Roche 454 amplicon sequencing, modified by the addition of a degenerate region at the 3’ end. It utilizes a PCR reaction, which resulted in no amplification from blanks, even after 50 cycles of PCR. After efforts to optimize experimental conditions, the method was tested with DNA extracted from cultured E. coli cells, and genome coverage was estimated after sequencing on three different occasions. Coverage did not vary greatly with the different experimental conditions tested, and was around 62% with a sequencing effort equivalent to a theoretical genome coverage of 14.10X. The GC content of the sequenced amplification product was within 2% of the predicted values for this strain of E. coli. The method was also applied to DNA extracted from marine subsurface samples from ODP Leg 201 site 1229 (Peru Margin, and results of a taxonomic analysis revealed microbial communities dominated by Proteobacteria, Chloroflexi, Firmicutes, Euryarchaeota, and Crenarchaeota, among others. These results were similar to those obtained previously for those samples; however, variations in the proportions of taxa show that community analysis can be sensitive to both the amplification technique used and the method of assigning sequences to taxonomic groups. Overall, we find that RAMP represents a valid methodology for amplifying metagenomes from low biomass samples.

  3. Novel Degenerate PCR Method for Whole-Genome Amplification Applied to Peru Margin (ODP Leg 201) Subsurface Samples

    Science.gov (United States)

    Martino, Amanda J.; Rhodes, Matthew E.; Biddle, Jennifer F.; Brandt, Leah D.; Tomsho, Lynn P.; House, Christopher H.

    2011-01-01

    A degenerate polymerase chain reaction (PCR)-based method of whole-genome amplification, designed to work fluidly with 454 sequencing technology, was developed and tested for use on deep marine subsurface DNA samples. While optimized here for use with Roche 454 technology, the general framework presented may be applicable to other next generation sequencing systems as well (e.g., Illumina, Ion Torrent). The method, which we have called random amplification metagenomic PCR (RAMP), involves the use of specific primers from Roche 454 amplicon sequencing, modified by the addition of a degenerate region at the 3′ end. It utilizes a PCR reaction, which resulted in no amplification from blanks, even after 50 cycles of PCR. After efforts to optimize experimental conditions, the method was tested with DNA extracted from cultured E. coli cells, and genome coverage was estimated after sequencing on three different occasions. Coverage did not vary greatly with the different experimental conditions tested, and was around 62% with a sequencing effort equivalent to a theoretical genome coverage of 14.10×. The GC content of the sequenced amplification product was within 2% of the predicted values for this strain of E. coli. The method was also applied to DNA extracted from marine subsurface samples from ODP Leg 201 site 1229 (Peru Margin), and results of a taxonomic analysis revealed microbial communities dominated by Proteobacteria, Chloroflexi, Firmicutes, Euryarchaeota, and Crenarchaeota, among others. These results were similar to those obtained previously for those samples; however, variations in the proportions of taxa identified illustrates well the generally accepted view that community analysis is sensitive to both the amplification technique used and the method of assigning sequences to taxonomic groups. Overall, we find that RAMP represents a valid methodology for amplifying metagenomes from low-biomass samples. PMID:22319519

  4. Single-tube linear DNA amplification for genome-wide studies using a few thousand cells

    NARCIS (Netherlands)

    Shankaranarayanan, P.; Mendoza-Parra, M.A.; Gool, van W.; Trindade, L.M.; Gronemeyer, H.

    2012-01-01

    Linear amplification of DNA (LinDA) by T7 polymerase is a versatile and robust method for generating sufficient amounts of DNA for genome-wide studies with minute amounts of cells. LinDA can be coupled to a great number of global profiling technologies. Indeed, chromatin immunoprecipitation coupled

  5. Single Cell Analysis of Dystrophin and SRY Gene by Using Whole Genome Amplification

    Institute of Scientific and Technical Information of China (English)

    徐晨明; 金帆; 黄荷凤; 陶冶; 叶英辉

    2001-01-01

    Objective To develop a reliable and sensitive method for detection of sex and multiloci of Duchenne muscular dystrophy (DMD) gene in single cell Materials & methods Whole genome of single cell were amplified by using 15-base random primers (primer extension preamplification, PEP), then a small aliquot of PEP product were analyzed by using locus-specific nest PCR amplification. The procedure was evaluated by detection dystrophin exons 8, 17, 19, 44, 45, 48 and human testis-determining gene (SRY)in single lymphocytes from known sources and single blastomeres from the couples with no family history of DMD.Results The amplification efficiency rate of six dystrophin exons from single lymphocytes and single blastomeres were 97. 2% (175/180) and 100% (60/60) respectively.Results of SRY showed that 100% (15/15) amplification in single male-derived lymphocytes and 0% (0/15) amplification in single female-derived lymphocytes. Conclusion The technique of single cell PEP-nest PCR for dystrophin exons 8, 17,19, 44, 45, 48 and SRY is highly specifc. PEP-nest PCR is suitable for Preimplantation genetic diagnosis (PGD) of DMD at single cell level.

  6. Rolling circle amplification of metazoan mitochondrialgenomes

    Energy Technology Data Exchange (ETDEWEB)

    Simison, W. Brian; Lindberg, D.R.; Boore, J.L.

    2005-07-31

    Here we report the successful use of rolling circle amplification (RCA) for the amplification of complete metazoan mt genomes to make a product that is amenable to high-throughput genome sequencing techniques. The benefits of RCA over PCR are many and with further development and refinement of RCA, the sequencing of organellar genomics will require far less time and effort than current long PCR approaches.

  7. Evaluation of WGA and Concanavalin A (Con A lectin as biomarkers of hepatosplenic schistosomiasis in human biopsies with no evidence of egg-granuloma system Avaliação das lectinas WGA e Con A como biomarcadoras de esquistossomose hepatoesplênica em biopsias humanas sem evidência do sistema ovo-granuloma

    Directory of Open Access Journals (Sweden)

    Moacyr Jesus Barreto de Melo Rêgo

    2013-06-01

    Full Text Available Introduction: Colonic lesions are predominant in patients with schistosomiasis. However, carbohydrate alterations in colonic schistosomiasis remain unclear. Lectin-ligands allow us to identify changes in the saccharide patterns of cells. Methods: Biopsies of descending and rectosigmoid colon of patients were submitted to WGA and Con A lectin histochemistry. Results: WGA stained stroma and gland cells of descending colon and rectosigmoid tissues in a granular strong cytoplasmatic pattern in schistosomiasis specimens differing from normal control and Con A failing to recognize all samples analyzed. Conclusions: WGA ligands are expressed differently in patients with hepatosplenic schistosomiasis and no evidence of egg-granuloma system. Introdução: Lesões do cólon são predominantes em pacientes com esquistossomose, entretanto alterações dos carboidratos no cólon com esquistossomose permanecem desconhecidas. Ligantes de lectinas permitem a identificação das mudanças no padrão dos carboidratos celulares. Métodos: Biópsias do cólon descendente e sigmóide dos pacientes foram submetidas a histoquímica com as lectinas WGA e Con A. Resultados: WGA marcou o estroma dos tecidos das células glandulares do cólon descendente e sigmóide com um padrão citoplasmático intenso e granular em espécimes com esquistossomose diferindo do controle normal e da Con A, a qual não reconheceu nenhum tecido estudado. Cólon sem esquistossomose não apresentou marcação. Conclusões: ligantes de WGA são diferentemente expressos em pacientes com esquistossomose hepatoesplênica sem evidência de sistema ovo-granuloma.

  8. Simultaneous genomic identification and profiling of a single cell using semiconductor-based next generation sequencing

    Directory of Open Access Journals (Sweden)

    Manabu Watanabe

    2014-09-01

    Full Text Available Combining single-cell methods and next-generation sequencing should provide a powerful means to understand single-cell biology and obviate the effects of sample heterogeneity. Here we report a single-cell identification method and seamless cancer gene profiling using semiconductor-based massively parallel sequencing. A549 cells (adenocarcinomic human alveolar basal epithelial cell line were used as a model. Single-cell capture was performed using laser capture microdissection (LCM with an Arcturus® XT system, and a captured single cell and a bulk population of A549 cells (≈106 cells were subjected to whole genome amplification (WGA. For cell identification, a multiplex PCR method (AmpliSeq™ SNP HID panel was used to enrich 136 highly discriminatory SNPs with a genotype concordance probability of 1031–35. For cancer gene profiling, we used mutation profiling that was performed in parallel using a hotspot panel for 50 cancer-related genes. Sequencing was performed using a semiconductor-based bench top sequencer. The distribution of sequence reads for both HID and Cancer panel amplicons was consistent across these samples. For the bulk population of cells, the percentages of sequence covered at coverage of more than 100× were 99.04% for the HID panel and 98.83% for the Cancer panel, while for the single cell percentages of sequence covered at coverage of more than 100× were 55.93% for the HID panel and 65.96% for the Cancer panel. Partial amplification failure or randomly distributed non-amplified regions across samples from single cells during the WGA procedures or random allele drop out probably caused these differences. However, comparative analyses showed that this method successfully discriminated a single A549 cancer cell from a bulk population of A549 cells. Thus, our approach provides a powerful means to overcome tumor sample heterogeneity when searching for somatic mutations.

  9. Whole Genome Amplification and Reduced-Representation Genome Sequencing of Schistosoma japonicum Miracidia.

    Directory of Open Access Journals (Sweden)

    Jonathan A Shortt

    2017-01-01

    Full Text Available In areas where schistosomiasis control programs have been implemented, morbidity and prevalence have been greatly reduced. However, to sustain these reductions and move towards interruption of transmission, new tools for disease surveillance are needed. Genomic methods have the potential to help trace the sources of new infections, and allow us to monitor drug resistance. Large-scale genotyping efforts for schistosome species have been hindered by cost, limited numbers of established target loci, and the small amount of DNA obtained from miracidia, the life stage most readily acquired from humans. Here, we present a method using next generation sequencing to provide high-resolution genomic data from S. japonicum for population-based studies.We applied whole genome amplification followed by double digest restriction site associated DNA sequencing (ddRADseq to individual S. japonicum miracidia preserved on Whatman FTA cards. We found that we could effectively and consistently survey hundreds of thousands of variants from 10,000 to 30,000 loci from archived miracidia as old as six years. An analysis of variation from eight miracidia obtained from three hosts in two villages in Sichuan showed clear population structuring by village and host even within this limited sample.This high-resolution sequencing approach yields three orders of magnitude more information than microsatellite genotyping methods that have been employed over the last decade, creating the potential to answer detailed questions about the sources of human infections and to monitor drug resistance. Costs per sample range from $50-$200, depending on the amount of sequence information desired, and we expect these costs can be reduced further given continued reductions in sequencing costs, improvement of protocols, and parallelization. This approach provides new promise for using modern genome-scale sampling to S. japonicum surveillance, and could be applied to other schistosome species

  10. Whole Genome Amplification and Reduced-Representation Genome Sequencing of Schistosoma japonicum Miracidia.

    Science.gov (United States)

    Shortt, Jonathan A; Card, Daren C; Schield, Drew R; Liu, Yang; Zhong, Bo; Castoe, Todd A; Carlton, Elizabeth J; Pollock, David D

    2017-01-01

    In areas where schistosomiasis control programs have been implemented, morbidity and prevalence have been greatly reduced. However, to sustain these reductions and move towards interruption of transmission, new tools for disease surveillance are needed. Genomic methods have the potential to help trace the sources of new infections, and allow us to monitor drug resistance. Large-scale genotyping efforts for schistosome species have been hindered by cost, limited numbers of established target loci, and the small amount of DNA obtained from miracidia, the life stage most readily acquired from humans. Here, we present a method using next generation sequencing to provide high-resolution genomic data from S. japonicum for population-based studies. We applied whole genome amplification followed by double digest restriction site associated DNA sequencing (ddRADseq) to individual S. japonicum miracidia preserved on Whatman FTA cards. We found that we could effectively and consistently survey hundreds of thousands of variants from 10,000 to 30,000 loci from archived miracidia as old as six years. An analysis of variation from eight miracidia obtained from three hosts in two villages in Sichuan showed clear population structuring by village and host even within this limited sample. This high-resolution sequencing approach yields three orders of magnitude more information than microsatellite genotyping methods that have been employed over the last decade, creating the potential to answer detailed questions about the sources of human infections and to monitor drug resistance. Costs per sample range from $50-$200, depending on the amount of sequence information desired, and we expect these costs can be reduced further given continued reductions in sequencing costs, improvement of protocols, and parallelization. This approach provides new promise for using modern genome-scale sampling to S. japonicum surveillance, and could be applied to other schistosome species and other

  11. Multiple displacement amplification of whole genomic DNA from urediospores of Puccinia striiformis f. sp. tritici.

    Science.gov (United States)

    Zhang, R; Ma, Z H; Wu, B M

    2015-05-01

    Biotrophic fungi, such as Puccinia striiformis f. sp. tritici, because they cannot be cultured on nutrient media, to obtain adequate quantity of DNA for molecular genetic analysis, are usually propagated on living hosts, wheat plants in case of P. striiformis f. sp. tritici. The propagation process is time-, space- and labor-consuming and has been a bottleneck to molecular genetic analysis of this pathogen. In this study we evaluated multiple displacement amplification (MDA) of pathogen genomic DNA from urediospores as an alternative approach to traditional propagation of urediospores followed by DNA extraction. The quantities of pathogen genomic DNA in the products were further determined via real-time PCR with a pair of primers specific for the β-tubulin gene of P. striiformis f. sp. tritici. The amplified fragment length polymorphism (AFLP) fingerprints were also compared between the DNA products. The results demonstrated that adequate genomic DNA at fragment size larger than 23 Kb could be amplified from 20 to 30 urediospores via MDA method. The real-time PCR results suggested that although fresh urediospores collected from diseased leaves were the best, spores picked from diseased leaves stored for a prolonged period could also be used for amplification. AFLP fingerprints exhibited no significant differences between amplified DNA and DNA extracted with CTAB method, suggesting amplified DNA can represent the pathogen's genomic DNA very well. Therefore, MDA could be used to obtain genomic DNA from small precious samples (dozens of spores) for molecular genetic analysis of wheat stripe rust pathogen, and other fungi that are difficult to propagate.

  12. Comparing sequencing assays and human-machine analyses in actionable genomics for glioblastoma.

    Science.gov (United States)

    Wrzeszczynski, Kazimierz O; Frank, Mayu O; Koyama, Takahiko; Rhrissorrakrai, Kahn; Robine, Nicolas; Utro, Filippo; Emde, Anne-Katrin; Chen, Bo-Juen; Arora, Kanika; Shah, Minita; Vacic, Vladimir; Norel, Raquel; Bilal, Erhan; Bergmann, Ewa A; Moore Vogel, Julia L; Bruce, Jeffrey N; Lassman, Andrew B; Canoll, Peter; Grommes, Christian; Harvey, Steve; Parida, Laxmi; Michelini, Vanessa V; Zody, Michael C; Jobanputra, Vaidehi; Royyuru, Ajay K; Darnell, Robert B

    2017-08-01

    To analyze a glioblastoma tumor specimen with 3 different platforms and compare potentially actionable calls from each. Tumor DNA was analyzed by a commercial targeted panel. In addition, tumor-normal DNA was analyzed by whole-genome sequencing (WGS) and tumor RNA was analyzed by RNA sequencing (RNA-seq). The WGS and RNA-seq data were analyzed by a team of bioinformaticians and cancer oncologists, and separately by IBM Watson Genomic Analytics (WGA), an automated system for prioritizing somatic variants and identifying drugs. More variants were identified by WGS/RNA analysis than by targeted panels. WGA completed a comparable analysis in a fraction of the time required by the human analysts. The development of an effective human-machine interface in the analysis of deep cancer genomic datasets may provide potentially clinically actionable calls for individual patients in a more timely and efficient manner than currently possible. NCT02725684.

  13. Detection of recombinant EPO in blood and urine samples with EPO WGA MAIIA, IEF and SAR-PAGE after microdose injections.

    Science.gov (United States)

    Dehnes, Yvette; Shalina, Alexandra; Myrvold, Linda

    2013-01-01

    The misuse of microdoses of performance enhancing drugs like erythropoietin (EPO) constitutes a major challenge in doping analysis. When injected intravenously, the half-life of recombinant human EPO (rhEPO) like epoetin alfa, beta, and zeta is only a few hours and hence, the window for direct detection of rhEPO in urine is small. In order to investigate the detection window for rhEPO directly in blood and urine with a combined affinity chromatography and lateral flow immunoassay (EPO WGA MAIIA), we recruited nine healthy people who each received six intravenously injected microdoses (7.5 IU/kg) of NeoRecormon (epoetin beta) over a period of three weeks. Blood and urine samples were collected in the days following the injections and analyzed with EPO WGA MAIIA as well as the current validated methods for rhEPO; isoelectric focusing (IEF) and sarcosyl polyacrylamide gel electrophoresis (SAR-PAGE). For samples collected 18 h after a microdose, the sensitivity of the EPO WGA MAIIA assay was 100% in plasma and 87.5% in urine samples at the respective 98% specificity threshold levels. In comparison, the sensitivity in plasma and urine was 75% and 100%, respectively, with IEF, and 87.5% in plasma and 100% in urine when analyzed with SAR-PAGE. We conclude that EPO WGA MAIIA is a sensitive assay for the detection of rhEPO, with the potential of being a fast, supplemental screening assay for use in doping analysis.

  14. Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing.

    Science.gov (United States)

    Hu, Jiazhi; Meyers, Robin M; Dong, Junchao; Panchakshari, Rohit A; Alt, Frederick W; Frock, Richard L

    2016-05-01

    Unbiased, high-throughput assays for detecting and quantifying DNA double-stranded breaks (DSBs) across the genome in mammalian cells will facilitate basic studies of the mechanisms that generate and repair endogenous DSBs. They will also enable more applied studies, such as those to evaluate the on- and off-target activities of engineered nucleases. Here we describe a linear amplification-mediated high-throughput genome-wide sequencing (LAM-HTGTS) method for the detection of genome-wide 'prey' DSBs via their translocation in cultured mammalian cells to a fixed 'bait' DSB. Bait-prey junctions are cloned directly from isolated genomic DNA using LAM-PCR and unidirectionally ligated to bridge adapters; subsequent PCR steps amplify the single-stranded DNA junction library in preparation for Illumina Miseq paired-end sequencing. A custom bioinformatics pipeline identifies prey sequences that contribute to junctions and maps them across the genome. LAM-HTGTS differs from related approaches because it detects a wide range of broken end structures with nucleotide-level resolution. Familiarity with nucleic acid methods and next-generation sequencing analysis is necessary for library generation and data interpretation. LAM-HTGTS assays are sensitive, reproducible, relatively inexpensive, scalable and straightforward to implement with a turnaround time of <1 week.

  15. Preparation of genomic DNA from a single species of uncultured magnetotactic bacterium by multiple-displacement amplification.

    Science.gov (United States)

    Arakaki, Atsushi; Shibusawa, Mie; Hosokawa, Masahito; Matsunaga, Tadashi

    2010-03-01

    Magnetotactic bacteria comprise a phylogenetically diverse group that is capable of synthesizing intracellular magnetic particles. Although various morphotypes of magnetotactic bacteria have been observed in the environment, bacterial strains available in pure culture are currently limited to a few genera due to difficulties in their enrichment and cultivation. In order to obtain genetic information from uncultured magnetotactic bacteria, a genome preparation method that involves magnetic separation of cells, flow cytometry, and multiple displacement amplification (MDA) using phi29 polymerase was used in this study. The conditions for the MDA reaction using samples containing 1 to 100 cells were evaluated using a pure-culture magnetotactic bacterium, "Magnetospirillum magneticum AMB-1," whose complete genome sequence is available. Uniform gene amplification was confirmed by quantitative PCR (Q-PCR) when 100 cells were used as a template. This method was then applied for genome preparation of uncultured magnetotactic bacteria from complex bacterial communities in an aquatic environment. A sample containing 100 cells of the uncultured magnetotactic coccus was prepared by magnetic cell separation and flow cytometry and used as an MDA template. 16S rRNA sequence analysis of the MDA product from these 100 cells revealed that the amplified genomic DNA was from a single species of magnetotactic bacterium that was phylogenetically affiliated with magnetotactic cocci in the Alphaproteobacteria. The combined use of magnetic separation, flow cytometry, and MDA provides a new strategy to access individual genetic information from magnetotactic bacteria in environmental samples.

  16. An efficient and high fidelity method for amplification, cloning and sequencing of complete tospovirus genomic RNA segments

    Science.gov (United States)

    Amplification and sequencing of the complete M- and S-RNA segments of Tomato spotted wilt virus and Impatiens necrotic spot virus as a single fragment is useful for whole genome sequencing of tospoviruses co-infecting a single host plant. It avoids issues associated with overlapping amplicon-based ...

  17. Whole-genome amplified DNA from stored dried blood spots is reliable in high resolution melting curve and sequencing analysis

    DEFF Research Database (Denmark)

    Winkel, Bo G; Hollegaard, Mads V; Olesen, Morten S

    2011-01-01

    BACKGROUND: The use of dried blood spots (DBS) samples in genomic workup has been limited by the relative low amounts of genomic DNA (gDNA) they contain. It remains to be proven that whole genome amplified DNA (wgaDNA) from stored DBS samples, constitutes a reliable alternative to gDNA.We wanted...

  18. Improved multiple displacement amplification (iMDA) and ultraclean reagents.

    Science.gov (United States)

    Motley, S Timothy; Picuri, John M; Crowder, Chris D; Minich, Jeremiah J; Hofstadler, Steven A; Eshoo, Mark W

    2014-06-06

    Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; however, many relevant samples are comprised of only a few cells. Genomic analysis of these samples requires a whole genome amplification method that is unbiased and free of exogenous DNA contamination. To address these challenges we have developed protocols for the production of DNA-free consumables including reagents and have improved upon multiple displacement amplification (iMDA). A specialized ethylene oxide treatment was developed that renders free DNA and DNA present within Gram positive bacterial cells undetectable by qPCR. To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed. Our multiple displacement amplification protocol employs a second strand-displacing DNA polymerase, improved buffers, improved reaction conditions and DNA free reagents. The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA. The sensitivity and specificity of sequencing of amplified DNA prepared using iMDA was compared to that of DNA obtained with two commercial whole genome amplification kits using 10 fg (~1-2 bacterial cells worth) of bacterial genomic DNA as a template. Analysis showed >99% of the iMDA reads mapped to the template organism whereas only 0.02% of the reads from the commercial kits mapped to the template. To assess the ability of iMDA to achieve balanced genomic coverage, a non-stochastic amount of bacterial genomic DNA (1 pg) was amplified and sequenced, and data obtained were compared to sequencing data obtained directly from genomic DNA. The iMDA DNA and genomic DNA sequencing had comparable coverage 99.98% of the reference genome at ≥1X coverage and 99.9% at ≥5X coverage while maintaining both balance

  19. Asexual populations of the human malaria parasite, Plasmodium falciparum, use a two-step genomic strategy to acquire accurate, beneficial DNA amplifications.

    Directory of Open Access Journals (Sweden)

    Jennifer L Guler

    Full Text Available Malaria drug resistance contributes to up to a million annual deaths. Judicious deployment of new antimalarials and vaccines could benefit from an understanding of early molecular events that promote the evolution of parasites. Continuous in vitro challenge of Plasmodium falciparum parasites with a novel dihydroorotate dehydrogenase (DHODH inhibitor reproducibly selected for resistant parasites. Genome-wide analysis of independently-derived resistant clones revealed a two-step strategy to evolutionary success. Some haploid blood-stage parasites first survive antimalarial pressure through fortuitous DNA duplications that always included the DHODH gene. Independently-selected parasites had different sized amplification units but they were always flanked by distant A/T tracks. Higher level amplification and resistance was attained using a second, more efficient and more accurate, mechanism for head-to-tail expansion of the founder unit. This second homology-based process could faithfully tune DNA copy numbers in either direction, always retaining the unique DNA amplification sequence from the original A/T-mediated duplication for that parasite line. Pseudo-polyploidy at relevant genomic loci sets the stage for gaining additional mutations at the locus of interest. Overall, we reveal a population-based genomic strategy for mutagenesis that operates in human stages of P. falciparum to efficiently yield resistance-causing genetic changes at the correct locus in a successful parasite. Importantly, these founding events arise with precision; no other new amplifications are seen in the resistant haploid blood stage parasite. This minimizes the need for meiotic genetic cleansing that can only occur in sexual stage development of the parasite in mosquitoes.

  20. Genomic gains and losses are similar in genetic and histologic subsets of rhabdomyosarcoma, whereas amplification predominates in embryonal with anaplasia and alveolar subtypes.

    Science.gov (United States)

    Bridge, Julia A; Liu, Jian; Qualman, Stephen J; Suijkerbuijk, Ron; Wenger, Gail; Zhang, Ji; Wan, Xiaoying; Baker, K Scott; Sorensen, Poul; Barr, Frederic G

    2002-03-01

    In this investigation, we selected PAX3/FKHR and PAX7/FKHR fusion transcript-positive and -negative alveolar rhabdomyosarcomas (ARMSs) and embryonal rhabdomyosarcomas (ERMSs) with and without anaplastic features, to ascertain genomic imbalance differences and/or similarities within these histopathologic and genetic rhabdomyosarcoma (RMS) variants. Comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) studies were performed on 45 rhabdomyosarcoma specimens consisting of 23 ARMSs and 22 ERMSs (12 ERMS cases were included from an earlier study). The anaplastic variant of RMS has not previously been subjected to CGH analysis. Overall, the most prominent imbalances were gain of chromosomes or chromosomal regions 2/2q (40%), 7/7q (31%), 8/8p (53%), 11/11q (31%), 12q13-15 (49%), 13q14 (22%), and 20/20p (31%), and loss of 1p36 (27%), 3p14-21 (22%), 9q21-22 (33%), 10q22-qter (18%), 16q (27%), 17p (22%), and 22 (22%). These gains and losses were distributed equally between ARMS and ERMS histologic subtypes (excluding 7/7q and 11/11q gain that were observed chiefly in ERMS), demonstrating that these entities are similar with respect to recurrent genomic imbalances. Moreover, genomic imbalances were also evenly distributed among the ARMS fusion transcript subtypes, providing evidence for a genetic kinship despite the absence of a fusion transcript in some cases. Genomic amplification was detected in 26% and 23% of the ARMS and ERMS cases, respectively (with nearly all of the latter subset exhibiting anaplastic features). One amplicon, involving 15q25-26, corresponds to the locus of the insulin-like growth factor type I receptor (IGF1R) gene. Amplification of IGF1R was confirmed molecularly in the cases exhibiting a 15q25-26 amplicon. In summary, these results indicate that genomic gains and losses involve alike chromosomes with similar frequencies within the histopathologic and genetic subtypes of rhabdomyosarcoma, that genomic amplification is

  1. Modeling the amplification dynamics of human Alu retrotransposons.

    Directory of Open Access Journals (Sweden)

    Dale J Hedges

    2005-09-01

    Full Text Available Retrotransposons have had a considerable impact on the overall architecture of the human genome. Currently, there are three lineages of retrotransposons (Alu, L1, and SVA that are believed to be actively replicating in humans. While estimates of their copy number, sequence diversity, and levels of insertion polymorphism can readily be obtained from existing genomic sequence data and population sampling, a detailed understanding of the temporal pattern of retrotransposon amplification remains elusive. Here we pose the question of whether, using genomic sequence and population frequency data from extant taxa, one can adequately reconstruct historical amplification patterns. To this end, we developed a computer simulation that incorporates several known aspects of primate Alu retrotransposon biology and accommodates sampling effects resulting from the methods by which mobile elements are typically discovered and characterized. By modeling a number of amplification scenarios and comparing simulation-generated expectations to empirical data gathered from existing Alu subfamilies, we were able to statistically reject a number of amplification scenarios for individual subfamilies, including that of a rapid expansion or explosion of Alu amplification at the time of human-chimpanzee divergence.

  2. Modeling the amplification dynamics of human alu retrotransposons.

    Directory of Open Access Journals (Sweden)

    2005-09-01

    Full Text Available Retrotransposons have had a considerable impact on the overall architecture of the human genome. Currently, there are three lineages of retrotransposons (Alu, L1, and SVA that are believed to be actively replicating in humans. While estimates of their copy number, sequence diversity, and levels of insertion polymorphism can readily be obtained from existing genomic sequence data and population sampling, a detailed understanding of the temporal pattern of retrotransposon amplification remains elusive. Here we pose the question of whether, using genomic sequence and population frequency data from extant taxa, one can adequately reconstruct historical amplification patterns. To this end, we developed a computer simulation that incorporates several known aspects of primate Alu retrotransposon biology and accommodates sampling effects resulting from the methods by which mobile elements are typically discovered and characterized. By modeling a number of amplification scenarios and comparing simulation-generated expectations to empirical data gathered from existing Alu subfamilies, we were able to statistically reject a number of amplification scenarios for individual subfamilies, including that of a rapid expansion or explosion of Alu amplification at the time of human-chimpanzee divergence.

  3. High-Quality Exome Sequencing of Whole-Genome Amplified Neonatal Dried Blood Spot DNA

    DEFF Research Database (Denmark)

    Poulsen, Jesper Buchhave; Lescai, Francesco; Grove, Jakob

    2016-01-01

    Stored neonatal dried blood spot (DBS) samples from neonatal screening programmes are a valuable diagnostic and research resource. Combined with information from national health registries they can be used in population-based studies of genetic diseases. DNA extracted from neonatal DBSs can...... be amplified to obtain micrograms of an otherwise limited resource, referred to as whole-genome amplified DNA (wgaDNA). Here we investigate the robustness of exome sequencing of wgaDNA of neonatal DBS samples. We conducted three pilot studies of seven, eight and seven subjects, respectively. For each subject...... we analysed a neonatal DBS sample and corresponding adult whole-blood (WB) reference sample. Different DNA sample types were prepared for each of the subjects. Pilot 1: wgaDNA of 2x3.2mm neonatal DBSs (DBS_2x3.2) and raw DNA extract of the WB reference sample (WB_ref). Pilot 2: DBS_2x3.2, WB...

  4. Mutation and genomic amplification of the PIK3CA proto-oncogene in pituitary adenomas

    International Nuclear Information System (INIS)

    Murat, C.B.; Braga, P.B.S.; Fortes, M.A.H.Z.; Bronstein, M.D.; Corrêa-Giannella, M.L.C.; Giorgi, R.R.

    2012-01-01

    The tumorigenesis of pituitary adenomas is poorly understood. Mutations of the PIK3CA proto-oncogene, which encodes the p110-α catalytic subunit of PI3K, have been reported in various types of human cancers regarding the role of the gene in cell proliferation and survival through activation of the PI3K/Akt signaling pathway. Only one Chinese study described somatic mutations and amplification of the PIK3CA gene in a large series of pituitary adenomas. The aim of the present study was to determine genetic alterations of PIK3CA in a second series that consisted of 33 pituitary adenomas of different subtypes diagnosed by immunohistochemistry: 6 adrenocorticotropic hormone-secreting microadenomas, 5 growth hormone-secreting macroadenomas, 7 prolactin-secreting macroadenomas, and 15 nonfunctioning macroadenomas. Direct sequencing of exons 9 and 20 assessed by qPCR was employed to investigate the presence of mutations and genomic amplification defined as a copy number ≥4. Previously identified PIK3CA mutations (exon 20) were detected in four cases (12.1%). Interestingly, the Chinese study reported mutations only in invasive tumors, while we found a PIK3CA mutation in one noninvasive corticotroph microadenoma. PIK3CA amplification was observed in 21.2% (7/33) of the cases. This study demonstrates the presence of somatic mutations and amplifications of the PIK3CA gene in a second series of pituitary adenomas, corroborating the previously described involvement of the PI3K/Akt signaling pathway in the tumorigenic process of this gland

  5. Mutation and genomic amplification of the PIK3CA proto-oncogene in pituitary adenomas

    Energy Technology Data Exchange (ETDEWEB)

    Murat, C.B.; Braga, P.B.S.; Fortes, M.A.H.Z. [Laboratório de Endocrinologia Celular e Molecular (LIM-25), Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil); Bronstein, M.D. [Unidade de Neuroendocrinologia, Serviço de Endocrinologia, Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil); Corrêa-Giannella, M.L.C.; Giorgi, R.R. [Laboratório de Endocrinologia Celular e Molecular (LIM-25), Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil)

    2012-07-13

    The tumorigenesis of pituitary adenomas is poorly understood. Mutations of the PIK3CA proto-oncogene, which encodes the p110-α catalytic subunit of PI3K, have been reported in various types of human cancers regarding the role of the gene in cell proliferation and survival through activation of the PI3K/Akt signaling pathway. Only one Chinese study described somatic mutations and amplification of the PIK3CA gene in a large series of pituitary adenomas. The aim of the present study was to determine genetic alterations of PIK3CA in a second series that consisted of 33 pituitary adenomas of different subtypes diagnosed by immunohistochemistry: 6 adrenocorticotropic hormone-secreting microadenomas, 5 growth hormone-secreting macroadenomas, 7 prolactin-secreting macroadenomas, and 15 nonfunctioning macroadenomas. Direct sequencing of exons 9 and 20 assessed by qPCR was employed to investigate the presence of mutations and genomic amplification defined as a copy number ≥4. Previously identified PIK3CA mutations (exon 20) were detected in four cases (12.1%). Interestingly, the Chinese study reported mutations only in invasive tumors, while we found a PIK3CA mutation in one noninvasive corticotroph microadenoma. PIK3CA amplification was observed in 21.2% (7/33) of the cases. This study demonstrates the presence of somatic mutations and amplifications of the PIK3CA gene in a second series of pituitary adenomas, corroborating the previously described involvement of the PI3K/Akt signaling pathway in the tumorigenic process of this gland.

  6. Copy number and loss of heterozygosity detected by SNP array of formalin-fixed tissues using whole-genome amplification.

    Directory of Open Access Journals (Sweden)

    Angela Stokes

    Full Text Available The requirement for large amounts of good quality DNA for whole-genome applications prohibits their use for small, laser capture micro-dissected (LCM, and/or rare clinical samples, which are also often formalin-fixed and paraffin-embedded (FFPE. Whole-genome amplification of DNA from these samples could, potentially, overcome these limitations. However, little is known about the artefacts introduced by amplification of FFPE-derived DNA with regard to genotyping, and subsequent copy number and loss of heterozygosity (LOH analyses. Using a ligation adaptor amplification method, we present data from a total of 22 Affymetrix SNP 6.0 experiments, using matched paired amplified and non-amplified DNA from 10 LCM FFPE normal and dysplastic oral epithelial tissues, and an internal method control. An average of 76.5% of SNPs were called in both matched amplified and non-amplified DNA samples, and concordance was a promising 82.4%. Paired analysis for copy number, LOH, and both combined, showed that copy number changes were reduced in amplified DNA, but were 99.5% concordant when detected, amplifications were the changes most likely to be 'missed', only 30% of non-amplified LOH changes were identified in amplified pairs, and when copy number and LOH are combined ∼50% of gene changes detected in the unamplified DNA were also detected in the amplified DNA and within these changes, 86.5% were concordant for both copy number and LOH status. However, there are also changes introduced as ∼20% of changes in the amplified DNA are not detected in the non-amplified DNA. An integrative network biology approach revealed that changes in amplified DNA of dysplastic oral epithelium localize to topologically critical regions of the human protein-protein interaction network, suggesting their functional implication in the pathobiology of this disease. Taken together, our results support the use of amplification of FFPE-derived DNA, provided sufficient samples are used

  7. Rapid detection of microbial DNA by a novel isothermal genome exponential amplification reaction (GEAR) assay.

    Science.gov (United States)

    Prithiviraj, Jothikumar; Hill, Vincent; Jothikumar, Narayanan

    2012-04-20

    In this study we report the development of a simple target-specific isothermal nucleic acid amplification technique, termed genome exponential amplification reaction (GEAR). Escherichia coli was selected as the microbial target to demonstrate the GEAR technique as a proof of concept. The GEAR technique uses a set of four primers; in the present study these primers targeted 5 regions on the 16S rRNA gene of E. coli. The outer forward and reverse Tab primer sequences are complementary to each other at their 5' end, whereas their 3' end sequences are complementary to their respective target nucleic acid sequences. The GEAR assay was performed at a constant temperature 60 °C and monitored continuously in a real-time PCR instrument in the presence of an intercalating dye (SYTO 9). The GEAR assay enabled amplification of as few as one colony forming units of E. coli per reaction within 30 min. We also evaluated the GEAR assay for rapid identification of bacterial colonies cultured on agar media directly in the reaction without DNA extraction. Cells from E. coli colonies were picked and added directly to GEAR assay mastermix without prior DNA extraction. DNA in the cells could be amplified, yielding positive results within 15 min. Published by Elsevier Inc.

  8. Amplification and chromosomal dispersion of human endogenous retroviral sequences

    International Nuclear Information System (INIS)

    Steele, P.E.; Martin, M.A.; Rabson, A.B.; Bryan, T.; O'Brien, S.J.

    1986-01-01

    Endogenous retroviral sequences have undergone amplification events involving both viral and flanking cellular sequences. The authors cloned members of an amplified family of full-length endogenous retroviral sequences. Genomic blotting, employing a flanking cellular DNA probe derived from a member of this family, revealed a similar array of reactive bands in both humans and chimpanzees, indicating that an amplification event involving retroviral and associated cellular DNA sequences occurred before the evolutionary separation of these two primates. Southern analyses of restricted somatic cell hybrid DNA preparations suggested that endogenous retroviral segments are widely dispersed in the human genome and that amplification and dispersion events may be linked

  9. The rolling circle amplification and next generation sequencing ...

    African Journals Online (AJOL)

    Rolling circle amplification is a simple approach of enriching populations of single-stranded DNA plant begomovirus genomes (genus, Begomovirus; family, Geminiviridae). This is an innovative approach that utilizes the robustness of the bacteriophage phi29 DNA polymerase used in circle amplification, together with deep ...

  10. RNA amplification for successful gene profiling analysis

    Directory of Open Access Journals (Sweden)

    Wang Ena

    2005-07-01

    Full Text Available Abstract The study of clinical samples is often limited by the amount of material available to study. While proteins cannot be multiplied in their natural form, DNA and RNA can be amplified from small specimens and used for high-throughput analyses. Therefore, genetic studies offer the best opportunity to screen for novel insights of human pathology when little material is available. Precise estimates of DNA copy numbers in a given specimen are necessary. However, most studies investigate static variables such as the genetic background of patients or mutations within pathological specimens without a need to assess proportionality of expression among different genes throughout the genome. Comparative genomic hybridization of DNA samples represents a crude exception to this rule since genomic amplification or deletion is compared among different specimens directly. For gene expression analysis, however, it is critical to accurately estimate the proportional expression of distinct RNA transcripts since such proportions directly govern cell function by modulating protein expression. Furthermore, comparative estimates of relative RNA expression at different time points portray the response of cells to environmental stimuli, indirectly informing about broader biological events affecting a particular tissue in physiological or pathological conditions. This cognitive reaction of cells is similar to the detection of electroencephalographic patterns which inform about the status of the brain in response to external stimuli. As our need to understand human pathophysiology at the global level increases, the development and refinement of technologies for high fidelity messenger RNA amplification have become the focus of increasing interest during the past decade. The need to increase the abundance of RNA has been met not only for gene specific amplification, but, most importantly for global transcriptome wide, unbiased amplification. Now gene

  11. Archived neonatal dried blood spot samples can be used for accurate whole genome and exome-targeted next-generation sequencing

    DEFF Research Database (Denmark)

    Hollegaard, Mads Vilhelm; Grauholm, Jonas; Nielsen, Ronni

    2013-01-01

    Dried blood spot samples (DBSS) have been collected and stored for decades as part of newborn screening programmes worldwide. Representing almost an entire population under a certain age and collected with virtually no bias, the Newborn Screening Biobanks are of immense value in medical studies......, for example, to examine the genetics of various disorders. We have previously demonstrated that DNA extracted from a fraction (2×3.2mm discs) of an archived DBSS can be whole genome amplified (wgaDNA) and used for accurate array genotyping. However, until now, it has been uncertain whether wgaDNA from DBSS...... can be used for accurate whole genome sequencing (WGS) and exome sequencing (WES). This study examined two individuals represented by three different types of samples each: whole-blood (reference samples), 3-year-old DBSS spotted with reference material (refDBSS), and 27- to 29-year-old archived...

  12. Diagnostic Devices for Isothermal Nucleic Acid Amplification

    Directory of Open Access Journals (Sweden)

    Chia-Chen Chang

    2012-06-01

    Full Text Available Since the development of the polymerase chain reaction (PCR technique, genomic information has been retrievable from lesser amounts of DNA than previously possible. PCR-based amplifications require high-precision instruments to perform temperature cycling reactions; further, they are cumbersome for routine clinical use. However, the use of isothermal approaches can eliminate many complications associated with thermocycling. The application of diagnostic devices for isothermal DNA amplification has recently been studied extensively. In this paper, we describe the basic concepts of several isothermal amplification approaches and review recent progress in diagnostic device development.

  13. Diagnostic devices for isothermal nucleic acid amplification.

    Science.gov (United States)

    Chang, Chia-Chen; Chen, Chien-Cheng; Wei, Shih-Chung; Lu, Hui-Hsin; Liang, Yang-Hung; Lin, Chii-Wann

    2012-01-01

    Since the development of the polymerase chain reaction (PCR) technique, genomic information has been retrievable from lesser amounts of DNA than previously possible. PCR-based amplifications require high-precision instruments to perform temperature cycling reactions; further, they are cumbersome for routine clinical use. However, the use of isothermal approaches can eliminate many complications associated with thermocycling. The application of diagnostic devices for isothermal DNA amplification has recently been studied extensively. In this paper, we describe the basic concepts of several isothermal amplification approaches and review recent progress in diagnostic device development.

  14. MYC and MYCN amplification can be reliably assessed by aCGH in medulloblastoma.

    Science.gov (United States)

    Bourdeaut, Franck; Grison, Camille; Maurage, Claude-Alain; Laquerriere, Annie; Vasiljevic, Alexandre; Delisle, Marie-Bernadette; Michalak, Sophie; Figarella-Branger, Dominique; Doz, François; Richer, Wilfrid; Pierron, Gaelle; Miquel, Catherine; Delattre, Olivier; Couturier, Jérôme

    2013-04-01

    As prognostic factors, MYC and MYCN amplifications are routinely assessed in medulloblastomas. Fluorescence in situ hybridization (FISH) is currently considered as the technique of reference. Recently, array comparative genomic hybridization (aCGH) has been developed as an alternative technique to evaluate genomic abnormalities in other tumor types; however, this technique has not been widely adopted as a replacement for FISH in medulloblastoma. In this study, 34 tumors were screened by both FISH and aCGH. In all cases showing amplification by FISH, aCGH also unambiguously revealed the abnormality. The aCGH technique was also performed on tumors showing no amplification by FISH, and the absence of amplification was confirmed in all cases. Interestingly, one tumor showed a subclonal MYC amplification by FISH. This subclonal amplification was observed in approximately 20% of tumor cells and was clearly evident on aCGH. In conclusion, our analysis confirms that aCGH is as safe as FISH for the detection of MYC/MYCN gene amplification. Given its cost efficiency in comparison to two FISH tests and the global genomic information additionally provided by an aCGH experiment, this reproducible technique can be safely retained as an alternative to FISH for routine investigation of medulloblastoma. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Particle infectivity of HIV-1 full-length genome infectious molecular clones in a subtype C heterosexual transmission pair following high fidelity amplification and unbiased cloning

    Energy Technology Data Exchange (ETDEWEB)

    Deymier, Martin J., E-mail: mdeymie@emory.edu [Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329 (United States); Claiborne, Daniel T., E-mail: dclaibo@emory.edu [Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329 (United States); Ende, Zachary, E-mail: zende@emory.edu [Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329 (United States); Ratner, Hannah K., E-mail: hannah.ratner@emory.edu [Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329 (United States); Kilembe, William, E-mail: wkilembe@rzhrg-mail.org [Zambia-Emory HIV Research Project (ZEHRP), B22/737 Mwembelelo, Emmasdale Post Net 412, P/BagE891, Lusaka (Zambia); Allen, Susan, E-mail: sallen5@emory.edu [Zambia-Emory HIV Research Project (ZEHRP), B22/737 Mwembelelo, Emmasdale Post Net 412, P/BagE891, Lusaka (Zambia); Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA (United States); Hunter, Eric, E-mail: eric.hunter2@emory.edu [Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329 (United States); Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA (United States)

    2014-11-15

    The high genetic diversity of HIV-1 impedes high throughput, large-scale sequencing and full-length genome cloning by common restriction enzyme based methods. Applying novel methods that employ a high-fidelity polymerase for amplification and an unbiased fusion-based cloning strategy, we have generated several HIV-1 full-length genome infectious molecular clones from an epidemiologically linked transmission pair. These clones represent the transmitted/founder virus and phylogenetically diverse non-transmitted variants from the chronically infected individual's diverse quasispecies near the time of transmission. We demonstrate that, using this approach, PCR-induced mutations in full-length clones derived from their cognate single genome amplicons are rare. Furthermore, all eight non-transmitted genomes tested produced functional virus with a range of infectivities, belying the previous assumption that a majority of circulating viruses in chronic HIV-1 infection are defective. Thus, these methods provide important tools to update protocols in molecular biology that can be universally applied to the study of human viral pathogens. - Highlights: • Our novel methodology demonstrates accurate amplification and cloning of full-length HIV-1 genomes. • A majority of plasma derived HIV variants from a chronically infected individual are infectious. • The transmitted/founder was more infectious than the majority of the variants from the chronically infected donor.

  16. Particle infectivity of HIV-1 full-length genome infectious molecular clones in a subtype C heterosexual transmission pair following high fidelity amplification and unbiased cloning

    International Nuclear Information System (INIS)

    Deymier, Martin J.; Claiborne, Daniel T.; Ende, Zachary; Ratner, Hannah K.; Kilembe, William; Allen, Susan; Hunter, Eric

    2014-01-01

    The high genetic diversity of HIV-1 impedes high throughput, large-scale sequencing and full-length genome cloning by common restriction enzyme based methods. Applying novel methods that employ a high-fidelity polymerase for amplification and an unbiased fusion-based cloning strategy, we have generated several HIV-1 full-length genome infectious molecular clones from an epidemiologically linked transmission pair. These clones represent the transmitted/founder virus and phylogenetically diverse non-transmitted variants from the chronically infected individual's diverse quasispecies near the time of transmission. We demonstrate that, using this approach, PCR-induced mutations in full-length clones derived from their cognate single genome amplicons are rare. Furthermore, all eight non-transmitted genomes tested produced functional virus with a range of infectivities, belying the previous assumption that a majority of circulating viruses in chronic HIV-1 infection are defective. Thus, these methods provide important tools to update protocols in molecular biology that can be universally applied to the study of human viral pathogens. - Highlights: • Our novel methodology demonstrates accurate amplification and cloning of full-length HIV-1 genomes. • A majority of plasma derived HIV variants from a chronically infected individual are infectious. • The transmitted/founder was more infectious than the majority of the variants from the chronically infected donor

  17. Effect of the lectins wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA-I) on the alpha-amylase secretion of rat pancreas in vitro and in vivo.

    Science.gov (United States)

    Mikkat, U; Damm, I; Schröder, G; Schmidt, K; Wirth, C; Weber, H; Jonas, L

    1998-05-01

    Lectins are able to bind to cholecystokinin (CCK) receptors and other glycosylated membrane proteins. The lectins wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA-I) are used for affinity chromatography to isolate the highly glycosylated CCK-A receptor of pancreatic acinar cells. According to the working hypothesis that lectin binding to the CCK receptor should alter the ligand-receptor interaction, the effect of WGA and UEA-I on CCK-8-induced enzyme secretion was studied on isolated rat pancreatic acini in vitro. In vitro both lectins showed a dosage-dependent inhibition of CCK-8-induced alpha-amylase secretion of acini over 60 min. WGA showed a strong inhibitory effect on amylase secretion, approximately 40%, in vitro. UEA-I caused a smaller, but significant decrease, approximately 20%, in enzyme secretion of isolated acini. Additionally, both lectins inhibited cerulein/secretin- or cerulein-induced pancreatic secretion of rats in vivo, but not after secretin alone. The results are discussed with respect to a possible influence of both lectins on the interaction of CCK or cerulein with the CCK-A receptor.

  18. Classification of human cancers based on DNA copy number amplification modeling

    Directory of Open Access Journals (Sweden)

    Knuutila Sakari

    2008-05-01

    Full Text Available Abstract Background DNA amplifications alter gene dosage in cancer genomes by multiplying the gene copy number. Amplifications are quintessential in a considerable number of advanced cancers of various anatomical locations. The aims of this study were to classify human cancers based on their amplification patterns, explore the biological and clinical fundamentals behind their amplification-pattern based classification, and understand the characteristics in human genomic architecture that associate with amplification mechanisms. Methods We applied a machine learning approach to model DNA copy number amplifications using a data set of binary amplification records at chromosome sub-band resolution from 4400 cases that represent 82 cancer types. Amplification data was fused with background data: clinical, histological and biological classifications, and cytogenetic annotations. Statistical hypothesis testing was used to mine associations between the data sets. Results Probabilistic clustering of each chromosome identified 111 amplification models and divided the cancer cases into clusters. The distribution of classification terms in the amplification-model based clustering of cancer cases revealed cancer classes that were associated with specific DNA copy number amplification models. Amplification patterns – finite or bounded descriptions of the ranges of the amplifications in the chromosome – were extracted from the clustered data and expressed according to the original cytogenetic nomenclature. This was achieved by maximal frequent itemset mining using the cluster-specific data sets. The boundaries of amplification patterns were shown to be enriched with fragile sites, telomeres, centromeres, and light chromosome bands. Conclusions Our results demonstrate that amplifications are non-random chromosomal changes and specifically selected in tumor tissue microenvironment. Furthermore, statistical evidence showed that specific chromosomal features

  19. PCR amplification of repetitive sequences as a possible approach in relative species quantification

    DEFF Research Database (Denmark)

    Ballin, Nicolai Zederkopff; Vogensen, Finn Kvist; Karlsson, Anders H

    2012-01-01

    Abstract Both relative and absolute quantifications are possible in species quantification when single copy genomic DNA is used. However, amplification of single copy genomic DNA does not allow a limit of detection as low as one obtained from amplification of repetitive sequences. Amplification...... of repetitive sequences is therefore frequently used in absolute quantification but problems occur in relative quantification as the number of repetitive sequences is unknown. A promising approach was developed where data from amplification of repetitive sequences were used in relative quantification of species...... to relatively quantify the amount of chicken DNA in a binary mixture of chicken DNA and pig DNA. However, the designed PCR primers lack the specificity required for regulatory species control....

  20. Gene amplification in carcinogenesis

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    Lucimari Bizari

    2006-01-01

    Full Text Available Gene amplification increases the number of genes in a genome and can give rise to karyotype abnormalities called double minutes (DM and homogeneously staining regions (HSR, both of which have been widely observed in human tumors but are also known to play a major role during embryonic development due to the fact that they are responsible for the programmed increase of gene expression. The etiology of gene amplification during carcinogenesis is not yet completely understood but can be considered a result of genetic instability. Gene amplification leads to an increase in protein expression and provides a selective advantage during cell growth. Oncogenes such as CCND1, c-MET, c-MYC, ERBB2, EGFR and MDM2 are amplified in human tumors and can be associated with increased expression of their respective proteins or not. In general, gene amplification is associated with more aggressive tumors, metastases, resistance to chemotherapy and a decrease in the period during which the patient stays free of the disease. This review discusses the major role of gene amplification in the progression of carcinomas, formation of genetic markers and as possible therapeutic targets for the development of drugs for the treatment of some types of tumors.

  1. Methylation-Sensitive Amplification Length Polymorphism (MS-AFLP) Microarrays for Epigenetic Analysis of Human Genomes.

    Science.gov (United States)

    Alonso, Sergio; Suzuki, Koichi; Yamamoto, Fumiichiro; Perucho, Manuel

    2018-01-01

    Somatic, and in a minor scale also germ line, epigenetic aberrations are fundamental to carcinogenesis, cancer progression, and tumor phenotype. DNA methylation is the most extensively studied and arguably the best understood epigenetic mechanisms that become altered in cancer. Both somatic loss of methylation (hypomethylation) and gain of methylation (hypermethylation) are found in the genome of malignant cells. In general, the cancer cell epigenome is globally hypomethylated, while some regions-typically gene-associated CpG islands-become hypermethylated. Given the profound impact that DNA methylation exerts on the transcriptional profile and genomic stability of cancer cells, its characterization is essential to fully understand the complexity of cancer biology, improve tumor classification, and ultimately advance cancer patient management and treatment. A plethora of methods have been devised to analyze and quantify DNA methylation alterations. Several of the early-developed methods relied on the use of methylation-sensitive restriction enzymes, whose activity depends on the methylation status of their recognition sequences. Among these techniques, methylation-sensitive amplification length polymorphism (MS-AFLP) was developed in the early 2000s, and successfully adapted from its original gel electrophoresis fingerprinting format to a microarray format that notably increased its throughput and allowed the quantification of the methylation changes. This array-based platform interrogates over 9500 independent loci putatively amplified by the MS-AFLP technique, corresponding to the NotI sites mapped throughout the human genome.

  2. From human monocytes to genome-wide binding sites--a protocol for small amounts of blood: monocyte isolation/ChIP-protocol/library amplification/genome wide computational data analysis.

    Directory of Open Access Journals (Sweden)

    Sebastian Weiterer

    Full Text Available Chromatin immunoprecipitation in combination with a genome-wide analysis via high-throughput sequencing is the state of the art method to gain genome-wide representation of histone modification or transcription factor binding profiles. However, chromatin immunoprecipitation analysis in the context of human experimental samples is limited, especially in the case of blood cells. The typically extremely low yields of precipitated DNA are usually not compatible with library amplification for next generation sequencing. We developed a highly reproducible protocol to present a guideline from the first step of isolating monocytes from a blood sample to analyse the distribution of histone modifications in a genome-wide manner.The protocol describes the whole work flow from isolating monocytes from human blood samples followed by a high-sensitivity and small-scale chromatin immunoprecipitation assay with guidance for generating libraries compatible with next generation sequencing from small amounts of immunoprecipitated DNA.

  3. Analysis of genomic alterations in neuroblastoma by multiplex ligation-dependent probe amplification and array comparative genomic hybridization: a comparison of results.

    Science.gov (United States)

    Combaret, Valérie; Iacono, Isabelle; Bréjon, Stéphanie; Schleiermacher, Gudrun; Pierron, Gäelle; Couturier, Jérôme; Bergeron, Christophe; Blay, Jean-Yves

    2012-12-01

    In cases of neuroblastoma, recurring genetic alterations--losses of the 1p, 3p, 4p, and 11q and/or gains of 1q, 2p, and 17q chromosome arms--are currently used to define the therapeutic strategy in therapeutic protocols for low- and intermediate-risk patients. Different genome-wide analysis techniques, such as array comparative genomic hybridization (aCGH) or multiplex ligation-dependent probe amplification (MLPA), have been suggested for detecting chromosome segmental abnormalities. In this study, we compared the results of the two technologies in the analyses of the DNA of tumor samples from 91 neuroblastoma patients. Similar results were obtained with the two techniques for 75 samples (82%). In five cases (5.5%), the MLPA results were not interpretable. Discrepancies between the aCGH and MLPA results were observed in 11 cases (12%). Among the discrepancies, a 18q21.2-qter gain and 16p11.2 and 11q14.1-q14.3 losses were detected only by aCGH. The MLPA results showed that the 7p, 7q, and 14q chromosome arms were affected in six cases, while in two cases, 2p and 17q gains were observed; these results were confirmed by neither aCGH nor fluorescence in situ hybridization (FISH) analysis. Because of the higher sensitivity and specificity of genome-wide information, reasonable cost, and shorter time of aCGH analysis, we recommend the aCGH procedure for the analysis of genomic alterations in neuroblastoma. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. Genomic DNA fingerprinting of clinical Haemophilus influenzae isolates by polymerase chain reaction amplification: comparison with major outer-membrane protein and restriction fragment length polymorphism analysis

    NARCIS (Netherlands)

    van Belkum, A.; Duim, B.; Regelink, A.; Möller, L.; Quint, W.; van Alphen, L.

    1994-01-01

    Non-capsulate strains of Haemophilus influenzae were genotyped by analysis of variable DNA segments obtained by amplification of genomic DNA with the polymerase chain reaction (PCR fingerprinting). Discrete fragments of 100-2000 bp were obtained. The reproducibility of the procedure was assessed by

  5. GENOMIC DNA-FINGERPRINTING OF CLINICAL HAEMOPHILUS-INFLUENZAE ISOLATES BY POLYMERASE CHAIN-REACTION AMPLIFICATION - COMPARISON WITH MAJOR OUTER-MEMBRANE PROTEIN AND RESTRICTION-FRAGMENT-LENGTH-POLYMORPHISM ANALYSIS

    NARCIS (Netherlands)

    VANBELKUM, A; DUIM, B; REGELINK, A; MOLLER, L; QUINT, W; VANALPHEN, L

    Non-capsulate strains of Haemophilus influenzae were genotyped by analysis of variable DNA segments obtained by amplification of genomic DNA with the polymerase chain reaction (PCR fingerprinting). Discrete fragments of 100-2000 bp were obtained. The reproducibility of the procedure was assessed by

  6. Combining Amplification Typing of L1 Active Subfamilies (ATLAS) with High-Throughput Sequencing.

    Science.gov (United States)

    Rahbari, Raheleh; Badge, Richard M

    2016-01-01

    With the advent of new generations of high-throughput sequencing technologies, the catalog of human genome variants created by retrotransposon activity is expanding rapidly. However, despite these advances in describing L1 diversity and the fact that L1 must retrotranspose in the germline or prior to germline partitioning to be evolutionarily successful, direct assessment of de novo L1 retrotransposition in the germline or early embryogenesis has not been achieved for endogenous L1 elements. A direct study of de novo L1 retrotransposition into susceptible loci within sperm DNA (Freeman et al., Hum Mutat 32(8):978-988, 2011) suggested that the rate of L1 retrotransposition in the germline is much lower than previously estimated (ATLAS L1 display technique (Badge et al., Am J Hum Genet 72(4):823-838, 2003) to investigate de novo L1 retrotransposition in human genomes. In this chapter, we describe how we combined a high-coverage ATLAS variant with high-throughput sequencing, achieving 11-25× sequence depth per single amplicon, to study L1 retrotransposition in whole genome amplified (WGA) DNAs.

  7. Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)-A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes.

    Science.gov (United States)

    Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

    2017-01-01

    Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare . However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop

  8. Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes

    Directory of Open Access Journals (Sweden)

    Karolina Chwialkowska

    2017-11-01

    Full Text Available Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq. We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare. However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation

  9. Successful application of the strategy of blastocyst biopsy, vitrification, whole genome amplification, and thawed embryo transfer for preimplantation genetic diagnosis of neurofibromatosis type 1

    Directory of Open Access Journals (Sweden)

    Yi-Lin Chen

    2011-03-01

    Conclusion: We first demonstrate successful application of blastocyst biopsy, vitrification, WGA, and thawed embryo transfer for PGD of a monogenic disease. Vitrification of blastocysts after biopsy permits sufficient time for shipment of samples and operation of molecular diagnosis.

  10. MDM2 and CDK4 amplifications are rare events in salivary duct carcinomas.

    Science.gov (United States)

    Grünewald, Inga; Trautmann, Marcel; Busch, Alina; Bauer, Larissa; Huss, Sebastian; Schweinshaupt, Petra; Vollbrecht, Claudia; Odenthal, Margarete; Quaas, Alexander; Büttner, Reinhard; Meyer, Moritz F; Beutner, Dirk; Hüttenbrink, Karl-Bernd; Wardelmann, Eva; Stenner, Markus; Hartmann, Wolfgang

    2016-11-15

    Salivary duct carcinoma (SDC) is an aggressive adenocarcinoma of the salivary glands associated with poor clinical outcome. SDCs are known to carry TP53 mutations in about 50%, however, only little is known about alternative pathogenic mechanisms within the p53 regulatory network. Particularly, data on alterations of the oncogenes MDM2 and CDK4 located in the chromosomal region 12q13-15 are limited in SDC, while genomic rearrangements of the adjacent HMGA2 gene locus are well documented in subsets of SDCs. We here analyzed the mutational status of the TP53 gene, genomic amplification of MDM2, CDK4 and HMGA2 rearrangement/amplification as well as protein expression of TP53 (p53), MDM2 and CDK4 in 51 de novo and ex pleomorphic adenoma SDCs.25 of 51 cases were found to carry TP53 mutations, associated with extreme positive immunohistochemical p53 staining levels in 13 cases. Three out of 51 tumors had an MDM2 amplification, one of them coinciding with a CDK4 amplification and two with a HMGA2 rearrangement/amplification. Two of the MDM2 amplifications occurred in the setting of a TP53 mutation. Two out of 51 cases showed a CDK4 amplification, one synchronously being MDM2 amplified and the other one displaying concurrent low copy number increases of both, MDM2 and HMGA2.In summary, we here show that subgroups of SDCs display genomic amplifications of MDM2 and/or CDK4, partly in association with TP53 mutations and rearrangement/amplification of HMGA2. Further research is necessary to clarify the role of chromosomal region 12q13-15 alterations in SDC tumorigenesis and their potential prognostic and therapeutic relevance.

  11. DNA Barcoding: Amplification and sequence analysis of rbcl and matK genome regions in three divergent plant species

    Directory of Open Access Journals (Sweden)

    Javed Iqbal Wattoo

    2016-11-01

    Full Text Available Background: DNA barcoding is a novel method of species identification based on nucleotide diversity of conserved sequences. The establishment and refining of plant DNA barcoding systems is more challenging due to high genetic diversity among different species. Therefore, targeting the conserved nuclear transcribed regions would be more reliable for plant scientists to reveal genetic diversity, species discrimination and phylogeny. Methods: In this study, we amplified and sequenced the chloroplast DNA regions (matk+rbcl of Solanum nigrum, Euphorbia helioscopia and Dalbergia sissoo to study the functional annotation, homology modeling and sequence analysis to allow a more efficient utilization of these sequences among different plant species. These three species represent three families; Solanaceae, Euphorbiaceae and Fabaceae respectively. Biological sequence homology and divergence of amplified sequences was studied using Basic Local Alignment Tool (BLAST. Results: Both primers (matk+rbcl showed good amplification in three species. The sequenced regions reveled conserved genome information for future identification of different medicinal plants belonging to these species. The amplified conserved barcodes revealed different levels of biological homology after sequence analysis. The results clearly showed that the use of these conserved DNA sequences as barcode primers would be an accurate way for species identification and discrimination. Conclusion: The amplification and sequencing of conserved genome regions identified a novel sequence of matK in native species of Solanum nigrum. The findings of the study would be applicable in medicinal industry to establish DNA based identification of different medicinal plant species to monitor adulteration.

  12. Single virus genomics: a new tool for virus discovery.

    Directory of Open Access Journals (Sweden)

    Lisa Zeigler Allen

    Full Text Available Whole genome amplification and sequencing of single microbial cells has significantly influenced genomics and microbial ecology by facilitating direct recovery of reference genome data. However, viral genomics continues to suffer due to difficulties related to the isolation and characterization of uncultivated viruses. We report here on a new approach called 'Single Virus Genomics', which enabled the isolation and complete genome sequencing of the first single virus particle. A mixed assemblage comprised of two known viruses; E. coli bacteriophages lambda and T4, were sorted using flow cytometric methods and subsequently immobilized in an agarose matrix. Genome amplification was then achieved in situ via multiple displacement amplification (MDA. The complete lambda phage genome was recovered with an average depth of coverage of approximately 437X. The isolation and genome sequencing of uncultivated viruses using Single Virus Genomics approaches will enable researchers to address questions about viral diversity, evolution, adaptation and ecology that were previously unattainable.

  13. Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes

    Science.gov (United States)

    Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

    2017-01-01

    Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare. However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop

  14. Caught in the middle with multiple displacement amplification: the myth of pooling for avoiding multiple displacement amplification bias in a metagenome.

    Science.gov (United States)

    Marine, Rachel; McCarren, Coleen; Vorrasane, Vansay; Nasko, Dan; Crowgey, Erin; Polson, Shawn W; Wommack, K Eric

    2014-01-30

    Shotgun metagenomics has become an important tool for investigating the ecology of microorganisms. Underlying these investigations is the assumption that metagenome sequence data accurately estimates the census of microbial populations. Multiple displacement amplification (MDA) of microbial community DNA is often used in cases where it is difficult to obtain enough DNA for sequencing; however, MDA can result in amplification biases that may impact subsequent estimates of population census from metagenome data. Some have posited that pooling replicate MDA reactions negates these biases and restores the accuracy of population analyses. This assumption has not been empirically tested. Using mock viral communities, we examined the influence of pooling on population-scale analyses. In pooled and single reaction MDA treatments, sequence coverage of viral populations was highly variable and coverage patterns across viral genomes were nearly identical, indicating that initial priming biases were reproducible and that pooling did not alleviate biases. In contrast, control unamplified sequence libraries showed relatively even coverage across phage genomes. MDA should be avoided for metagenomic investigations that require quantitative estimates of microbial taxa and gene functional groups. While MDA is an indispensable technique in applications such as single-cell genomics, amplification biases cannot be overcome by combining replicate MDA reactions. Alternative library preparation techniques should be utilized for quantitative microbial ecology studies utilizing metagenomic sequencing approaches.

  15. Linear and exponential TAIL-PCR: a method for efficient and quick amplification of flanking sequences adjacent to Tn5 transposon insertion sites.

    Science.gov (United States)

    Jia, Xianbo; Lin, Xinjian; Chen, Jichen

    2017-11-02

    Current genome walking methods are very time consuming, and many produce non-specific amplification products. To amplify the flanking sequences that are adjacent to Tn5 transposon insertion sites in Serratia marcescens FZSF02, we developed a genome walking method based on TAIL-PCR. This PCR method added a 20-cycle linear amplification step before the exponential amplification step to increase the concentration of the target sequences. Products of the linear amplification and the exponential amplification were diluted 100-fold to decrease the concentration of the templates that cause non-specific amplification. Fast DNA polymerase with a high extension speed was used in this method, and an amplification program was used to rapidly amplify long specific sequences. With this linear and exponential TAIL-PCR (LETAIL-PCR), we successfully obtained products larger than 2 kb from Tn5 transposon insertion mutant strains within 3 h. This method can be widely used in genome walking studies to amplify unknown sequences that are adjacent to known sequences.

  16. Dihydrofolate reductase amplification and sensitization to methotrexate of methotrexate-resistant colon cancer cells

    DEFF Research Database (Denmark)

    Morales Torres, Christina; García, Maria J; Ribas, Maria

    2009-01-01

    Gene amplification is one of the most frequent manifestations of genomic instability in human tumors and plays an important role in tumor progression and acquisition of drug resistance. To better understand the factors involved in acquired resistance to cytotoxic drugs via gene amplification, we ...

  17. Direct squencing from the minimal number of DNA molecules needed to fill a 454 picotiterplate.

    Directory of Open Access Journals (Sweden)

    Mária Džunková

    Full Text Available The large amount of DNA needed to prepare a library in next generation sequencing protocols hinders direct sequencing of small DNA samples. This limitation is usually overcome by the enrichment of such samples with whole genome amplification (WGA, mostly by multiple displacement amplification (MDA based on φ29 polymerase. However, this technique can be biased by the GC content of the sample and is prone to the development of chimeras as well as contamination during enrichment, which contributes to undesired noise during sequence data analysis, and also hampers the proper functional and/or taxonomic assignments. An alternative to MDA is direct DNA sequencing (DS, which represents the theoretical gold standard in genome sequencing. In this work, we explore the possibility of sequencing the genome of Escherichia coli fs 24 from the minimum number of DNA molecules required for pyrosequencing, according to the notion of one-bead-one-molecule. Using an optimized protocol for DS, we constructed a shotgun library containing the minimum number of DNA molecules needed to fill a selected region of a picotiterplate. We gathered most of the reference genome extension with uniform coverage. We compared the DS method with MDA applied to the same amount of starting DNA. As expected, MDA yielded a sparse and biased read distribution, with a very high amount of unassigned and unspecific DNA amplifications. The optimized DS protocol allows unbiased sequencing to be performed from samples with a very small amount of DNA.

  18. Comparative genomic analysis reveals multiple long terminal repeats, lineage-specific amplification, and frequent interelement recombination for Cassandra retrotransposon in pear (Pyrus bretschneideri Rehd.).

    Science.gov (United States)

    Yin, Hao; Du, Jianchang; Li, Leiting; Jin, Cong; Fan, Lian; Li, Meng; Wu, Jun; Zhang, Shaoling

    2014-06-04

    Cassandra transposable elements belong to a specific group of terminal-repeat retrotransposons in miniature (TRIM). Although Cassandra TRIM elements have been found in almost all vascular plants, detailed investigations on the nature, abundance, amplification timeframe, and evolution have not been performed in an individual genome. We therefore conducted a comprehensive analysis of Cassandra retrotransposons using the newly sequenced pear genome along with four other Rosaceae species, including apple, peach, mei, and woodland strawberry. Our data reveal several interesting findings for this particular retrotransposon family: 1) A large number of the intact copies contain three, four, or five long terminal repeats (LTRs) (∼20% in pear); 2) intact copies and solo LTRs with or without target site duplications are both common (∼80% vs. 20%) in each genome; 3) the elements exhibit an overall unbiased distribution among the chromosomes; 4) the elements are most successfully amplified in pear (5,032 copies); and 5) the evolutionary relationships of these elements vary among different lineages, species, and evolutionary time. These results indicate that Cassandra retrotransposons contain more complex structures (elements with multiple LTRs) than what we have known previously, and that frequent interelement unequal recombination followed by transposition may play a critical role in shaping and reshaping host genomes. Thus this study provides insights into the property, propensity, and molecular mechanisms governing the formation and amplification of Cassandra retrotransposons, and enhances our understanding of the structural variation, evolutionary history, and transposition process of LTR retrotransposons in plants. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  19. Diagnosis of brugian filariasis by loop-mediated isothermal amplification.

    Directory of Open Access Journals (Sweden)

    Catherine B Poole

    Full Text Available In this study we developed and evaluated a Brugia Hha I repeat loop-mediated isothermal amplification (LAMP assay for the rapid detection of Brugia genomic DNA. Amplification was detected using turbidity or fluorescence as readouts. Reactions generated a turbidity threshold value or a clear visual positive within 30 minutes using purified genomic DNA equivalent to one microfilaria. Similar results were obtained using DNA isolated from blood samples containing B. malayi microfilariae. Amplification was specific to B. malayi and B. timori, as no turbidity was observed using DNA from the related filarial parasites Wuchereria bancrofti, Onchocerca volvulus or Dirofilaria immitis, or from human or mosquito. Furthermore, the assay was most robust using a new strand-displacing DNA polymerase termed Bst 2.0 compared to wild-type Bst DNA polymerase, large fragment. The results indicate that the Brugia Hha I repeat LAMP assay is rapid, sensitive and Brugia-specific with the potential to be developed further as a field tool for diagnosis and mapping of brugian filariasis.

  20. Whole genome amplification: Use of advanced isothermal method

    African Journals Online (AJOL)

    Yomi

    2010-12-29

    Dec 29, 2010 ... 1Ph.D. Student, Department of Animal Science, Science and Research Branch, Islamic Azad University(IAU), ... sequence has a large effect on both the denaturation of ..... performance of multiple displacement amplification and OmniPlex ... Dean FB, Hosono S, Fang L, Wu L, Faruqi AF, Bray-Ward P, Sun Z,.

  1. Hormonal Involvement in Breast Cancer Gene Amplification

    Science.gov (United States)

    2010-10-01

    been shown to induce DN A amplification in yeast (Gopalakrishnan et al., 2001; Nguy en et al., 2001; Green et al., 2006) an d increased Cdt1 results in...re-replication in human cells (Dorn et al., 2008). The N- terminus of Cdt1 is important for re-replication, perhaps through interactions with PCNA...evolution of a cancer genome. Genome Res. (Epub. Dec. 3, 2008). Harris TD, Buzby PR, Babcock H, Beer E, Bowers J, Bras lavsky I, Causey M

  2. Comparative Genomic Hybridization of Human Malignant Gliomas Reveals Multiple Amplification Sites and Nonrandom Chromosomal Gains and Losses

    Science.gov (United States)

    Schròck, Evelin; Thiel, Gundula; Lozanova, Tanka; du Manoir, Stanislas; Meffert, Marie-Christine; Jauch, Anna; Speicher, Michael R.; Nürnberg, Peter; Vogel, Siegfried; Janisch, Werner; Donis-Keller, Helen; Ried, Thomas; Witkowski, Regine; Cremer, Thomas

    1994-01-01

    Nine human malignant gliomas (2 astrocytomas grade III and 7 glioblastomas) were analyzed using comparative genomic hybridization (CGH). In addition to the amplification of the EGFR gene at 7p12 in 4 of 9 cases, six new amplification sites were mapped to 1q32, 4q12, 7q21.1, 7q21.2-3, 12p, and 22q12. Nonrandom chromosomal gains and losses were identified with overrepresentation of chromosome 7 and underrepresentation of chromosome 10 as the most frequent events (1 of 2 astrocytomas, 7 of 7 glioblastomas). Gain of a part or the whole chromosome 19 and losses of chromosome bands 9pter-23 and 22q13 were detected each in five cases. Loss of chromosome band 17p13 and gain of chromosome 20 were revealed each in three cases. The validity of the CGH data was confirmed using interphase cytogenetics with YAC clones, chromosome painting in tumor metaphase spreads, and DNA fingerprinting. A comparison of CGH data with the results of chromosome banding analyses indicates that metaphase spreads accessible in primary tumor cell cultures may not represent the clones predominant in the tumor tissue ImagesFigure 1Figure 4Figure 6 PMID:8203461

  3. Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes

    OpenAIRE

    Karolina Chwialkowska; Urszula Korotko; Joanna Kosinska; Iwona Szarejko; Miroslaw Kwasniewski

    2017-01-01

    Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing ...

  4. DNA Amplification by Breakage/Fusion/Bridge Cycles Initiated by Spontaneous Telomere Loss in a Human Cancer Cell Line

    Directory of Open Access Journals (Sweden)

    Anthony W.l. Lo

    2002-01-01

    Full Text Available The development of genomic instability is an important step in generatingthe multiple genetic changes required for cancer. One consequence of genomic instability is the overexpression of oncogenes due to gene amplification. One mechanism for gene amplification is the breakagelfusionlbridge (B/F/Bcyclethatinvolvesthe repeated fusion and breakage of chromosomes following the loss of a telomere. B/F/B cycles have been associated with low-copy gene amplification in human cancer cells, and have been proposed to be an initiating event in high-copy gene amplification. We have found that spontaneous telomere loss on a marker chromosome 16 in a human tumor cell line results in sister chromatid fusion and prolonged periods of chromosome instability. The high rate of anaphase bridges involving chromosome 16 demonstrates that this instability results from B/F/B cycles. The amplification of subtelomeric DNA on the marker chromosome provides conclusive evidence that B/F/B cycles initiated by spontaneous telomere loss are a mechanism for gene amplification in human cancer cells.

  5. Chromosome copy analysis by single-cell comparative genomic hybridization technique based on primer extension preamplification and degenerate oligonucleotide primed-PCR%引物延伸预扩增结合简并引物PCR在单细胞比较基因组杂交分析染色体异常中的应用

    Institute of Scientific and Technical Information of China (English)

    谭珂; 狄玉芬; 程德华; 徐芳; 卢光绣; 谭跃球

    2010-01-01

    Objective To establish a single-cell whole genome amplification (WGA) technique, in combination with comparative genomic hybridization (CGH), for analyzing chromosomal copy number changes, and to explore its clinical application in preimplantation genetic diagnosis (PGD). Methods Twelve single cell samples with known karyotypes, including 5 chorionic villus samples, 4 human embryonic stem cell (hESC) samples and 3 peripheral lymphocyte samples, and 4 single blastomere samples carrying chromosomal abnormalities detected by PGD, were collected for whole genome amplification by combining primer extension preamplification (PEP) with degenerate oligonucleotide primed-PCR (DOP-PCR)amplification. The amplified products labeled by red fluorescence were mixed with control DNA labeled by green fluorescence, and then the mixture was analyzed by CGH. As a comparison, 10 single cell samples were amplified by DOP-PCR only and then CGH analysis was performed. Results The amplification using PEP-DOP-PCR was more stable than traditional DOP-PCR. The products of PEP-DOP-PCR range from 100 bp to 1000 bp, with the mean size being about 400 bp. The CGH results were consistent with analyses by other methods. However, only 6 out of 10 single cell samples were successfully amplified by DOP-PCR,and CGH analysis showed a high background and 2 samples showed inconsistent results from other methods. Conclusion PEP-DOP-PCR can effectively amplify the whole genome DNA of single cell.Combined with CGH, this WGA method can successfully detect single-cell chromosomal copy number changes, while DOP-PCR was easy to fail to amplify and amplify inhomogeneousty, and CGH analysis using this PCR product usually showed high background. These results suggest that PEP-DOP-CGH is a promising method for preimplantation genetic diagnosis.%目的 建立一种可信的单细胞全基因组扩增(whole genome amplification.WGA)技术,结合比较基因组杂交(comparative genomic hybridization

  6. Anterograde axonal transport and intercellular transfer of WGA-HRP in trigeminal-innervated sensory receptors of rat incisive papilla.

    Science.gov (United States)

    Chan, K Y; Byers, M R

    1985-04-08

    The ultrastructure and identification of WGA-HRP-labeled sensory receptors in the rat incisive papilla (the most anterior part of hard palate) were studied using semiserial thin sections. Various sensory receptors were organized according to three locations: dome region (ventral), chemosensory corpuscle region (medial to orifice of incisive canal), and lateral labium (apposing the incisive canal). In the dome region, the sensory receptors were localized in three sensory zones that were associated with surface ridges (one medial and two lateral). In each of these zones, intraepithelial receptor axons and Merkel receptors occurred in the epithelium, while simple unencapsulated corpuscles, glomerular-Meissner corpuscles, and incisive (encapsulated) corpuscles occurred in the lamina propria. In the chemosensory corpuscle region, chemosensory corpuscles and intraepithelial receptor axons were located in the epithelium, and incisive corpuscles were present in the lamina propria. In the lateral labium, only intraepithelial receptor axons were prominent. In all these sensory receptors, the preterminal axons and axon terminals were labeled with the tracer protein. In addition, some nonneuronal cells closely associated with the axon terminals were selectively labeled, e.g., terminal Schwann cells, lamellar Schwann cells, Merkel cells, corpuscular basal cells and chemosensory cells. Other adjacent cells were not labeled, e.g., unspecialized epithelial cells, capsular cells, corpuscular sustentacular cells, and fibroblasts. In both labeled axons and cells, WGA-HRP was incorporated into vesicles, tubules, and vacuolar organelles. The specific intercellular transfer of tracer protein may indicate trophic interactions between axon terminals and support cells in sensory receptors. The specific organization of multiple sensory receptors in the rat incisive papilla may provide a useful alternative system for studying somatosensory physiology.

  7. Amplification of an ancestral mammalian L1 family of long interspersed repeated DNA occurred just before the murine radiation

    International Nuclear Information System (INIS)

    Pascale, E.; Valle, E.; Furano, A.V.

    1990-01-01

    Each mammalian genus examined so far contains 50,000-100,000 members of an L1 (LINE 1) family of long interspersed repeated DNA elements. Current knowledge on the evolution of L1 families presents a paradox because, although L1 families have been in mammalian genomes since before the mammalian radiation ∼80 million years ago, most members of the L1 families are only a few million years old. Accordingly it has been suggested either that the extensive amplification that characterizes present-day L1 families did not occur in the past or that old members were removed as new one were generated. However, the authors show here that an ancestral rodent L1 family was extensively amplified ∼10 million years ago and that the relics of this amplification have persisted in modern murine genomes. This amplification occurred just before the divergence of modern murine genera from their common ancestor and identifies the murine node in the lineage of modern muroid rodents The results suggest that repeated amplification of L1 elements is a feature of the evaluation of mammalian genomes and that ancestral amplification events could provide a useful tool for determining mammalian lineages

  8. Sequencing intractable DNA to close microbial genomes.

    Directory of Open Access Journals (Sweden)

    Richard A Hurt

    Full Text Available Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome sequencing projects, providing the genetic blueprint for in-depth studies. Oftentimes, difficult to sequence regions in microbial genomes are ruled "intractable" resulting in a growing number of genomes with sequence gaps deposited in databases. A procedure was developed to sequence such problematic regions in the "non-contiguous finished" Desulfovibrio desulfuricans ND132 genome (6 intractable gaps and the Desulfovibrio africanus genome (1 intractable gap. The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to amplification and sequencing. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle supported amplification and closure of all gap regions in both genomes. The developed procedures support accurate gene annotation, and provide a step-wise method that reduces the effort required for genome finishing.

  9. Sequencing Intractable DNA to Close Microbial Genomes

    Energy Technology Data Exchange (ETDEWEB)

    Hurt, Jr., Richard Ashley [ORNL; Brown, Steven D [ORNL; Podar, Mircea [ORNL; Palumbo, Anthony Vito [ORNL; Elias, Dwayne A [ORNL

    2012-01-01

    Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome sequencing projects, providing the genetic blueprint for for in-depth studies. Oftentimes, difficult to sequence regions in microbial genomes are ruled intractable resulting in a growing number of genomes with sequence gaps deposited in databases. A procedure was developed to sequence such difficult regions in the non-contiguous finished Desulfovibrio desulfuricans ND132 genome (6 intractable gaps) and the Desulfovibrio africanus genome (1 intractable gap). The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to amplification and sequencing. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle supported amplification and closure of all gap regions in both genomes. These developed procedures support accurate gene annotation, and provide a step-wise method that reduces the effort required for genome finishing.

  10. Development and validation of concurrent preimplantation genetic diagnosis for single gene disorders and comprehensive chromosomal aneuploidy screening without whole genome amplification.

    Science.gov (United States)

    Zimmerman, Rebekah S; Jalas, Chaim; Tao, Xin; Fedick, Anastasia M; Kim, Julia G; Pepe, Russell J; Northrop, Lesley E; Scott, Richard T; Treff, Nathan R

    2016-02-01

    To develop a novel and robust protocol for multifactorial preimplantation genetic testing of trophectoderm biopsies using quantitative polymerase chain reaction (qPCR). Prospective and blinded. Not applicable. Couples indicated for preimplantation genetic diagnosis (PGD). None. Allele dropout (ADO) and failed amplification rate, genotyping consistency, chromosome screening success rate, and clinical outcomes of qPCR-based screening. The ADO frequency on a single cell from a fibroblast cell line was 1.64% (18/1,096). When two or more cells were tested, the ADO frequency dropped to 0.02% (1/4,426). The rate of amplification failure was 1.38% (55/4,000) overall, with 2.5% (20/800) for single cells and 1.09% (35/3,200) for samples that had two or more cells. Among 152 embryos tested in 17 cases by qPCR-based PGD and CCS, 100% were successfully given a diagnosis, with 0% ADO or amplification failure. Genotyping consistency with reference laboratory results was >99%. Another 304 embryos from 43 cases were included in the clinical application of qPCR-based PGD and CCS, for which 99.7% (303/304) of the embryos were given a definitive diagnosis, with only 0.3% (1/304) having an inconclusive result owing to recombination. In patients receiving a transfer with follow-up, the pregnancy rate was 82% (27/33). This study demonstrates that the use of qPCR for PGD testing delivers consistent and more reliable results than existing methods and that single gene disorder PGD can be run concurrently with CCS without the need for additional embryo biopsy or whole genome amplification. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  11. Effects of sample treatments on genome recovery via single-cell genomics

    Energy Technology Data Exchange (ETDEWEB)

    Clingenpeel, Scott [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Schwientek, Patrick [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Hugenholtz, Philip [Univ. of Queensland, Brisbane (Australia); Woyke, Tanja [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States)

    2014-06-13

    It is known that single-cell genomics is a powerful tool for accessing genetic information from uncultivated microorganisms. Methods of handling samples before single-cell genomic amplification may affect the quality of the genomes obtained. Using three bacterial strains we demonstrate that, compared to cryopreservation, lower-quality single-cell genomes are recovered when the sample is preserved in ethanol or if the sample undergoes fluorescence in situ hybridization, while sample preservation in paraformaldehyde renders it completely unsuitable for sequencing.

  12. Radiation-induced gene amplification in rodent and human cells

    International Nuclear Information System (INIS)

    Luecke-Huhle, C.; Gloss, B.; Herrlich, P.

    1990-01-01

    Ionizing and UV radiations induce amplification of SV40 DNA sequences integrated in the genome of Chinese hamster cells and increase amplification of the dihydrofolate reductase (DHFR) gene during methotrexate selection in human skin fibroblasts of a patient with ataxia telangiectasia. Various types of external (60-Co-γ-rays, 241-Am-α-particles, UV) or internal radiation (caused by the decay of 125 I incorporated into DNA in form of I-UdR) were applied. By cell fusion experiments it could be shown that SV40 gene amplification is mediated by one or several diffusible trans-acting factors induced or activated in a dose dependent manner by all types of radiation. One of these factors binds to a 10 bp sequence within the minimal origin of replication of SV40. In vivo competition with an excess of a synthetic oligonucleotide comprising this sequence blocks radiation-induced amplification. (author) 25 refs.; 8 figs

  13. Evaluation of three methods of DNA extraction from paraffin-embedded material for the amplification of genomic DNA by means of the PCR technique

    Directory of Open Access Journals (Sweden)

    MESQUITA Ricardo Alves

    2001-01-01

    Full Text Available There are several protocols reported in the literature for the extraction of genomic DNA from formalin-fixed paraffin-embedded samples. Genomic DNA is utilized in molecular analyses, including PCR. This study compares three different methods for the extraction of genomic DNA from formalin-fixed paraffin-embedded (inflammatory fibrous hyperplasia and non-formalin-fixed (normal oral mucosa samples: phenol with enzymatic digestion, and silica with and without enzymatic digestion. The amplification of DNA by means of the PCR technique was carried out with primers for the exon 7 of human keratin type 14. Amplicons were analyzed by means of electrophoresis in an 8% polyacrylamide gel with 5% glycerol, followed by silver-staining visualization. The phenol/enzymatic digestion and the silica/enzymatic digestion methods provided amplicons from both tissue samples. The method described is a potential aid in the establishment of the histopathologic diagnosis and in retrospective studies with archival paraffin-embedded samples.

  14. Ultrasmall volume molecular isothermal amplification in microfluidic chip with advanced surface processing

    International Nuclear Information System (INIS)

    Huang Guoliang; Yang Xiaoyong; Ma Li; Yang Xu

    2011-01-01

    In this paper, we developed a metal micro-fluidic chip with advanced surface processing for ultra-small volume molecular isothermal amplification. This method takes advantages of the nucleic acid amplification with good stability and consistency, high sensitivity about 31 genomic DNA copies and bacteria specific gene identification. Based on the advanced surface processing, the bioreaction assays of nucleic acid amplification was dropped about 392nl in volume. A high numerical aperture confocal optical detection system was advanced to sensitively monitor the DNA amplification with low noise and high power collecting fluorescence near to the optical diffraction limit. A speedy nucleic acid isothermal amplification was performed in the ultra-small volume microfluidic chip, where the time at the inflexions of second derivative to DNA exponential amplified curves was brought forward and the sensitivity was improved about 65 folds to that of in current 25μl Ep-tube amplified reaction, which indicates a promising clinic molecular diagnostics in the droplet amplification.

  15. Sex and PRNP genotype determination in preimplantation caprine embryos.

    Science.gov (United States)

    Guignot, F; Perreau, C; Cavarroc, C; Touzé, J-L; Pougnard, J-L; Dupont, F; Beckers, J-F; Rémy, B; Babilliot, J-M; Bed'Hom, B; Lamorinière, J M; Mermillod, P; Baril, G

    2011-08-01

    The objective of this study was to test the accuracy of genotype diagnosis after whole amplification of DNA extracted from biopsies obtained by trimming goat embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer. Whole genome amplification (WGA) was performed using Multiple Displacement Amplification (MDA). Sex and prion protein (PRNP) genotypes were determined. Sex diagnosis was carried out by PCR amplification of ZFX/ZFY and Y chromosome-specific sequences. Prion protein genotype determination was performed on codons 142, 154, 211, 222 and 240. Embryos were collected at day 7 after oestrus and biopsied either immediately after collection (blastocysts and expanded blastocysts) or after 24 h of in vitro culture (compacted morulae). Biopsied embryos were frozen by vitrification. Vitrified whole embryos were kept as control. DNA of biopsies was extracted and amplified using MDA. Sex diagnosis was efficient for 97.4% of biopsies and PRNP genotyping was determined in 78.7% of biopsies. After embryo transfer, no significant difference was observed in kidding rate between biopsied and vitrified control embryos, whereas embryo survival rate was different between biopsied and whole vitrified embryos (p = 0.032). At birth, 100% of diagnosed sex and 98.2% of predetermined codons were correct. Offspring PRNP profiles were in agreement with parental genotype. Whole genome amplification with MDA kit coupled with sex diagnosis and PRNP genotype predetermination are very accurate techniques to genotype goat embryos before transfer. These novel results allow us to plan selection of scrapie-resistant genotypes and kid sex before transfer of cryopreserved embryo. © 2010 Blackwell Verlag GmbH.

  16. DNA-based identification of spices: DNA isolation, whole genome amplification, and polymerase chain reaction.

    Science.gov (United States)

    Focke, Felix; Haase, Ilka; Fischer, Markus

    2011-01-26

    Usually spices are identified morphologically using simple methods like magnifying glasses or microscopic instruments. On the other hand, molecular biological methods like the polymerase chain reaction (PCR) enable an accurate and specific detection also in complex matrices. Generally, the origins of spices are plants with diverse genetic backgrounds and relationships. The processing methods used for the production of spices are complex and individual. Consequently, the development of a reliable DNA-based method for spice analysis is a challenging intention. However, once established, this method will be easily adapted to less difficult food matrices. In the current study, several alternative methods for the isolation of DNA from spices have been developed and evaluated in detail with regard to (i) its purity (photometric), (ii) yield (fluorimetric methods), and (iii) its amplifiability (PCR). Whole genome amplification methods were used to preamplify isolates to improve the ratio between amplifiable DNA and inhibiting substances. Specific primer sets were designed, and the PCR conditions were optimized to detect 18 spices selectively. Assays of self-made spice mixtures were performed to proof the applicability of the developed methods.

  17. Population diversity of ammonium oxidizers investigated by specific PCR amplification

    Science.gov (United States)

    Ward, B.B.; Voytek, M.A.; Witzel, K.-P.

    1997-01-01

    The species composition of ammonia-oxidizing bacteria in aquatic environments was investigated using PCR primers for 16S rRNA genes to amplify specific subsets of the total ammonia-oxidizer population. The specificity of the amplification reactions was determined using total genomic DNA from known nitrifying strains and non-nitrifying strains identified as having similar rDNA sequences. Specificity of amplification was determined both for direct amplification, using the nitrifier specific primers, and with nested amplification, in which the nitrifier primers were used to reamplify a fragment obtained from direct amplification with Eubacterial universal primers. The present level of specificity allows the distinction between Nitrosomonas europaea, Nitrosomonas sp. (marine) and the other known ammonia-oxidizers in the beta subclass of the Proteobacteria. Using total DNA extracted from natural samples, we used direct amplification to determine presence/absence of different species groups. Species composition was found to differ among depths in vertical profiles of lake samples and among samples and enrichments from various other aquatic environments. Nested PCR yielded several more positive reactions, which implies that nitrifier DNA was present in most samples, but often at very low levels.

  18. SNP high-throughput screening in grapevine using the SNPlex™ genotyping system

    Directory of Open Access Journals (Sweden)

    Velasco Riccardo

    2008-01-01

    Full Text Available Abstract Background Until recently, only a small number of low- and mid-throughput methods have been used for single nucleotide polymorphism (SNP discovery and genotyping in grapevine (Vitis vinifera L.. However, following completion of the sequence of the highly heterozygous genome of Pinot Noir, it has been possible to identify millions of electronic SNPs (eSNPs thus providing a valuable source for high-throughput genotyping methods. Results Herein we report the first application of the SNPlex™ genotyping system in grapevine aiming at the anchoring of an eukaryotic genome. This approach combines robust SNP detection with automated assay readout and data analysis. 813 candidate eSNPs were developed from non-repetitive contigs of the assembled genome of Pinot Noir and tested in 90 progeny of Syrah × Pinot Noir cross. 563 new SNP-based markers were obtained and mapped. The efficiency rate of 69% was enhanced to 80% when multiple displacement amplification (MDA methods were used for preparation of genomic DNA for the SNPlex assay. Conclusion Unlike other SNP genotyping methods used to investigate thousands of SNPs in a few genotypes, or a few SNPs in around a thousand genotypes, the SNPlex genotyping system represents a good compromise to investigate several hundred SNPs in a hundred or more samples simultaneously. Therefore, the use of the SNPlex assay, coupled with whole genome amplification (WGA, is a good solution for future applications in well-equipped laboratories.

  19. Simple system for isothermal DNA amplification coupled to lateral flow detection.

    Directory of Open Access Journals (Sweden)

    Kristina Roskos

    Full Text Available Infectious disease diagnosis in point-of-care settings can be greatly improved through integrated, automated nucleic acid testing devices. We have developed an early prototype for a low-cost system which executes isothermal DNA amplification coupled to nucleic acid lateral flow (NALF detection in a mesofluidic cartridge attached to a portable instrument. Fluid handling inside the cartridge is facilitated through one-way passive valves, flexible pouches, and electrolysis-driven pumps, which promotes a compact and inexpensive instrument design. The closed-system disposable prevents workspace amplicon contamination. The cartridge design is based on standard scalable manufacturing techniques such as injection molding. Nucleic acid amplification occurs in a two-layer pouch that enables efficient heat transfer. We have demonstrated as proof of principle the amplification and detection of Mycobacterium tuberculosis (M.tb genomic DNA in the cartridge, using either Loop Mediated Amplification (LAMP or the Exponential Amplification Reaction (EXPAR, both coupled to NALF detection. We envision that a refined version of this cartridge, including upstream sample preparation coupled to amplification and detection, will enable fully-automated sample-in to answer-out infectious disease diagnosis in primary care settings of low-resource countries with high disease burden.

  20. Common fur and mystacial vibrissae parallel sensory pathways: 14C 2-deoxyglucose and WGA-HRP studies in the rat

    International Nuclear Information System (INIS)

    Sharp, F.R.; Gonzalez, M.F.; Morgan, C.W.; Morton, M.T.; Sharp, J.W.

    1988-01-01

    Stimulation of mystacial vibrissae in rows A,B, and C increased (14C) 2-deoxyglucose (2DG) uptake in spinal trigeminal nucleus pars caudalis (Sp5c) mostly in ventral portions of laminae III-IV with less activation of II and V. Stimulation of common fur above the whiskers mainly activated lamina II, with less activation in deeper layers. The patterns of activation were compatible with an inverted head, onion skin Sp5c somatotopy. Wheatgerm Agglutinin-Horseradish Peroxidase (WGA-HRP) injections into common fur between mystacial vibrissae rows A-B and B-C led to anterograde transganglionic labeling only of Sp5c, mainly of lamina II with less label in layer V, and very sparse label in III and IV. WGA-HRP skin injections appear to primarily label small fibers, which along with larger fibers, were metabolically activated during common fur stimulation. Mystacial vibrissae stimulation increased 2DG uptake in ventral ipsilateral spinal trigeminal nuclei pars interpolaris (Sp5i) and oralis (Sp5o) and principal trigeminal sensory nucleus (Pr5). Common fur stimulation above the whiskers slightly increased 2DG uptake in ventral Sp5i, Sp5o, and possibly Pr5. The most dorsal aspect of the ventroposteromedial (VPM) nucleus of thalamus was activated contralateral to whisker stimulation. Stimulation of the common fur dorsal to the whiskers activated a region of dorsal VPM caudal to the VPM region activated during whisker stimulation. This is consistent with previous data showing that ventral whiskers and portions of the face are represented rostrally in VPM, and more dorsal whiskers and dorsal portions of the face are represented progressively more caudally in VPM. Mystacial vibrissae stimulation activated the contralateral primary sensory SI barrelfield cortex and a separate region in the second somatosensory SII cortex

  1. Real-time monitoring of mycobacterium genomic DNA with target-primed rolling circle amplification by a Au nanoparticle-embedded SPR biosensor.

    Science.gov (United States)

    Xiang, Yang; Zhu, Xiaoyan; Huang, Qing; Zheng, Junsong; Fu, Weiling

    2015-04-15

    In this study, we developed a surface plasmon resonance (SPR) DNA biosensor array based on target-primed rolling circle amplification (RCA) for isothermal and rapid detection of two pathogenic mycobacteria, Mycobacterium tuberculosis complex (MTBC) and Mycobacterium avium complex (MAC).The species-specific padlock probe (PLP) was designed to target the sequence in 16S-23S rRNA gene internal transcribed spacer (ITS). After ligation, the circularized PLP could be primed by the target sequence to initial RCA. The RCA performed simultaneously with the cleavage reaction to produce small fragments of single strand DNA which immediately hybridized with the probe immobilized on the sensor chip without denaturation. This process caused SPR angle changes on the chip surface, which made the detection for analysis from the solution achievable, and dynamic real-time RCA monitoring of mycobacterium possible. Besides, Au nanoparticles (AuNPs) were directly assembled onto the surface of the sensor chip via hexanedithiol (HDT) for the enhancement of sensitivity as a label-free detection system. Experimental results show that the signal enhancement by the target-primed RCA together with AuNPs-embedded surface caused at least10-fold increased sensitivity as compared with conventional RCA on bare SPR chip method. Within 40min amplification duration as low as 20amol of synthetic targets and 10(4)CFUmL(-1) of genomic DNA from clinical samples can be detected. The proposed method not only provides a simple design idea for liquid-phase amplification monitoring, but also apply it in clinical pathogen detection, which holds great promise in ultrasensitive bioassay in the future. Copyright © 2014. Published by Elsevier B.V.

  2. Evidence of high-elevation amplification versus Arctic amplification.

    Science.gov (United States)

    Wang, Qixiang; Fan, Xiaohui; Wang, Mengben

    2016-01-12

    Elevation-dependent warming in high-elevation regions and Arctic amplification are of tremendous interest to many scientists who are engaged in studies in climate change. Here, using annual mean temperatures from 2781 global stations for the 1961-2010 period, we find that the warming for the world's high-elevation stations (>500 m above sea level) is clearly stronger than their low-elevation counterparts; and the high-elevation amplification consists of not only an altitudinal amplification but also a latitudinal amplification. The warming for the high-elevation stations is linearly proportional to the temperature lapse rates along altitudinal and latitudinal gradients, as a result of the functional shape of Stefan-Boltzmann law in both vertical and latitudinal directions. In contrast, neither altitudinal amplification nor latitudinal amplification is found within the Arctic region despite its greater warming than lower latitudes. Further analysis shows that the Arctic amplification is an integrated part of the latitudinal amplification trend for the low-elevation stations (≤500 m above sea level) across the entire low- to high-latitude Northern Hemisphere, also a result of the mathematical shape of Stefan-Boltzmann law but only in latitudinal direction.

  3. Complete Genomes of Classical Swine Fever Virus Cloned into Bacterial Artificial Chromosomes

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Reimann, I.; Uttenthal, Åse

    Complete genome amplification of viral RNA provides a new tool for the generation of modified pestiviruses. We have used our full-genome amplification strategy for generation of amplicons representing complete genomes of classical swine fever virus. The amplicons were cloned directly into a stabl...... single-copy bacterial artificial chromosome (BAC) generating full-length pestivirus DNAs from which infectious RNA transcripts could be also derived. Our strategy allows construction of stable infectious BAC DNAs from a single full-length PCR product....

  4. Genomic Sequencing of Single Microbial Cells from Environmental Samples

    Energy Technology Data Exchange (ETDEWEB)

    Ishoey, Thomas; Woyke, Tanja; Stepanauskas, Ramunas; Novotny, Mark; Lasken, Roger S.

    2008-02-01

    Recently developed techniques allow genomic DNA sequencing from single microbial cells [Lasken RS: Single-cell genomic sequencing using multiple displacement amplification, Curr Opin Microbiol 2007, 10:510-516]. Here, we focus on research strategies for putting these methods into practice in the laboratory setting. An immediate consequence of single-cell sequencing is that it provides an alternative to culturing organisms as a prerequisite for genomic sequencing. The microgram amounts of DNA required as template are amplified from a single bacterium by a method called multiple displacement amplification (MDA) avoiding the need to grow cells. The ability to sequence DNA from individual cells will likely have an immense impact on microbiology considering the vast numbers of novel organisms, which have been inaccessible unless culture-independent methods could be used. However, special approaches have been necessary to work with amplified DNA. MDA may not recover the entire genome from the single copy present in most bacteria. Also, some sequence rearrangements can occur during the DNA amplification reaction. Over the past two years many research groups have begun to use MDA, and some practical approaches to single-cell sequencing have been developed. We review the consensus that is emerging on optimum methods, reliability of amplified template, and the proper interpretation of 'composite' genomes which result from the necessity of combining data from several single-cell MDA reactions in order to complete the assembly. Preferred laboratory methods are considered on the basis of experience at several large sequencing centers where >70% of genomes are now often recovered from single cells. Methods are reviewed for preparation of bacterial fractions from environmental samples, single-cell isolation, DNA amplification by MDA, and DNA sequencing.

  5. DNA sequence responsible for the amplification of adjacent genes.

    Science.gov (United States)

    Pasion, S G; Hartigan, J A; Kumar, V; Biswas, D K

    1987-10-01

    A 10.3-kb DNA fragment in the 5'-flanking region of the rat prolactin (rPRL) gene was isolated from F1BGH(1)2C1, a strain of rat pituitary tumor cells (GH cells) that produces prolactin in response to 5-bromodeoxyuridine (BrdU). Following transfection and integration into genomic DNA of recipient mouse L cells, this DNA induced amplification of the adjacent thymidine kinase gene from Herpes simplex virus type 1 (HSV1TK). We confirmed the ability of this "Amplicon" sequence to induce amplification of other linked or unlinked genes in DNA-mediated gene transfer studies. When transferred into the mouse L cells with the 10.3-5'rPRL gene sequence of BrdU-responsive cells, both the human growth hormone and the HSV1TK genes are amplified in response to 5-bromodeoxyuridine. This observation is substantiated by BrdU-induced amplification of the cotransferred bacterial Neo gene. Cotransfection studies reveal that the BrdU-induced amplification capability is associated with a 4-kb DNA sequence in the 5'-flanking region of the rPRL gene of BrdU-responsive cells. These results demonstrate that genes of heterologous origin, linked or unlinked, and selected or unselected, can be coamplified when located within the amplification boundary of the Amplicon sequence.

  6. Reliable single cell array CGH for clinical samples.

    Directory of Open Access Journals (Sweden)

    Zbigniew T Czyż

    Full Text Available BACKGROUND: Disseminated cancer cells (DCCs and circulating tumor cells (CTCs are extremely rare, but comprise the precursors cells of distant metastases or therapy resistant cells. The detailed molecular analysis of these cells may help to identify key events of cancer cell dissemination, metastatic colony formation and systemic therapy escape. METHODOLOGY/PRINCIPAL FINDINGS: Using the Ampli1™ whole genome amplification (WGA technology and high-resolution oligonucleotide aCGH microarrays we optimized conditions for the analysis of structural copy number changes. The protocol presented here enables reliable detection of numerical genomic alterations as small as 0.1 Mb in a single cell. Analysis of single cells from well-characterized cell lines and single normal cells confirmed the stringent quantitative nature of the amplification and hybridization protocol. Importantly, fixation and staining procedures used to detect DCCs showed no significant impact on the outcome of the analysis, proving the clinical usability of our method. In a proof-of-principle study we tracked the chromosomal changes of single DCCs over a full course of high-dose chemotherapy treatment by isolating and analyzing DCCs of an individual breast cancer patient at four different time points. CONCLUSIONS/SIGNIFICANCE: The protocol enables detailed genome analysis of DCCs and thereby assessment of the clonal evolution during the natural course of the disease and under selection pressures. The results from an exemplary patient provide evidence that DCCs surviving selective therapeutic conditions may be recruited from a pool of genomically less advanced cells, which display a stable subset of specific genomic alterations.

  7. Isothermal multiple displacement amplification: a methodical approach enhancing molecular routine diagnostics of microcarcinomas and small biopsies

    Directory of Open Access Journals (Sweden)

    Mairinger FD

    2014-08-01

    Full Text Available Fabian D Mairinger,1 Robert FH Walter,2 Claudia Vollbrecht,3 Thomas Hager,1 Karl Worm,1 Saskia Ting,1 Jeremias Wohlschläger,1 Paul Zarogoulidis,4 Konstantinos Zarogoulidis,4 Kurt W Schmid1 1Institute of Pathology, 2Ruhrlandklinik, West German Lung Center, University Hospital Essen, Essen, 3Institute of Pathology, University Hospital Cologne, Cologne, Germany; 4Pulmonary Department, Oncology Unit, G Papanikolaou General Hospital, Aristotle University of Thessaloniki, Thessaloniki, Greece Background and methods: Isothermal multiple displacement amplification (IMDA can be a powerful tool in molecular routine diagnostics for homogeneous and sequence-independent whole-genome amplification of notably small tumor samples, eg, microcarcinomas and biopsies containing a small amount of tumor. Currently, this method is not well established in pathology laboratories. We designed a study to confirm the feasibility and convenience of this method for routine diagnostics with formalin-fixed, paraffin-embedded samples prepared by laser-capture microdissection. Results: A total of 250 µg DNA (concentration 5 µg/µL was generated by amplification over a period of 8 hours with a material input of approximately 25 cells, approximately equivalent to 175 pg of genomic DNA. In the generated DNA, a representation of all chromosomes could be shown and the presence of elected genes relevant for diagnosis in clinical samples could be proven. Mutational analysis of clinical samples could be performed without any difficulty and showed concordance with earlier diagnostic findings. Conclusion: We established the feasibility and convenience of IMDA for routine diagnostics. We also showed that small amounts of DNA, which were not analyzable with current molecular methods, could be sufficient for a wide field of applications in molecular routine diagnostics when they are preamplified with IMDA. Keywords: isothermal multiple displacement amplification, isothermal, whole-genome

  8. Approaches to genotyping individual miracidia of Schistosoma japonicum.

    Science.gov (United States)

    Xiao, Ning; Remais, Justin V; Brindley, Paul J; Qiu, Dong-Chuan; Carlton, Elizabeth J; Li, Rong-Zhi; Lei, Yang; Blair, David

    2013-12-01

    Molecular genetic tools are needed to address questions as to the source and dynamics of transmission of the human blood fluke Schistosoma japonicum in regions where human infections have reemerged, and to characterize infrapopulations in individual hosts. The life stage that interests us as a target for collecting genotypic data is the miracidium, a very small larval stage that consequently yields very little DNA for analysis. Here, we report the successful development of a multiplex format permitting genotyping of 17 microsatellite loci in four sequential multiplex reactions using a single miracidium held on a Whatman Classic FTA indicating card. This approach was successful after short storage periods, but after long storage (>4 years), considerable difficulty was encountered in multiplex genotyping, necessitating the use of whole genome amplification (WGA) methods. WGA applied to cards stored for long periods of time resulted in sufficient DNA for accurate and repeatable genotyping. Trials and tests of these methods, as well as application to some field-collected samples, are reported, along with the discussion of the potential insights to be gained from such techniques. These include recognition of sibships among miracidia from a single host, and inference of the minimum number of worm pairs that might be present in a host.

  9. Single-tube linear DNA amplification (LinDA) for robust ChIP-seq

    NARCIS (Netherlands)

    Shankaranarayanan, P.; Mendoza-Parra, M.A.; Walia, M.; Wang, L.; Li, N.; Trindade, L.M.; Gronemeyer, H.

    2011-01-01

    Genome-wide profiling of transcription factors based on massive parallel sequencing of immunoprecipitated chromatin (ChIP-seq) requires nanogram amounts of DNA. Here we describe a high-fidelity, single-tube linear DNA amplification method (LinDA) for ChIP-seq and reChIP-seq with picogram DNA amounts

  10. A mechanism of gene amplification driven by small DNA fragments.

    Directory of Open Access Journals (Sweden)

    Kuntal Mukherjee

    Full Text Available DNA amplification is a molecular process that increases the copy number of a chromosomal tract and often causes elevated expression of the amplified gene(s. Although gene amplification is frequently observed in cancer and other degenerative disorders, the molecular mechanisms involved in the process of DNA copy number increase remain largely unknown. We hypothesized that small DNA fragments could be the trigger of DNA amplification events. Following our findings that small fragments of DNA in the form of DNA oligonucleotides can be highly recombinogenic, we have developed a system in the yeast Saccharomyces cerevisiae to capture events of chromosomal DNA amplification initiated by small DNA fragments. Here we demonstrate that small DNAs can amplify a chromosomal region, generating either tandem duplications or acentric extrachromosomal DNA circles. Small fragment-driven DNA amplification (SFDA occurs with a frequency that increases with the length of homology between the small DNAs and the target chromosomal regions. SFDA events are triggered even by small single-stranded molecules with as little as 20-nt homology with the genomic target. A double-strand break (DSB external to the chromosomal amplicon region stimulates the amplification event up to a factor of 20 and favors formation of extrachromosomal circles. SFDA is dependent on Rad52 and Rad59, partially dependent on Rad1, Rad10, and Pol32, and independent of Rad51, suggesting a single-strand annealing mechanism. Our results reveal a novel molecular model for gene amplification, in which small DNA fragments drive DNA amplification and define the boundaries of the amplicon region. As DNA fragments are frequently found both inside cells and in the extracellular environment, such as the serum of patients with cancer or other degenerative disorders, we propose that SFDA may be a common mechanism for DNA amplification in cancer cells, as well as a more general cause of DNA copy number variation

  11. Analysis of the giant genomes of Fritillaria (Liliaceae) indicates that a lack of DNA removal characterizes extreme expansions in genome size.

    Science.gov (United States)

    Kelly, Laura J; Renny-Byfield, Simon; Pellicer, Jaume; Macas, Jiří; Novák, Petr; Neumann, Pavel; Lysak, Martin A; Day, Peter D; Berger, Madeleine; Fay, Michael F; Nichols, Richard A; Leitch, Andrew R; Leitch, Ilia J

    2015-10-01

    Plants exhibit an extraordinary range of genome sizes, varying by > 2000-fold between the smallest and largest recorded values. In the absence of polyploidy, changes in the amount of repetitive DNA (transposable elements and tandem repeats) are primarily responsible for genome size differences between species. However, there is ongoing debate regarding the relative importance of amplification of repetitive DNA versus its deletion in governing genome size. Using data from 454 sequencing, we analysed the most repetitive fraction of some of the largest known genomes for diploid plant species, from members of Fritillaria. We revealed that genomic expansion has not resulted from the recent massive amplification of just a handful of repeat families, as shown in species with smaller genomes. Instead, the bulk of these immense genomes is composed of highly heterogeneous, relatively low-abundance repeat-derived DNA, supporting a scenario where amplified repeats continually accumulate due to infrequent DNA removal. Our results indicate that a lack of deletion and low turnover of repetitive DNA are major contributors to the evolution of extremely large genomes and show that their size cannot simply be accounted for by the activity of a small number of high-abundance repeat families. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  12. A novel method, digital genome scanning detects KRAS gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth

    Directory of Open Access Journals (Sweden)

    Yanagihara Kazuyoshi

    2009-06-01

    Full Text Available Abstract Background Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of KRAS are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS, which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells. Methods DGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for KRAS amplification by quantitative PCR, and investigated KRAS amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of KRAS knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively. Results DGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the KRAS gene locus. Amplification of the KRAS locus was detected in 15% (3/20 of gastric cancer cell lines (8–18-fold amplification and 4.7% (4/86 of primary gastric tumors (8–50-fold amplification. KRAS mutations were identified in two of the three cell lines in which KRAS was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased KRAS copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type KRAS, but not in cells with amplified mutant KRAS. Knock-down of KRAS in gastric cancer cells that carried amplified wild

  13. A novel method, digital genome scanning detects KRAS gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth

    International Nuclear Information System (INIS)

    Mita, Hiroaki; Yanagihara, Kazuyoshi; Fujita, Masahiro; Hosokawa, Masao; Kusano, Masanobu; Sabau, Sorin Vasile; Tatsumi, Haruyuki; Imai, Kohzoh; Shinomura, Yasuhisa; Tokino, Takashi; Toyota, Minoru; Aoki, Fumio; Akashi, Hirofumi; Maruyama, Reo; Sasaki, Yasushi; Suzuki, Hiromu; Idogawa, Masashi; Kashima, Lisa

    2009-01-01

    Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of KRAS are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS), which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells. DGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for KRAS amplification by quantitative PCR, and investigated KRAS amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of KRAS knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively. DGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the KRAS gene locus. Amplification of the KRAS locus was detected in 15% (3/20) of gastric cancer cell lines (8–18-fold amplification) and 4.7% (4/86) of primary gastric tumors (8–50-fold amplification). KRAS mutations were identified in two of the three cell lines in which KRAS was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased KRAS copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type KRAS, but not in cells with amplified mutant KRAS. Knock-down of KRAS in gastric cancer cells that carried amplified wild-type KRAS resulted in the inhibition of cell growth and

  14. Whole-genome sequencing of a laboratory-evolved yeast strain

    Directory of Open Access Journals (Sweden)

    Dunham Maitreya J

    2010-02-01

    Full Text Available Abstract Background Experimental evolution of microbial populations provides a unique opportunity to study evolutionary adaptation in response to controlled selective pressures. However, until recently it has been difficult to identify the precise genetic changes underlying adaptation at a genome-wide scale. New DNA sequencing technologies now allow the genome of parental and evolved strains of microorganisms to be rapidly determined. Results We sequenced >93.5% of the genome of a laboratory-evolved strain of the yeast Saccharomyces cerevisiae and its ancestor at >28× depth. Both single nucleotide polymorphisms and copy number amplifications were found, with specific gains over array-based methodologies previously used to analyze these genomes. Applying a segmentation algorithm to quantify structural changes, we determined the approximate genomic boundaries of a 5× gene amplification. These boundaries guided the recovery of breakpoint sequences, which provide insights into the nature of a complex genomic rearrangement. Conclusions This study suggests that whole-genome sequencing can provide a rapid approach to uncover the genetic basis of evolutionary adaptations, with further applications in the study of laboratory selections and mutagenesis screens. In addition, we show how single-end, short read sequencing data can provide detailed information about structural rearrangements, and generate predictions about the genomic features and processes that underlie genome plasticity.

  15. Single-Cell-Based Platform for Copy Number Variation Profiling through Digital Counting of Amplified Genomic DNA Fragments.

    Science.gov (United States)

    Li, Chunmei; Yu, Zhilong; Fu, Yusi; Pang, Yuhong; Huang, Yanyi

    2017-04-26

    We develop a novel single-cell-based platform through digital counting of amplified genomic DNA fragments, named multifraction amplification (mfA), to detect the copy number variations (CNVs) in a single cell. Amplification is required to acquire genomic information from a single cell, while introducing unavoidable bias. Unlike prevalent methods that directly infer CNV profiles from the pattern of sequencing depth, our mfA platform denatures and separates the DNA molecules from a single cell into multiple fractions of a reaction mix before amplification. By examining the sequencing result of each fraction for a specific fragment and applying a segment-merge maximum likelihood algorithm to the calculation of copy number, we digitize the sequencing-depth-based CNV identification and thus provide a method that is less sensitive to the amplification bias. In this paper, we demonstrate a mfA platform through multiple displacement amplification (MDA) chemistry. When performing the mfA platform, the noise of MDA is reduced; therefore, the resolution of single-cell CNV identification can be improved to 100 kb. We can also determine the genomic region free of allelic drop-out with mfA platform, which is impossible for conventional single-cell amplification methods.

  16. Early embryogenesis-specific expression of the rice transposon Ping enhances amplification of the MITE mPing.

    Directory of Open Access Journals (Sweden)

    Shota Teramoto

    2014-06-01

    Full Text Available Miniature inverted-repeat transposable elements (MITEs are numerically predominant transposable elements in the rice genome, and their activities have influenced the evolution of genes. Very little is known about how MITEs can rapidly amplify to thousands in the genome. The rice MITE mPing is quiescent in most cultivars under natural growth conditions, although it is activated by various stresses, such as tissue culture, gamma-ray irradiation, and high hydrostatic pressure. Exceptionally in the temperate japonica rice strain EG4 (cultivar Gimbozu, mPing has reached over 1000 copies in the genome, and is amplifying owing to its active transposition even under natural growth conditions. Being the only active MITE, mPing in EG4 is an appropriate material to study how MITEs amplify in the genome. Here, we provide important findings regarding the transposition and amplification of mPing in EG4. Transposon display of mPing using various tissues of a single EG4 plant revealed that most de novo mPing insertions arise in embryogenesis during the period from 3 to 5 days after pollination (DAP, and a large majority of these insertions are transmissible to the next generation. Locus-specific PCR showed that mPing excisions and insertions arose at the same time (3 to 5 DAP. Moreover, expression analysis and in situ hybridization analysis revealed that Ping, an autonomous partner for mPing, was markedly up-regulated in the 3 DAP embryo of EG4, whereas such up-regulation of Ping was not observed in the mPing-inactive cultivar Nipponbare. These results demonstrate that the early embryogenesis-specific expression of Ping is responsible for the successful amplification of mPing in EG4. This study helps not only to elucidate the whole mechanism of mPing amplification but also to further understand the contribution of MITEs to genome evolution.

  17. Amplification of PVT1 contributes to the pathophysiology ofovarian and breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Guan, Yinghui; Kuo, Wen-Lin; Stilwell, Jackie; Takano, Hirokuni; Lapuk, Anna; Fridlyand, Jane; Mao, Jian-Hua; Yu, Mami; Ginzinger, David; Gray, Joe W.

    2007-10-09

    Purpose. This study was designed to elucidate the role of amplification at 8q24 in the pathophysiology of ovarian and breast cancer since increased copy number at this locus is one of the most frequent genomic abnormalities in these cancers. Experimental Design. To accomplish this, we assessed the association of amplification at 8q24 with outcome in ovarian cancers using FISH to tissue microarrays and measured responses of ovarian and breast cancer cell lines to specific small interfering RNAs (siRNA) against the oncogene, MYC, and a putative noncoding RNA, PVT1, both of which map to 8q24. Results. Amplification of 8q24 was associated with significantly reduced survival duration. In addition, siRNA-mediated reduction in either PVT1 or MYC expression inhibited proliferation in breast and ovarian cancer cell lines in which they were both amplified and over expressed but not in lines in which they were not amplified/over expressed. Inhibition of PVT1 expression also induced a strong apoptotic response in cell lines in which it was over expressed but not in lines in which it was not amplified/over expressed. Inhibition of MYC, on the other hand, did not induce an apoptotic response in cell lines in which MYC was amplified and over expressed. Conclusions. These results suggest that MYC and PVT1 contribute independently to ovarian and breast pathogenesis when over expressed because of genomic abnormalities. They also suggest that PVT1 mediated inhibition of apoptosis may explain why amplification of 8q24 is associated with reduced survival duration in patients treated with agents that act through apoptotic mechanisms.

  18. Quantitative high-resolution genomic analysis of single cancer cells.

    Science.gov (United States)

    Hannemann, Juliane; Meyer-Staeckling, Sönke; Kemming, Dirk; Alpers, Iris; Joosse, Simon A; Pospisil, Heike; Kurtz, Stefan; Görndt, Jennifer; Püschel, Klaus; Riethdorf, Sabine; Pantel, Klaus; Brandt, Burkhard

    2011-01-01

    During cancer progression, specific genomic aberrations arise that can determine the scope of the disease and can be used as predictive or prognostic markers. The detection of specific gene amplifications or deletions in single blood-borne or disseminated tumour cells that may give rise to the development of metastases is of great clinical interest but technically challenging. In this study, we present a method for quantitative high-resolution genomic analysis of single cells. Cells were isolated under permanent microscopic control followed by high-fidelity whole genome amplification and subsequent analyses by fine tiling array-CGH and qPCR. The assay was applied to single breast cancer cells to analyze the chromosomal region centred by the therapeutical relevant EGFR gene. This method allows precise quantitative analysis of copy number variations in single cell diagnostics.

  19. The full-length E1-circumflexE4 protein of human papillomavirus type 18 modulates differentiation-dependent viral DNA amplification and late gene expression

    International Nuclear Information System (INIS)

    Wilson, Regina; Ryan, Gordon B.; Knight, Gillian L.; Laimins, Laimonis A.; Roberts, Sally

    2007-01-01

    Activation of the productive phase of the human papillomavirus (HPV) life cycle in differentiated keratinocytes is coincident with high-level expression of E1-circumflexE4 protein. To determine the role of E1-circumflexE4 in the HPV replication cycle, we constructed HPV18 mutant genomes in which expression of the full-length E1-circumflexE4 protein was abrogated. Undifferentiated keratinocytes containing mutant genomes showed enhanced proliferation when compared to cells containing wildtype genomes, but there were no differences in maintenance of viral episomes. Following differentiation, cells with mutant genomes exhibited reduced levels of viral DNA amplification and late gene expression, compared to wildtype genome-containing cells. This indicates that HPV18 E1-circumflexE4 plays an important role in regulating HPV late functions, and it may also function in the early phase of the replication cycle. Our finding that full-length HPV18 E1-circumflexE4 protein plays a significant role in promoting viral genome amplification concurs with a similar report with HPV31, but is in contrast to an HPV11 study where viral DNA amplification was not dependent on full-length E1-circumflexE4 expression, and to HPV16 where only C-terminal truncations in E1-circumflexE4 abrogated vegetative genome replication. This suggests that type-specific differences exist between various E1-circumflexE4 proteins

  20. Homozygous Deletions and Recurrent Amplifications Implicate New Genes Involved in Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Wennuan Liu

    2008-08-01

    Full Text Available Prostate cancer cell lines provide ideal in vitro systems for the identification and analysis of prostate tumor suppressors and oncogenes. A detailed characterization of the architecture of prostate cancer cell line genomes would facilitate the study of precise roles of various genes in prostate tumorigenesis in general. To contribute to such a characterization, we used the GeneChip 500K single nucleotide polymorphic (SNP array for analysis of genotypes and relative DNA copy number changes across the genome of 11 cell lines derived from both normal and cancerous prostate tissues. For comparison purposes, we also examined the alterations observed in the cell lines in tumor/normal pairs of clinical samples from 72 patients. Along with genome-wide maps of DNA copy number changes and loss of heterozygosity for these cell lines, we report previously unreported homozygous deletions and recurrent amplifications in prostate cancers in this study. The homozygous deletions affected a number of biologically important genes, including PPP2R2A and BNIP3L identified in this study and CDKN2A/CDKN2B reported previously. Although most amplified genomic regions tended to be large, amplifications at 8q24.21 were of particular interest because the affected regions are relatively small, are found in multiple cell lines, are located near MYC, an oncogene strongly implicated in prostate tumorigenesis, and are known to harbor SNPs that are associated with inherited susceptibility for prostate cancer. The genomic alterations revealed in this study provide an important catalog of positional information relevant to efforts aimed at deciphering the molecular genetic basis of prostate cancer.

  1. Microsatellite loci in Japanese quail and cross-species amplification in chicken and guinea fowl

    Directory of Open Access Journals (Sweden)

    Mizutani Makoto

    2002-03-01

    Full Text Available Abstract In line with the Gifu University's initiative to map the Japanese quail genome, a total of 100 Japanese quail microsatellite markers isolated in our laboratory were evaluated in a population of 20 unrelated quails randomly sampled from a colony of wild quail origin. Ninety-eight markers were polymorphic with an average of 3.7 alleles per locus and a mean heterozygosity of 0.423. To determine the utility of these markers for comparative genome mapping in Phasianidae, cross-species amplification of all the markers was tested with chicken and guinea fowl DNA. Amplification products similar in size to the orthologous loci in quail were observed in 42 loci in chicken and 20 loci in guinea fowl. Of the cross-reactive markers, 57.1% in chicken and 55.0% in guinea fowl were polymorphic when tested in 20 birds from their respective populations. Five of 15 markers that could cross-amplify Japanese quail, chicken, and guinea fowl DNA were polymorphic in all three species. Amplification of orthologous loci was confirmed by sequencing 10 loci each from chicken and guinea fowl and comparing with them the corresponding quail sequence. The microsatellite markers reported would serve as a useful resource base for genetic mapping in quail and comparative mapping in Phasianidae.

  2. Locked nucleic acid inhibits amplification of contaminating DNA in real-time PCR

    DEFF Research Database (Denmark)

    Hummelshoj, Lone; Ryder, Lars P; Madsen, Hans O

    2005-01-01

    and real-time PCR, the addition of LNA showed blocking of the amplification of genomic XBP1 but not cDNA XBP1. To test the effect of melting temperature (Tm) on the LNA, we investigated the number of LNA nucleotides that could be replaced with DNA nucleotides and still retain the blocking activity. More...

  3. Detecting single DNA copy number variations in complex genomes using one nanogram of starting DNA and BAC-array CGH.

    Science.gov (United States)

    Guillaud-Bataille, Marine; Valent, Alexander; Soularue, Pascal; Perot, Christine; Inda, Maria Mar; Receveur, Aline; Smaïli, Sadek; Roest Crollius, Hugues; Bénard, Jean; Bernheim, Alain; Gidrol, Xavier; Danglot, Gisèle

    2004-07-29

    Comparative genomic hybridization to bacterial artificial chromosome (BAC)-arrays (array-CGH) is a highly efficient technique, allowing the simultaneous measurement of genomic DNA copy number at hundreds or thousands of loci, and the reliable detection of local one-copy-level variations. We report a genome-wide amplification method allowing the same measurement sensitivity, using 1 ng of starting genomic DNA, instead of the classical 1 microg usually necessary. Using a discrete series of DNA fragments, we defined the parameters adapted to the most faithful ligation-mediated PCR amplification and the limits of the technique. The optimized protocol allows a 3000-fold DNA amplification, retaining the quantitative characteristics of the initial genome. Validation of the amplification procedure, using DNA from 10 tumour cell lines hybridized to BAC-arrays of 1500 spots, showed almost perfectly superimposed ratios for the non-amplified and amplified DNAs. Correlation coefficients of 0.96 and 0.99 were observed for regions of low-copy-level variations and all regions, respectively (including in vivo amplified oncogenes). Finally, labelling DNA using two nucleotides bearing the same fluorophore led to a significant increase in reproducibility and to the correct detection of one-copy gain or loss in >90% of the analysed data, even for pseudotriploid tumour genomes.

  4. The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes

    Science.gov (United States)

    Liu, Shengyi; Liu, Yumei; Yang, Xinhua; Tong, Chaobo; Edwards, David; Parkin, Isobel A. P.; Zhao, Meixia; Ma, Jianxin; Yu, Jingyin; Huang, Shunmou; Wang, Xiyin; Wang, Junyi; Lu, Kun; Fang, Zhiyuan; Bancroft, Ian; Yang, Tae-Jin; Hu, Qiong; Wang, Xinfa; Yue, Zhen; Li, Haojie; Yang, Linfeng; Wu, Jian; Zhou, Qing; Wang, Wanxin; King, Graham J; Pires, J. Chris; Lu, Changxin; Wu, Zhangyan; Sampath, Perumal; Wang, Zhuo; Guo, Hui; Pan, Shengkai; Yang, Limei; Min, Jiumeng; Zhang, Dong; Jin, Dianchuan; Li, Wanshun; Belcram, Harry; Tu, Jinxing; Guan, Mei; Qi, Cunkou; Du, Dezhi; Li, Jiana; Jiang, Liangcai; Batley, Jacqueline; Sharpe, Andrew G; Park, Beom-Seok; Ruperao, Pradeep; Cheng, Feng; Waminal, Nomar Espinosa; Huang, Yin; Dong, Caihua; Wang, Li; Li, Jingping; Hu, Zhiyong; Zhuang, Mu; Huang, Yi; Huang, Junyan; Shi, Jiaqin; Mei, Desheng; Liu, Jing; Lee, Tae-Ho; Wang, Jinpeng; Jin, Huizhe; Li, Zaiyun; Li, Xun; Zhang, Jiefu; Xiao, Lu; Zhou, Yongming; Liu, Zhongsong; Liu, Xuequn; Qin, Rui; Tang, Xu; Liu, Wenbin; Wang, Yupeng; Zhang, Yangyong; Lee, Jonghoon; Kim, Hyun Hee; Denoeud, France; Xu, Xun; Liang, Xinming; Hua, Wei; Wang, Xiaowu; Wang, Jun; Chalhoub, Boulos; Paterson, Andrew H

    2014-01-01

    Polyploidization has provided much genetic variation for plant adaptive evolution, but the mechanisms by which the molecular evolution of polyploid genomes establishes genetic architecture underlying species differentiation are unclear. Brassica is an ideal model to increase knowledge of polyploid evolution. Here we describe a draft genome sequence of Brassica oleracea, comparing it with that of its sister species B. rapa to reveal numerous chromosome rearrangements and asymmetrical gene loss in duplicated genomic blocks, asymmetrical amplification of transposable elements, differential gene co-retention for specific pathways and variation in gene expression, including alternative splicing, among a large number of paralogous and orthologous genes. Genes related to the production of anticancer phytochemicals and morphological variations illustrate consequences of genome duplication and gene divergence, imparting biochemical and morphological variation to B. oleracea. This study provides insights into Brassica genome evolution and will underpin research into the many important crops in this genus. PMID:24852848

  5. Quantitative high-resolution genomic analysis of single cancer cells.

    Directory of Open Access Journals (Sweden)

    Juliane Hannemann

    Full Text Available During cancer progression, specific genomic aberrations arise that can determine the scope of the disease and can be used as predictive or prognostic markers. The detection of specific gene amplifications or deletions in single blood-borne or disseminated tumour cells that may give rise to the development of metastases is of great clinical interest but technically challenging. In this study, we present a method for quantitative high-resolution genomic analysis of single cells. Cells were isolated under permanent microscopic control followed by high-fidelity whole genome amplification and subsequent analyses by fine tiling array-CGH and qPCR. The assay was applied to single breast cancer cells to analyze the chromosomal region centred by the therapeutical relevant EGFR gene. This method allows precise quantitative analysis of copy number variations in single cell diagnostics.

  6. Characterizing the cancer genome in lung adenocarcinoma

    Science.gov (United States)

    Weir, Barbara A.; Woo, Michele S.; Getz, Gad; Perner, Sven; Ding, Li; Beroukhim, Rameen; Lin, William M.; Province, Michael A.; Kraja, Aldi; Johnson, Laura A.; Shah, Kinjal; Sato, Mitsuo; Thomas, Roman K.; Barletta, Justine A.; Borecki, Ingrid B.; Broderick, Stephen; Chang, Andrew C.; Chiang, Derek Y.; Chirieac, Lucian R.; Cho, Jeonghee; Fujii, Yoshitaka; Gazdar, Adi F.; Giordano, Thomas; Greulich, Heidi; Hanna, Megan; Johnson, Bruce E.; Kris, Mark G.; Lash, Alex; Lin, Ling; Lindeman, Neal; Mardis, Elaine R.; McPherson, John D.; Minna, John D.; Morgan, Margaret B.; Nadel, Mark; Orringer, Mark B.; Osborne, John R.; Ozenberger, Brad; Ramos, Alex H.; Robinson, James; Roth, Jack A.; Rusch, Valerie; Sasaki, Hidefumi; Shepherd, Frances; Sougnez, Carrie; Spitz, Margaret R.; Tsao, Ming-Sound; Twomey, David; Verhaak, Roel G. W.; Weinstock, George M.; Wheeler, David A.; Winckler, Wendy; Yoshizawa, Akihiko; Yu, Soyoung; Zakowski, Maureen F.; Zhang, Qunyuan; Beer, David G.; Wistuba, Ignacio I.; Watson, Mark A.; Garraway, Levi A.; Ladanyi, Marc; Travis, William D.; Pao, William; Rubin, Mark A.; Gabriel, Stacey B.; Gibbs, Richard A.; Varmus, Harold E.; Wilson, Richard K.; Lander, Eric S.; Meyerson, Matthew

    2008-01-01

    Somatic alterations in cellular DNA underlie almost all human cancers1. The prospect of targeted therapies2 and the development of high-resolution, genome-wide approaches3–8 are now spurring systematic efforts to characterize cancer genomes. Here we report a large-scale project to characterize copy-number alterations in primary lung adenocarcinomas. By analysis of a large collection of tumors (n = 371) using dense single nucleotide polymorphism arrays, we identify a total of 57 significantly recurrent events. We find that 26 of 39 autosomal chromosome arms show consistent large-scale copy-number gain or loss, of which only a handful have been linked to a specific gene. We also identify 31 recurrent focal events, including 24 amplifications and 7 homozygous deletions. Only six of these focal events are currently associated with known mutations in lung carcinomas. The most common event, amplification of chromosome 14q13.3, is found in ~12% of samples. On the basis of genomic and functional analyses, we identify NKX2-1 (NK2 homeobox 1, also called TITF1), which lies in the minimal 14q13.3 amplification interval and encodes a lineage-specific transcription factor, as a novel candidate proto-oncogene involved in a significant fraction of lung adenocarcinomas. More generally, our results indicate that many of the genes that are involved in lung adenocarcinoma remain to be discovered. PMID:17982442

  7. Amplification factor variable amplifier

    NARCIS (Netherlands)

    Akitsugu, Oshita; Nauta, Bram

    2007-01-01

    PROBLEM TO BE SOLVED: To provide an amplification factor variable amplifier capable of achieving temperature compensation of an amplification factor over a wide variable amplification factor range. ; SOLUTION: A Gilbert type amplification factor variable amplifier 11 amplifies an input signal and

  8. The successes and future prospects of the linear antisense RNA amplification methodology.

    Science.gov (United States)

    Li, Jifen; Eberwine, James

    2018-05-01

    It has been over a quarter of a century since the introduction of the linear RNA amplification methodology known as antisense RNA (aRNA) amplification. Whereas most molecular biology techniques are rapidly replaced owing to the fast-moving nature of development in the field, the aRNA procedure has become a base that can be built upon through varied uses of the technology. The technique was originally developed to assess RNA populations from small amounts of starting material, including single cells, but over time its use has evolved to include the detection of various cellular entities such as proteins, RNA-binding-protein-associated cargoes, and genomic DNA. In this Perspective we detail the linear aRNA amplification procedure and its use in assessing various components of a cell's chemical phenotype. This procedure is particularly useful in efforts to multiplex the simultaneous detection of various cellular processes. These efforts are necessary to identify the quantitative chemical phenotype of cells that underlies cellular function.

  9. Amplification factor variable amplifier

    NARCIS (Netherlands)

    Akitsugu, Oshita; Nauta, Bram

    2010-01-01

    PROBLEM TO BE SOLVED: To provide an amplification factor variable amplifier capable of achieving temperature compensation of an amplification factor over a wide variable amplification factor range. ;SOLUTION: A Gilbert type amplification factor variable amplifier 11 amplifies an input signal and can

  10. Genome U-Plot: a whole genome visualization.

    Science.gov (United States)

    Gaitatzes, Athanasios; Johnson, Sarah H; Smadbeck, James B; Vasmatzis, George

    2018-05-15

    The ability to produce and analyze whole genome sequencing (WGS) data from samples with structural variations (SV) generated the need to visualize such abnormalities in simplified plots. Conventional two-dimensional representations of WGS data frequently use either circular or linear layouts. There are several diverse advantages regarding both these representations, but their major disadvantage is that they do not use the two-dimensional space very efficiently. We propose a layout, termed the Genome U-Plot, which spreads the chromosomes on a two-dimensional surface and essentially quadruples the spatial resolution. We present the Genome U-Plot for producing clear and intuitive graphs that allows researchers to generate novel insights and hypotheses by visualizing SVs such as deletions, amplifications, and chromoanagenesis events. The main features of the Genome U-Plot are its layered layout, its high spatial resolution and its improved aesthetic qualities. We compare conventional visualization schemas with the Genome U-Plot using visualization metrics such as number of line crossings and crossing angle resolution measures. Based on our metrics, we improve the readability of the resulting graph by at least 2-fold, making apparent important features and making it easy to identify important genomic changes. A whole genome visualization tool with high spatial resolution and improved aesthetic qualities. An implementation and documentation of the Genome U-Plot is publicly available at https://github.com/gaitat/GenomeUPlot. vasmatzis.george@mayo.edu. Supplementary data are available at Bioinformatics online.

  11. Whole genome amplification and microsatellite genotyping of herbarium DNA revealed the identity of an ancient grapevine cultivar

    Science.gov (United States)

    Malenica, Nenad; Šimon, Silvio; Besendorfer, Višnja; Maletić, Edi; Karoglan Kontić, Jasminka; Pejić, Ivan

    2011-09-01

    Reconstruction of the grapevine cultivation history has advanced tremendously during the last decade. Identification of grapevine cultivars by using microsatellite DNA markers has mostly become a routine. The parentage of several renowned grapevine cultivars, like Cabernet Sauvignon and Chardonnay, has been elucidated. However, the assembly of a complete grapevine genealogy is not yet possible because missing links might no longer be in cultivation or are even extinct. This problem could be overcome by analyzing ancient DNA from grapevine herbarium specimens and other historical remnants of once cultivated varieties. Here, we present the first successful genotyping of a grapevine herbarium specimen and the identification of the corresponding grapevine cultivar. Using a set of nine grapevine microsatellite markers, in combination with a whole genome amplification procedure, we found the 90-year-old Tribidrag herbarium specimen to display the same microsatellite profile as the popular American cultivar Zinfandel. This work, together with information from several historical documents, provides a new clue of Zinfandel cultivation in Croatia as early as the beginning of fifteenth century, under the native name Tribidrag. Moreover, it emphasizes substantial information potential of existing grapevine and other herbarium collections worldwide.

  12. ASAP: Amplification, sequencing & annotation of plastomes

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    Folta Kevin M

    2005-12-01

    Full Text Available Abstract Background Availability of DNA sequence information is vital for pursuing structural, functional and comparative genomics studies in plastids. Traditionally, the first step in mining the valuable information within a chloroplast genome requires sequencing a chloroplast plasmid library or BAC clones. These activities involve complicated preparatory procedures like chloroplast DNA isolation or identification of the appropriate BAC clones to be sequenced. Rolling circle amplification (RCA is being used currently to amplify the chloroplast genome from purified chloroplast DNA and the resulting products are sheared and cloned prior to sequencing. Herein we present a universal high-throughput, rapid PCR-based technique to amplify, sequence and assemble plastid genome sequence from diverse species in a short time and at reasonable cost from total plant DNA, using the large inverted repeat region from strawberry and peach as proof of concept. The method exploits the highly conserved coding regions or intergenic regions of plastid genes. Using an informatics approach, chloroplast DNA sequence information from 5 available eudicot plastomes was aligned to identify the most conserved regions. Cognate primer pairs were then designed to generate ~1 – 1.2 kb overlapping amplicons from the inverted repeat region in 14 diverse genera. Results 100% coverage of the inverted repeat region was obtained from Arabidopsis, tobacco, orange, strawberry, peach, lettuce, tomato and Amaranthus. Over 80% coverage was obtained from distant species, including Ginkgo, loblolly pine and Equisetum. Sequence from the inverted repeat region of strawberry and peach plastome was obtained, annotated and analyzed. Additionally, a polymorphic region identified from gel electrophoresis was sequenced from tomato and Amaranthus. Sequence analysis revealed large deletions in these species relative to tobacco plastome thus exhibiting the utility of this method for structural and

  13. Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology.

    Science.gov (United States)

    Binga, Erik K; Lasken, Roger S; Neufeld, Josh D

    2008-03-01

    Microbial ecology is a field that applies molecular techniques to analyze genes and communities associated with a plethora of unique environments on this planet. In the past, low biomass and the predominance of a few abundant community members have impeded the application of techniques such as PCR, microarray analysis and metagenomics to complex microbial populations. In the absence of suitable cultivation methods, it was not possible to obtain DNA samples from individual microorganisms. Recently, a method called multiple displacement amplification (MDA) has been used to circumvent these limitations by amplifying DNA from microbial communities in low-biomass environments, individual cells from uncultivated microbial species and active organisms obtained through stable isotope probing incubations. This review describes the development and applications of MDA, discusses its strengths and limitations and highlights the impact of MDA on the field of microbial ecology. Whole genome amplification via MDA has increased access to the genomic DNA of uncultivated microorganisms and low-biomass environments and represents a 'power tool' in the molecular toolbox of microbial ecologists.

  14. GST-PRIME: an algorithm for genome-wide primer design.

    Science.gov (United States)

    Leister, Dario; Varotto, Claudio

    2007-01-01

    The profiling of mRNA expression based on DNA arrays has become a powerful tool to study genome-wide transcription of genes in a number of organisms. GST-PRIME is a software package created to facilitate large-scale primer design for the amplification of probes to be immobilized on arrays for transcriptome analyses, even though it can be also applied in low-throughput approaches. GST-PRIME allows highly efficient, direct amplification of gene-sequence tags (GSTs) from genomic DNA (gDNA), starting from annotated genome or transcript sequences. GST-PRIME provides a customer-friendly platform for automatic primer design, and despite the relative simplicity of the algorithm, experimental tests in the model plant species Arabidopsis thaliana confirmed the reliability of the software. This chapter describes the algorithm used for primer design, its input and output files, and the installation of the standalone package and its use.

  15. Epstein–Barr virus particles induce centrosome amplification and chromosomal instability

    Science.gov (United States)

    Shumilov, Anatoliy; Tsai, Ming-Han; Schlosser, Yvonne T.; Kratz, Anne-Sophie; Bernhardt, Katharina; Fink, Susanne; Mizani, Tuba; Lin, Xiaochen; Jauch, Anna; Mautner, Josef; Kopp-Schneider, Annette; Feederle, Regina; Hoffmann, Ingrid; Delecluse, Henri-Jacques

    2017-01-01

    Infections with Epstein–Barr virus (EBV) are associated with cancer development, and EBV lytic replication (the process that generates virus progeny) is a strong risk factor for some cancer types. Here we report that EBV infection of B-lymphocytes (in vitro and in a mouse model) leads to an increased rate of centrosome amplification, associated with chromosomal instability. This effect can be reproduced with virus-like particles devoid of EBV DNA, but not with defective virus-like particles that cannot infect host cells. Viral protein BNRF1 induces centrosome amplification, and BNRF1-deficient viruses largely lose this property. These findings identify a new mechanism by which EBV particles can induce chromosomal instability without establishing a chronic infection, thereby conferring a risk for development of tumours that do not necessarily carry the viral genome. PMID:28186092

  16. Fenton reaction induced cancer in wild type rats recapitulates genomic alterations observed in human cancer.

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    Shinya Akatsuka

    Full Text Available Iron overload has been associated with carcinogenesis in humans. Intraperitoneal administration of ferric nitrilotriacetate initiates a Fenton reaction in renal proximal tubules of rodents that ultimately leads to a high incidence of renal cell carcinoma (RCC after repeated treatments. We performed high-resolution microarray comparative genomic hybridization to identify characteristics in the genomic profiles of this oxidative stress-induced rat RCCs. The results revealed extensive large-scale genomic alterations with a preference for deletions. Deletions and amplifications were numerous and sometimes fragmented, demonstrating that a Fenton reaction is a cause of such genomic alterations in vivo. Frequency plotting indicated that two of the most commonly altered loci corresponded to a Cdkn2a/2b deletion and a Met amplification. Tumor sizes were proportionally associated with Met expression and/or amplification, and clustering analysis confirmed our results. Furthermore, we developed a procedure to compare whole genomic patterns of the copy number alterations among different species based on chromosomal syntenic relationship. Patterns of the rat RCCs showed the strongest similarity to the human RCCs among five types of human cancers, followed by human malignant mesothelioma, an iron overload-associated cancer. Therefore, an iron-dependent Fenton chemical reaction causes large-scale genomic alterations during carcinogenesis, which may result in distinct genomic profiles. Based on the characteristics of extensive genome alterations in human cancer, our results suggest that this chemical reaction may play a major role during human carcinogenesis.

  17. The JAK-STAT transcriptional regulator, STAT-5, activates the ATM DNA damage pathway to induce HPV 31 genome amplification upon epithelial differentiation.

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    Shiyuan Hong

    Full Text Available High-risk human papillomavirus (HPV must evade innate immune surveillance to establish persistent infections and to amplify viral genomes upon differentiation. Members of the JAK-STAT family are important regulators of the innate immune response and HPV proteins downregulate expression of STAT-1 to allow for stable maintenance of viral episomes. STAT-5 is another member of this pathway that modulates the inflammatory response and plays an important role in controlling cell cycle progression in response to cytokines and growth factors. Our studies show that HPV E7 activates STAT-5 phosphorylation without altering total protein levels. Inhibition of STAT-5 phosphorylation by the drug pimozide abolishes viral genome amplification and late gene expression in differentiating keratinocytes. In contrast, treatment of undifferentiated cells that stably maintain episomes has no effect on viral replication. Knockdown studies show that the STAT-5β isoform is mainly responsible for this activity and that this is mediated through the ATM DNA damage response. A downstream target of STAT-5, the peroxisome proliferator-activated receptor γ (PPARγ contributes to the effects on members of the ATM pathway. Overall, these findings identify an important new regulatory mechanism by which the innate immune regulator, STAT-5, promotes HPV viral replication through activation of the ATM DNA damage response.

  18. DNAzyme Feedback Amplification: Relaying Molecular Recognition to Exponential DNA Amplification.

    Science.gov (United States)

    Liu, Meng; Yin, Qingxin; McConnell, Erin M; Chang, Yangyang; Brennan, John D; Li, Yingfu

    2018-03-26

    Technologies capable of linking DNA amplification to molecular recognition are very desirable for ultrasensitive biosensing applications. We have developed a simple but powerful isothermal DNA amplification method, termed DNAzyme feedback amplification (DFA), that is capable of relaying molecular recognition to exponential DNA amplification. The method incorporates both an RNA-cleaving DNAzyme (RCD) and rolling circle amplification (RCA) carried out by a special DNA polymerase using a circular DNA template. DFA begins with a stimulus-dependent RCA reaction, producing tandemly linked RCDs in long-chain DNA products. These RCDs cleave an RNA-containing DNA sequence to form additional primers that hybridize to the circular DNA molecule, giving rise to DNA assemblies that act as the new inputs for RCA. The RCA reaction and the cleavage event keep on feeding each other autonomously, resulting in exponential growth of repetitive DNA sequences that can be easily detected. This method can be used for the detection of both nucleic acid based targets and non-nucleic acid analytes. In this article, we discuss the conceptual framework of the feedback amplification approach, the essential features of this method as well as remaining challenges and possible solutions. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Isothermal multiple displacement amplification: a methodical approach enhancing molecular routine diagnostics of microcarcinomas and small biopsies.

    Science.gov (United States)

    Mairinger, Fabian D; Walter, Robert Fh; Vollbrecht, Claudia; Hager, Thomas; Worm, Karl; Ting, Saskia; Wohlschläger, Jeremias; Zarogoulidis, Paul; Zarogoulidis, Konstantinos; Schmid, Kurt W

    2014-01-01

    Isothermal multiple displacement amplification (IMDA) can be a powerful tool in molecular routine diagnostics for homogeneous and sequence-independent whole-genome amplification of notably small tumor samples, eg, microcarcinomas and biopsies containing a small amount of tumor. Currently, this method is not well established in pathology laboratories. We designed a study to confirm the feasibility and convenience of this method for routine diagnostics with formalin-fixed, paraffin-embedded samples prepared by laser-capture microdissection. A total of 250 μg DNA (concentration 5 μg/μL) was generated by amplification over a period of 8 hours with a material input of approximately 25 cells, approximately equivalent to 175 pg of genomic DNA. In the generated DNA, a representation of all chromosomes could be shown and the presence of elected genes relevant for diagnosis in clinical samples could be proven. Mutational analysis of clinical samples could be performed without any difficulty and showed concordance with earlier diagnostic findings. We established the feasibility and convenience of IMDA for routine diagnostics. We also showed that small amounts of DNA, which were not analyzable with current molecular methods, could be sufficient for a wide field of applications in molecular routine diagnostics when they are preamplified with IMDA.

  20. Detection of MDM2/CDK4 amplification in lipomatous soft tissue tumors from formalin-fixed, paraffin-embedded tissue: comparison of multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH).

    Science.gov (United States)

    Creytens, David; van Gorp, Joost; Ferdinande, Liesbeth; Speel, Ernst-Jan; Libbrecht, Louis

    2015-02-01

    In this study, the detection of MDM2 and CDK4 amplification was evaluated in lipomatous soft tissue tumors using multiplex ligation-dependent probe amplification (MLPA), a PCR-based technique, in comparison with fluorescence in situ hybridization (FISH). These 2 techniques were evaluated in a series of 77 formalin-fixed, paraffin-embedded lipomatous tumors (27 benign adipose tumors, 28 atypical lipomatous tumors/well-differentiated liposarcomas, 18 dedifferentiated liposarcomas, and 4 pleomorphic liposarcomas). Using MLPA, with a cut-off ratio of >2, 36/71 samples (22 atypical lipomatous tumors/well-differentiated liposarcomas, and 14 dedifferentiated liposarcomas) showed MDM2 and CDK4 amplification. Using FISH as gold standard, MLPA showed a sensitivity of 90% (36/40) and a specificity of 100% (31/31) in detecting amplification of MDM2 and CDK4 in lipomatous soft tissue tumors. In case of high-level amplification (MDM2-CDK4/CEP12 ratio >5), concordance was 100%. Four cases of atypical lipomatous tumor/well-differentiated liposarcoma (4/26, 15%) with a low MDM2 and CDK4 amplification level (MDM2-CDK4/CEP12 ratio ranging between 2 and 2.5) detected by FISH showed no amplification by MLPA, although gain of MDM2 and CDK4 (ratios ranging between 1.6 and 1.9) was seen with MLPA. No amplification was detected in benign lipomatous tumors and pleomorphic liposarcomas. Furthermore, there was a very high concordance between the ratios obtained by FISH and MLPA. In conclusion, MLPA proves to be an appropriate and straightforward technique for screening MDM2/CDK4 amplification in lipomatous tumors, especially when a correct cut-off value and reference samples are chosen, and could be considered a good alternative to FISH to determine MDM2 and CDK4 amplification in liposarcomas. Moreover, because MLPA, as a multiplex technique, allows simultaneous detection of multiple chromosomal changes of interest, it could be in the future a very reliable and fast molecular analysis on

  1. Genomic amplification patterns of human telomerase RNA gene and C-MYC in liquid-based cytological specimens used for the detection of high-grade cervical intraepithelial neoplasia

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    Chen Shaomin

    2012-04-01

    Full Text Available Abstract Background The amplification of oncogenes initiated by high-risk human papillomavirus (HPV infection is an early event in cervical carcinogenesis and can be used for cervical lesion diagnosis. We measured the genomic amplification rates and the patterns of human telomerase RNA gene (TERC and C-MYC in the liquid-based cytological specimens to evaluate the diagnostic characteristics for the detection of high-grade cervical lesions. Methods Two hundred and forty-three residual cytological specimens were obtained from outpatients aged 25 to 64 years at Qilu Hospital, Shandong University. The specimens were evaluated by fluorescence in situ hybridization (FISH using chromosome probes to TERC (3q26 and C-MYC (8q24. All of the patients underwent colposcopic examination and histological evaluation. A Chi-square test was used for categorical data analysis. Results In the normal, cervical intraepithelial neoplasia grade 1 (CIN1, grade 2 (CIN2, grade 3 (CIN3 and squamous cervical cancer (SCC cases, the TERC positive rates were 9.2%, 17.2%, 76.2%, 100.0% and 100.0%, respectively; the C-MYC positive rates were 20.7%, 31.0%, 71.4%, 81.8% and 100.0%, respectively. The TERC and C-MYC positive rates were higher in the CIN2+ (CIN2, CIN3 and SCC cases than in the normal and CIN1 cases (p p p > 0.05. Conclusions The TERC test is highly sensitive and is therefore suitable for cervical cancer screening. The C-MYC test is not suitable for cancer screening because of its lower sensitivity. The amplification patterns of TERC become more diverse and complex as the severity of cervical diseases increases, whereas for C-MYC, the amplification patterns are similar between the normal/CIN1 and CIN2+ groups. Virtual slides The virtual slide(s for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1308004512669913.

  2. Integrated genomic and gene expression profiling identifies two major genomic circuits in urothelial carcinoma.

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    David Lindgren

    Full Text Available Similar to other malignancies, urothelial carcinoma (UC is characterized by specific recurrent chromosomal aberrations and gene mutations. However, the interconnection between specific genomic alterations, and how patterns of chromosomal alterations adhere to different molecular subgroups of UC, is less clear. We applied tiling resolution array CGH to 146 cases of UC and identified a number of regions harboring recurrent focal genomic amplifications and deletions. Several potential oncogenes were included in the amplified regions, including known oncogenes like E2F3, CCND1, and CCNE1, as well as new candidate genes, such as SETDB1 (1q21, and BCL2L1 (20q11. We next combined genome profiling with global gene expression, gene mutation, and protein expression data and identified two major genomic circuits operating in urothelial carcinoma. The first circuit was characterized by FGFR3 alterations, overexpression of CCND1, and 9q and CDKN2A deletions. The second circuit was defined by E3F3 amplifications and RB1 deletions, as well as gains of 5p, deletions at PTEN and 2q36, 16q, 20q, and elevated CDKN2A levels. TP53/MDM2 alterations were common for advanced tumors within the two circuits. Our data also suggest a possible RAS/RAF circuit. The tumors with worst prognosis showed a gene expression profile that indicated a keratinized phenotype. Taken together, our integrative approach revealed at least two separate networks of genomic alterations linked to the molecular diversity seen in UC, and that these circuits may reflect distinct pathways of tumor development.

  3. Gene amplification in Chinese hamster embryo cells by the decay of incorporated iodine-125

    International Nuclear Information System (INIS)

    Luecke-Huhle, Christine; Ehrfeld, Angelika; Rau, Waltraud

    1988-01-01

    Simian Virus 40-transformed Chinese hamster embryo cells (Co631) contain 5 viral copies integrated per cell genome. These SV40 sequences were used as an endogenous indicator gene to study response of mammalian cells to radiation at gene level. Cells were internally irradiated by Auger electrons emitted by Iodine-125 which was incorporated in cell DNA in form of 5-[ 125 I] iododeoxyuridine ( 125 IdU). An increase in gene copy number was measured using dispersed cell blotting and Southern analysis in combination with highly sensitive DNA hybridization. A 13-fold amplification of the SV40 sequences and a 2-fold amplification of two cellular oncogenes of the ras family were found. Other cellular genes, like the α-actin gene, are not amplified and no variation in gene copy number was observed after incubation of cells with cold IdU. Thus, specific gene amplification seems to be the consequence of radiation-induced DNA damage and the resulting cell cycle arrest. (author)

  4. Frequent amplification of CENPF, GMNN and CDK13 genes in hepatocellular carcinomas.

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    Hye-Eun Kim

    Full Text Available Genomic changes frequently occur in cancer cells during tumorigenesis from normal cells. Using the Illumina Human NS-12 single-nucleotide polymorphism (SNP chip to screen for gene copy number changes in primary hepatocellular carcinomas (HCCs, we initially detected amplification of 35 genes from four genomic regions (1q21-41, 6p21.2-24.1, 7p13 and 8q13-23. By integrated screening of these genes for both DNA copy number and gene expression in HCC and colorectal cancer, we selected CENPF (centromere protein F/mitosin, GMNN (geminin, DNA replication inhibitor, CDK13 (cyclin-dependent kinase 13, and FAM82B (family with sequence similarity 82, member B as common cancer genes. Each gene exhibited an amplification frequency of ~30% (range, 20-50% in primary HCC (n = 57 and colorectal cancer (n = 12, as well as in a panel of human cancer cell lines (n = 70. Clonogenic and invasion assays of NIH3T3 cells transfected with each of the four amplified genes showed that CENPF, GMNN, and CDK13 were highly oncogenic whereas FAM82B was not. Interestingly, the oncogenic activity of these genes (excluding FAM82B was highly correlated with gene-copy numbers in tumor samples (correlation coefficient, r>0.423, indicating that amplifications of CENPF, GMNN, and CDK13 genes are tightly linked and coincident in tumors. Furthermore, we confirmed that CDK13 gene copy number was significantly associated with clinical onset age in patients with HCC (P = 0.0037. Taken together, our results suggest that coincidently amplified CDK13, GMNN, and CENPF genes can play a role as common cancer-driver genes in human cancers.

  5. Multiplex amplification of large sets of human exons.

    Science.gov (United States)

    Porreca, Gregory J; Zhang, Kun; Li, Jin Billy; Xie, Bin; Austin, Derek; Vassallo, Sara L; LeProust, Emily M; Peck, Bill J; Emig, Christopher J; Dahl, Fredrik; Gao, Yuan; Church, George M; Shendure, Jay

    2007-11-01

    A new generation of technologies is poised to reduce DNA sequencing costs by several orders of magnitude. But our ability to fully leverage the power of these technologies is crippled by the absence of suitable 'front-end' methods for isolating complex subsets of a mammalian genome at a scale that matches the throughput at which these platforms will routinely operate. We show that targeting oligonucleotides released from programmable microarrays can be used to capture and amplify approximately 10,000 human exons in a single multiplex reaction. Additionally, we show integration of this protocol with ultra-high-throughput sequencing for targeted variation discovery. Although the multiplex capture reaction is highly specific, we found that nonuniform capture is a key issue that will need to be resolved by additional optimization. We anticipate that highly multiplexed methods for targeted amplification will enable the comprehensive resequencing of human exons at a fraction of the cost of whole-genome resequencing.

  6. Mechanism of chimera formation during the Multiple Displacement Amplification reaction

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    Stockwell Timothy B

    2007-04-01

    Full Text Available Abstract Background Multiple Displacement Amplification (MDA is a method used for amplifying limiting DNA sources. The high molecular weight amplified DNA is ideal for DNA library construction. While this has enabled genomic sequencing from one or a few cells of unculturable microorganisms, the process is complicated by the tendency of MDA to generate chimeric DNA rearrangements in the amplified DNA. Determining the source of the DNA rearrangements would be an important step towards reducing or eliminating them. Results Here, we characterize the major types of chimeras formed by carrying out an MDA whole genome amplification from a single E. coli cell and sequencing by the 454 Life Sciences method. Analysis of 475 chimeras revealed the predominant reaction mechanisms that create the DNA rearrangements. The highly branched DNA synthesized in MDA can assume many alternative secondary structures. DNA strands extended on an initial template can be displaced becoming available to prime on a second template creating the chimeras. Evidence supports a model in which branch migration can displace 3'-ends freeing them to prime on the new templates. More than 85% of the resulting DNA rearrangements were inverted sequences with intervening deletions that the model predicts. Intramolecular rearrangements were favored, with displaced 3'-ends reannealing to single stranded 5'-strands contained within the same branched DNA molecule. In over 70% of the chimeric junctions, the 3' termini had initiated priming at complimentary sequences of 2–21 nucleotides (nts in the new templates. Conclusion Formation of chimeras is an important limitation to the MDA method, particularly for whole genome sequencing. Identification of the mechanism for chimera formation provides new insight into the MDA reaction and suggests methods to reduce chimeras. The 454 sequencing approach used here will provide a rapid method to assess the utility of reaction modifications.

  7. High Genomic Instability Predicts Survival in Metastatic High-Risk Neuroblastoma

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    Sara Stigliani

    2012-09-01

    Full Text Available We aimed to identify novel molecular prognostic markers to better predict relapse risk estimate for children with high-risk (HR metastatic neuroblastoma (NB. We performed genome- and/or transcriptome-wide analyses of 129 stage 4 HR NBs. Children older than 1 year of age were categorized as “short survivors” (dead of disease within 5 years from diagnosis and “long survivors” (alive with an overall survival time ≥ 5 years. We reported that patients with less than three segmental copy number aberrations in their tumor represent a molecularly defined subgroup with a high survival probability within the current HR group of patients. The complex genomic pattern is a prognostic marker independent of NB-associated chromosomal aberrations, i.e., MYCN amplification, 1p and 11q losses, and 17q gain. Integrative analysis of genomic and expression signatures demonstrated that fatal outcome is mainly associated with loss of cell cycle control and deregulation of Rho guanosine triphosphates (GTPases functioning in neuritogenesis. Tumors with MYCN amplification show a lower chromosome instability compared to MYCN single-copy NBs (P = .0008, dominated by 17q gain and 1p loss. Moreover, our results suggest that the MYCN amplification mainly drives disruption of neuronal differentiation and reduction of cell adhesion process involved in tumor invasion and metastasis. Further validation studies are warranted to establish this as a risk stratification for patients.

  8. Analyses of Genotypes and Phenotypes of Ten Chinese Patients with Wolf-Hirschhorn Syndrome by Multiplex Ligation-dependent Probe Amplification and Array Comparative Genomic Hybridization.

    Science.gov (United States)

    Yang, Wen-Xu; Pan, Hong; Li, Lin; Wu, Hai-Rong; Wang, Song-Tao; Bao, Xin-Hua; Jiang, Yu-Wu; Qi, Yu

    2016-03-20

    Wolf-Hirschhorn syndrome (WHS) is a contiguous gene syndrome that is typically caused by a deletion of the distal portion of the short arm of chromosome 4. However, there are few reports about the features of Chinese WHS patients. This study aimed to characterize the clinical and molecular cytogenetic features of Chinese WHS patients using the combination of multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (array CGH). Clinical information was collected from ten patients with WHS. Genomic DNA was extracted from the peripheral blood of the patients. The deletions were analyzed by MLPA and array CGH. All patients exhibited the core clinical symptoms of WHS, including severe growth delay, a Greek warrior helmet facial appearance, differing degrees of intellectual disability, and epilepsy or electroencephalogram anomalies. The 4p deletions ranged from 2.62 Mb to 17.25 Mb in size and included LETM1, WHSC1, and FGFR3. The combined use of MLPA and array CGH is an effective and specific means to diagnose WHS and allows for the precise identification of the breakpoints and sizes of deletions. The deletion of genes in the WHS candidate region is closely correlated with the core WHS phenotype.

  9. Optimization of Saanen sperm genes amplification: evaluation of standardized protocols in genetically uncharacterized rural goats reared under a subtropical environment.

    Science.gov (United States)

    Barbour, Elie K; Saade, Maya F; Sleiman, Fawwak T; Hamadeh, Shady K; Mouneimne, Youssef; Kassaifi, Zeina; Kayali, Ghazi; Harakeh, Steve; Jaber, Lina S; Shaib, Houssam A

    2012-10-01

    The purpose of this research is to optimize quantitatively the amplification of specific sperm genes in reference genomically characterized Saanen goat and to evaluate the standardized protocols applicability on sperms of uncharacterized genome of rural goats reared under subtropical environment for inclusion in future selection programs. The optimization of the protocols in Saanen sperms included three production genes (growth hormone (GH) exons 2, 3, and 4, αS1-casein (CSN1S1), and α-lactalbumin) and two health genes (MHC class II DRB and prion (PrP)). The optimization was based on varying the primers concentrations and the inclusion of a PCR cosolvent (Triton X). The impact of the studied variables on statistically significant increase in the yield of amplicons was noticed in four out of five (80%) optimized protocols, namely in those related to GH, CSN1S1, α-lactalbumin, and PrP genes (P 0.05). The applicability of the optimized protocols of Saanen sperm genes on amplification of uncharacterized rural goat sperms revealed a 100% success in tested individuals for amplification of GH, CSN1S1, α-lactalbumin, and MHC class II DRB genes and a 75% success for the PrP gene. The significant success in applicability of the Saanen quantitatively optimized protocols to other uncharacterized genome of rural goats allows for their inclusion in future selection, targeting the sustainability of this farming system in a subtropical environment and the improvement of the farmers livelihood.

  10. Chemically induced DNA hypomethylation in breast carcinoma cells detected by the amplification of intermethylated sites

    International Nuclear Information System (INIS)

    Sadikovic, Bekim; Haines, Thomas R; Butcher, Darci T; Rodenhiser, David I

    2004-01-01

    Compromised patterns of gene expression result in genomic instability, altered patterns of gene expression and tumour formation. Specifically, aberrant DNA hypermethylation in gene promoter regions leads to gene silencing, whereas global hypomethylation events can result in chromosomal instability and oncogene activation. Potential links exist between environmental agents and DNA methylation, but the destabilizing effects of environmental exposures on the DNA methylation machinery are not understood within the context of breast cancer aetiology. We assessed genome-wide changes in methylation patterns using a unique methylation profiling technique called amplification of intermethylated sites (AIMS). This method generates easily readable fingerprints that represent the investigated cell line's methylation profile, based on the differential cleavage of DNA with methylation-specific isoschisomeric restriction endonucleases. We validated this approach by demonstrating both unique and reoccurring sites of genomic hypomethylation in four breast carcinoma cell lines treated with the cytosine analogue 5-azacytidine. Comparison of treated with control samples revealed individual bands that exhibited methylation changes, and these bands were excized and cloned, and the precise genomic location individually identified. In most cases, these regions of hypomethylation coincided with susceptible target regions previously associated with chromosome breakage, rearrangement and gene amplification. Similarly, we observed that acute benzopyrene exposure is associated with altered methylation patterns in these cell lines. These results reinforce the link between environmental exposures, DNA methylation and breast cancer, and support a role for AIMS as a rapid, affordable screening method to identify environmentally induced DNA methylation changes that occur in tumourigenesis

  11. Origin-Dependent Inverted-Repeat Amplification: Tests of a Model for Inverted DNA Amplification.

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    Bonita J Brewer

    2015-12-01

    Full Text Available DNA replication errors are a major driver of evolution--from single nucleotide polymorphisms to large-scale copy number variations (CNVs. Here we test a specific replication-based model to explain the generation of interstitial, inverted triplications. While no genetic information is lost, the novel inversion junctions and increased copy number of the included sequences create the potential for adaptive phenotypes. The model--Origin-Dependent Inverted-Repeat Amplification (ODIRA-proposes that a replication error at pre-existing short, interrupted, inverted repeats in genomic sequences generates an extrachromosomal, inverted dimeric, autonomously replicating intermediate; subsequent genomic integration of the dimer yields this class of CNV without loss of distal chromosomal sequences. We used a combination of in vitro and in vivo approaches to test the feasibility of the proposed replication error and its downstream consequences on chromosome structure in the yeast Saccharomyces cerevisiae. We show that the proposed replication error-the ligation of leading and lagging nascent strands to create "closed" forks-can occur in vitro at short, interrupted inverted repeats. The removal of molecules with two closed forks results in a hairpin-capped linear duplex that we show replicates in vivo to create an inverted, dimeric plasmid that subsequently integrates into the genome by homologous recombination, creating an inverted triplication. While other models have been proposed to explain inverted triplications and their derivatives, our model can also explain the generation of human, de novo, inverted amplicons that have a 2:1 mixture of sequences from both homologues of a single parent--a feature readily explained by a plasmid intermediate that arises from one homologue and integrates into the other homologue prior to meiosis. Our tests of key features of ODIRA lend support to this mechanism and suggest further avenues of enquiry to unravel the origins

  12. Origin-Dependent Inverted-Repeat Amplification: Tests of a Model for Inverted DNA Amplification.

    Science.gov (United States)

    Brewer, Bonita J; Payen, Celia; Di Rienzi, Sara C; Higgins, Megan M; Ong, Giang; Dunham, Maitreya J; Raghuraman, M K

    2015-12-01

    DNA replication errors are a major driver of evolution--from single nucleotide polymorphisms to large-scale copy number variations (CNVs). Here we test a specific replication-based model to explain the generation of interstitial, inverted triplications. While no genetic information is lost, the novel inversion junctions and increased copy number of the included sequences create the potential for adaptive phenotypes. The model--Origin-Dependent Inverted-Repeat Amplification (ODIRA)-proposes that a replication error at pre-existing short, interrupted, inverted repeats in genomic sequences generates an extrachromosomal, inverted dimeric, autonomously replicating intermediate; subsequent genomic integration of the dimer yields this class of CNV without loss of distal chromosomal sequences. We used a combination of in vitro and in vivo approaches to test the feasibility of the proposed replication error and its downstream consequences on chromosome structure in the yeast Saccharomyces cerevisiae. We show that the proposed replication error-the ligation of leading and lagging nascent strands to create "closed" forks-can occur in vitro at short, interrupted inverted repeats. The removal of molecules with two closed forks results in a hairpin-capped linear duplex that we show replicates in vivo to create an inverted, dimeric plasmid that subsequently integrates into the genome by homologous recombination, creating an inverted triplication. While other models have been proposed to explain inverted triplications and their derivatives, our model can also explain the generation of human, de novo, inverted amplicons that have a 2:1 mixture of sequences from both homologues of a single parent--a feature readily explained by a plasmid intermediate that arises from one homologue and integrates into the other homologue prior to meiosis. Our tests of key features of ODIRA lend support to this mechanism and suggest further avenues of enquiry to unravel the origins of interstitial

  13. Genome-wide comparative analysis reveals human-mouse regulatory landscape and evolution.

    Science.gov (United States)

    Denas, Olgert; Sandstrom, Richard; Cheng, Yong; Beal, Kathryn; Herrero, Javier; Hardison, Ross C; Taylor, James

    2015-02-14

    Because species-specific gene expression is driven by species-specific regulation, understanding the relationship between sequence and function of the regulatory regions in different species will help elucidate how differences among species arise. Despite active experimental and computational research, relationships among sequence, conservation, and function are still poorly understood. We compared transcription factor occupied segments (TFos) for 116 human and 35 mouse TFs in 546 human and 125 mouse cell types and tissues from the Human and the Mouse ENCODE projects. We based the map between human and mouse TFos on a one-to-one nucleotide cross-species mapper, bnMapper, that utilizes whole genome alignments (WGA). Our analysis shows that TFos are under evolutionary constraint, but a substantial portion (25.1% of mouse and 25.85% of human on average) of the TFos does not have a homologous sequence on the other species; this portion varies among cell types and TFs. Furthermore, 47.67% and 57.01% of the homologous TFos sequence shows binding activity on the other species for human and mouse respectively. However, 79.87% and 69.22% is repurposed such that it binds the same TF in different cells or different TFs in the same cells. Remarkably, within the set of repurposed TFos, the corresponding genome regions in the other species are preferred locations of novel TFos. These events suggest exaptation of some functional regulatory sequences into new function. Despite TFos repurposing, we did not find substantial changes in their predicted target genes, suggesting that CRMs buffer evolutionary events allowing little or no change in the TFos - target gene associations. Thus, the small portion of TFos with strictly conserved occupancy underestimates the degree of conservation of regulatory interactions. We mapped regulatory sequences from an extensive number of TFs and cell types between human and mouse using WGA. A comparative analysis of this correspondence unveiled the

  14. Evaluation of whole genome amplified DNA to decrease material expenditure and increase quality

    Directory of Open Access Journals (Sweden)

    Marie Bækvad-Hansen

    2017-06-01

    Discussion: Whole genome amplified DNA samples from dried blood spots is well suited for array genotyping and produces robust and reliable genotype data. However, the amplification process introduces additional noise to the data, making detection of structural variants such as copy number variants difficult. With this study, we explore ways of optimizing the amplification protocol in order to reduce noise and increase data quality. We found, that the amplification process was very robust, and that changes in amplification time or temperature did not alter the genotyping calls or quality of the array data. Adding additional replicates of each sample also lead to insignificant changes in the array data. Thus, the amount of noise introduced by the amplification process was consistent regardless of changes made to the amplification protocol. We also explored ways of decreasing material expenditure by reducing the spot size or the amplification reaction volume. The reduction did not affect the quality of the genotyping data.

  15. High-throughput genotyping of single nucleotide polymorphisms with rolling circle amplification

    Directory of Open Access Journals (Sweden)

    Sun Zhenyu

    2001-08-01

    Full Text Available Abstract Background Single nucleotide polymorphisms (SNPs are the foundation of powerful complex trait and pharmacogenomic analyses. The availability of large SNP databases, however, has emphasized a need for inexpensive SNP genotyping methods of commensurate simplicity, robustness, and scalability. We describe a solution-based, microtiter plate method for SNP genotyping of human genomic DNA. The method is based upon allele discrimination by ligation of open circle probes followed by rolling circle amplification of the signal using fluorescent primers. Only the probe with a 3' base complementary to the SNP is circularized by ligation. Results SNP scoring by ligation was optimized to a 100,000 fold discrimination against probe mismatched to the SNP. The assay was used to genotype 10 SNPs from a set of 192 genomic DNA samples in a high-throughput format. Assay directly from genomic DNA eliminates the need to preamplify the target as done for many other genotyping methods. The sensitivity of the assay was demonstrated by genotyping from 1 ng of genomic DNA. We demonstrate that the assay can detect a single molecule of the circularized probe. Conclusions Compatibility with homogeneous formats and the ability to assay small amounts of genomic DNA meets the exacting requirements of automated, high-throughput SNP scoring.

  16. GAB2 amplifications refine molecular classification of melanoma.

    Science.gov (United States)

    Chernoff, Karen A; Bordone, Lindsey; Horst, Basil; Simon, Katherine; Twadell, William; Lee, Keagan; Cohen, Jason A; Wang, Shuang; Silvers, David N; Brunner, Georg; Celebi, Julide Tok

    2009-07-01

    Gain-of-function mutations in BRAF, NRAS, or KIT are associated with distinct melanoma subtypes with KIT mutations and/or copy number changes frequently observed among melanomas arising from sun-protected sites, such as acral skin (palms, soles, and nail bed) and mucous membranes. GAB2 has recently been implicated in melanoma pathogenesis, and increased copy numbers are found in a subset of melanomas. We sought to determine the association of increased copy numbers of GAB2 among melanoma subtypes in the context of genetic alterations in BRAF, NRAS, and KIT. A total of 85 melanomas arising from sun-protected (n = 23) and sun-exposed sites (n = 62) were analyzed for copy number changes using array-based comparative genomic hybridization and for gain-of-function mutations in BRAF, NRAS, and KIT. GAB2 amplifications were found in 9% of the cases and were associated with melanomas arising from acral and mucosal sites (P = 0.005). Increased copy numbers of the KIT locus were observed in 6% of the cases. The overall mutation frequencies for BRAF and NRAS were 43.5% and 14%, respectively, and were mutually exclusive. Among the acral and mucosal melanomas studied, the genetic alteration frequency was 26% for GAB2, 13% for KIT, 30% for BRAF, and 4% for NRAS. Importantly, the majority of GAB2 amplifications occurred independent from genetic events in BRAF, NRAS, and KIT. GAB2 amplification is critical for melanomas arising from sun-protected sites. Genetic alterations in GAB2 will help refine the molecular classification of melanomas.

  17. Comparison of the Soil Dynamic Amplification Factor and Soil Amplification by Using Microtremor and MASW Methods Respectively

    Science.gov (United States)

    Tuncel, Aykut; Cevdet Özdag, Özkan; Pamuk, Eren; Akgün, Mustafa

    2017-12-01

    Single Station Microtremor method, which is widely used nowadays, is an effective and easy applicable method. In this study, dynamic amplification factor distributions of the study area were obtained using scenario earthquake parameters with single station microtremor data gathered at 112 points. In addition, a surface wave active method, which is known as MASW (Multichannel Analysis of Surface Waves), was applied at 43 profiles to calculate the soil amplification values. Dynamic amplification factor (DAF), soil amplification, the predominant soil period (PSP), geology and topography data of the study area were analysed together. Dynamic amplification factor and soil amplification values were obtained 2 or higher at about sea level parts of the study area which are generally composed of alluvial units. Additionally, in high altitude regions that are composed of volcanic rocks, relatively lower dynamic amplification factor and soil amplification values were obtained. The minimum amplification value in the study area was 1.15, while the maximum amplification value was 3.05 according to the dynamic amplification results and the soil amplification values were between 1.16 and 3.85 in harmony. It is seen that the obtained DAF values and the soil amplification values calculated from the seismic velocities are very similar to each other numerically and regionally. Because of this, it is concluded that the values of the soil amplification obtained by the MASW method and the calculated DAF values in this study are in harmony with each other. Although the depths of research in these two calculation methods are different from each other, the similarity of the results allows us to arrive at the result of how effective the ground layer is on the amplification. It has a great importance to calculate the amplification values and other dynamic parameters by in situ measurements for a planned plot because geological units can vary even at very short distances in heterogeneously

  18. PriFi - Using a Multiple Alignment of Related Sequences to Find Primers for  Amplification of Homologs

    DEFF Research Database (Denmark)

    Fredslund, Jakob; Schauser, Leif; Madsen, Lene Heegaard

    2005-01-01

    Using a comparative approach, the web program PriFi (http://cgi-www.daimi.au.dk/cgi-chili/PriFi/main) designs pairs of primers useful for PCR amplification of genomic DNA in species where prior sequence information is not available. The program works with an alignment of DNA sequences from phylog...

  19. [Complete genome sequencing and sequence analysis of BCG Tice].

    Science.gov (United States)

    Wang, Zhiming; Pan, Yuanlong; Wu, Jun; Zhu, Baoli

    2012-10-04

    The objective of this study is to obtain the complete genome sequence of Bacillus Calmette-Guerin Tice (BCG Tice), in order to provide more information about the molecular biology of BCG Tice and design more reasonable vaccines to prevent tuberculosis. We assembled the data from high-throughput sequencing with SOAPdenovo software, with many contigs and scaffolds obtained. There are many sequence gaps and physical gaps remained as a result of regional low coverage and low quality. We designed primers at the end of contigs and performed PCR amplification in order to link these contigs and scaffolds. With various enzymes to perform PCR amplification, adjustment of PCR reaction conditions, and combined with clone construction to sequence, all the gaps were finished. We obtained the complete genome sequence of BCG Tice and submitted it to GenBank of National Center for Biotechnology Information (NCBI). The genome of BCG Tice is 4334064 base pairs in length, with GC content 65.65%. The problems and strategies during the finishing step of BCG Tice sequencing are illuminated here, with the hope of affording some experience to those who are involved in the finishing step of genome sequencing. The microarray data were verified by our results.

  20. Intrachromosomal amplification, locus deletion and point mutation in the aquaglyceroporin AQP1 gene in antimony resistant Leishmania (Viannia guyanensis.

    Directory of Open Access Journals (Sweden)

    Rubens Monte-Neto

    2015-02-01

    Full Text Available Antimony resistance complicates the treatment of infections caused by the parasite Leishmania.Using next generation sequencing, we sequenced the genome of four independent Leishmania guyanensis antimony-resistant (SbR mutants and found different chromosomal alterations including aneuploidy, intrachromosomal gene amplification and gene deletion. A segment covering 30 genes on chromosome 19 was amplified intrachromosomally in three of the four mutants. The gene coding for the multidrug resistance associated protein A involved in antimony resistance was also amplified in the four mutants, most likely through chromosomal translocation. All mutants also displayed a reduced accumulation of antimony mainly due to genomic alterations at the level of the subtelomeric region of chromosome 31 harboring the gene coding for the aquaglyceroporin 1 (LgAQP1. Resistance involved the loss of LgAQP1 through subtelomeric deletions in three mutants. Interestingly, the fourth mutant harbored a single G133D point mutation in LgAQP1 whose role in resistance was functionality confirmed through drug sensitivity and antimony accumulation assays. In contrast to the Leishmania subspecies that resort to extrachromosomal amplification, the Viannia strains studied here used intrachromosomal amplification and locus deletion.This is the first report of a naturally occurred point mutation in AQP1 in antimony resistant parasites.

  1. Frequency of aneuploidy related to age in porcine oocytes.

    Directory of Open Access Journals (Sweden)

    Miroslav Hornak

    Full Text Available It is generally accepted that mammalian oocytes are frequently suffering from chromosome segregation errors during meiosis I, which have severe consequences, including pregnancy loss, developmental disorders and mental retardation. In a search for physiologically more relevant model than rodent oocytes to study this phenomenon, we have employed comparative genomic hybridization (CGH, combined with whole genome amplification (WGA, to study the frequency of aneuploidy in porcine oocytes, including rare cells obtained from aged animals. Using this method, we were able to analyze segregation pattern of each individual chromosome during meiosis I. In contrast to the previous reports where conventional methods, such as chromosome spreads or FISH, were used to estimate frequency of aneuploidy, our results presented here show, that the frequency of this phenomenon was overestimated in porcine oocytes. Surprisingly, despite the results from human and mouse showing an increase in the frequency of aneuploidy with advanced maternal age, our results obtained by the most accurate method currently available for scoring the aneuploidy in oocytes indicated no increase in the frequency of aneuploidy even in oocytes from animals, whose age was close to the life expectancy of the breed.

  2. Highly sensitive chemiluminescent point mutation detection by circular strand-displacement amplification reaction.

    Science.gov (United States)

    Shi, Chao; Ge, Yujie; Gu, Hongxi; Ma, Cuiping

    2011-08-15

    Single nucleotide polymorphism (SNP) genotyping is attracting extensive attentions owing to its direct connections with human diseases including cancers. Here, we have developed a highly sensitive chemiluminescence biosensor based on circular strand-displacement amplification and the separation by magnetic beads reducing the background signal for point mutation detection at room temperature. This method took advantage of both the T4 DNA ligase recognizing single-base mismatch with high selectivity and the strand-displacement reaction of polymerase to perform signal amplification. The detection limit of this method was 1.3 × 10(-16)M, which showed better sensitivity than that of most of those reported detection methods of SNP. Additionally, the magnetic beads as carrier of immobility was not only to reduce the background signal, but also may have potential apply in high through-put screening of SNP detection in human genome. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. The dynamics of diverse segmental amplifications in populations of Saccharomyces cerevisiae adapting to strong selection.

    Science.gov (United States)

    Payen, Celia; Di Rienzi, Sara C; Ong, Giang T; Pogachar, Jamie L; Sanchez, Joseph C; Sunshine, Anna B; Raghuraman, M K; Brewer, Bonita J; Dunham, Maitreya J

    2014-03-20

    Population adaptation to strong selection can occur through the sequential or parallel accumulation of competing beneficial mutations. The dynamics, diversity, and rate of fixation of beneficial mutations within and between populations are still poorly understood. To study how the mutational landscape varies across populations during adaptation, we performed experimental evolution on seven parallel populations of Saccharomyces cerevisiae continuously cultured in limiting sulfate medium. By combining quantitative polymerase chain reaction, array comparative genomic hybridization, restriction digestion and contour-clamped homogeneous electric field gel electrophoresis, and whole-genome sequencing, we followed the trajectory of evolution to determine the identity and fate of beneficial mutations. During a period of 200 generations, the yeast populations displayed parallel evolutionary dynamics that were driven by the coexistence of independent beneficial mutations. Selective amplifications rapidly evolved under this selection pressure, in particular common inverted amplifications containing the sulfate transporter gene SUL1. Compared with single clones, detailed analysis of the populations uncovers a greater complexity whereby multiple subpopulations arise and compete despite a strong selection. The most common evolutionary adaptation to strong selection in these populations grown in sulfate limitation is determined by clonal interference, with adaptive variants both persisting and replacing one another.

  4. Highly frequent promoter methylation and PIK3CA amplification in non-small cell lung cancer (NSCLC)

    International Nuclear Information System (INIS)

    Ji, Meiju; Guan, Haixia; Gao, Cuixia; Shi, Bingyin; Hou, Peng

    2011-01-01

    Lung cancer is the leading cause of cancer-related death worldwide. Genetic and epigenetic alterations have been identified frequently in lung cancer, such as promoter methylation, gene mutations and genomic amplification. However, the interaction between genetic and epigenetic events and their significance in lung tumorigenesis remains poorly understood. We determined the promoter methylation of 6 genes and PIK3CA amplification using quantitative methylation-specific PCR (Q-MSP) and real-time quantitative PCR, respectively, and explore the association of promoter methylation with PIK3CA amplification in a large cohort of clinically well-characterized non-small cell lung cancer (NSCLC). Highly frequent promoter methylation was observed in NSCLC. With 100% diagnostic specificity, excellent sensitivity, ranging from 45.8 to 84.1%, was found for each of the 6 genes. The promoter methylation was associated with histologic type. Methylation of CALCA, CDH1, DAPK1, and EVX2 was more common in squamous cell carcinomas (SCC) compared to adenocarcinomas (ADC). Conversely, there was a trend toward a higher frequency of RASSF1A methylation in ADC than SCC. In addition, PIK3CA amplification was frequently found in NSCLC, and was associated with certain clinicopathologic features, such as smoking history, histologic type and pleural indentation. Importantly, aberrant promoter methylation of certain genes was significantly associated with PIK3CA amplification. Our data showed highly frequent promoter methylation and PIK3CA amplification in Chinese NSCLC population, and first demonstrated the associations of gene methylation with PIK3CA amplification, suggesting that these epigenetic events may be a consequence of overactivation of PI3K/Akt pathway

  5. Characterization of genomic alterations in radiation-associated breast cancer among childhood cancer survivors, using comparative genomic hybridization (CGH arrays.

    Directory of Open Access Journals (Sweden)

    Xiaohong R Yang

    Full Text Available Ionizing radiation is an established risk factor for breast cancer. Epidemiologic studies of radiation-exposed cohorts have been primarily descriptive; molecular events responsible for the development of radiation-associated breast cancer have not been elucidated. In this study, we used array comparative genomic hybridization (array-CGH to characterize genome-wide copy number changes in breast tumors collected in the Childhood Cancer Survivor Study (CCSS. Array-CGH data were obtained from 32 cases who developed a second primary breast cancer following chest irradiation at early ages for the treatment of their first cancers, mostly Hodgkin lymphoma. The majority of these cases developed breast cancer before age 45 (91%, n = 29, had invasive ductal tumors (81%, n = 26, estrogen receptor (ER-positive staining (68%, n = 19 out of 28, and high proliferation as indicated by high Ki-67 staining (77%, n = 17 out of 22. Genomic regions with low-copy number gains and losses and high-level amplifications were similar to what has been reported in sporadic breast tumors, however, the frequency of amplifications of the 17q12 region containing human epidermal growth factor receptor 2 (HER2 was much higher among CCSS cases (38%, n = 12. Our findings suggest that second primary breast cancers in CCSS were enriched for an "amplifier" genomic subgroup with highly proliferative breast tumors. Future investigation in a larger irradiated cohort will be needed to confirm our findings.

  6. Acquiring Reference Genomes from Uncultured Microbes by Micromanipulation and Low-complexity Metagenomics

    DEFF Research Database (Denmark)

    Karst, Søren Michael; Albertsen, Mads; Nielsen, Jeppe Lund

    A pre-requisite for many of the –omics approaches applied in environmental microbiology today are high quality reference genomes. Until recently such genomes have been difficult to obtain from unculturable, complex microbial communities. However, lately the ‘single cell genomics’ approach based...... on isolation and amplification of genomic DNA from a single or few clonal cells has proven efficient for this purpose although very tedious. The aim of this study was to apply the methodology of single cell genomics to filamentous organisms and microcolonies of specific species from microbial communities...

  7. Genomic amplification of Fanconi anemia complementation group A (FancA) in head and neck squamous cell carcinoma (HNSCC): Cellular mechanisms of radioresistance and clinical relevance.

    Science.gov (United States)

    Hess, Julia; Unger, Kristian; Orth, Michael; Schötz, Ulrike; Schüttrumpf, Lars; Zangen, Verena; Gimenez-Aznar, Igor; Michna, Agata; Schneider, Ludmila; Stamp, Ramona; Selmansberger, Martin; Braselmann, Herbert; Hieber, Ludwig; Drexler, Guido A; Kuger, Sebastian; Klein, Diana; Jendrossek, Verena; Friedl, Anna A; Belka, Claus; Zitzelsberger, Horst; Lauber, Kirsten

    2017-02-01

    Radio (chemo) therapy is a crucial treatment modality for head and neck squamous cell carcinoma (HNSCC), but relapse is frequent, and the underlying mechanisms remain largely elusive. Therefore, novel biomarkers are urgently needed. Previously, we identified gains on 16q23-24 to be associated with amplification of the Fanconi anemia A (FancA) gene and to correlate with reduced progression-free survival after radiotherapy. Here, we analyzed the effects of FancA on radiation sensitivity in vitro, characterized the underlying mechanisms, and evaluated their clinical relevance. Silencing of FancA expression in HNSCC cell lines with genomic gains on 16q23-24 resulted in significantly impaired clonogenic survival upon irradiation. Conversely, overexpression of FancA in immortalized keratinocytes conferred increased survival accompanied by improved DNA repair, reduced accumulation of chromosomal translocations, but no hyperactivation of the FA/BRCA-pathway. Downregulation of interferon signaling as identified by microarray analyses, enforced irradiation-induced senescence, and elevated production of the senescence-associated secretory phenotype (SASP) appeared to be candidate mechanisms contributing to FancA-mediated radioresistance. Data of the TCGA HNSCC cohort confirmed the association of gains on 16q24.3 with FancA overexpression and impaired overall survival. Importantly, transcriptomic alterations similar to those observed upon FancA overexpression in vitro strengthened the clinical relevance. Overall, FancA amplification and overexpression appear to be crucial for radiotherapeutic failure in HNSCC. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  8. New Fpg probe chemistry for direct detection of recombinase polymerase amplification on lateral flow strips.

    Science.gov (United States)

    Powell, Michael L; Bowler, Frank R; Martinez, Aurore J; Greenwood, Catherine J; Armes, Niall; Piepenburg, Olaf

    2018-02-15

    Rapid, cost-effective and sensitive detection of nucleic acids has the ability to improve upon current practices employed for pathogen detection in diagnosis of infectious disease and food testing. Furthermore, if assay complexity can be reduced, nucleic acid amplification tests could be deployed in resource-limited and home use scenarios. In this study, we developed a novel Fpg (Formamidopyrimidine DNA glycosylase) probe chemistry, which allows lateral flow detection of amplification in undiluted recombinase polymerase amplification (RPA) reactions. The prototype nucleic acid lateral flow chemistry was applied to a human genomic target (rs1207445), Campylobacter jejuni 16S rDNA and two genetic markers of the important food pathogen E. coli O157:H7. All four assays have an analytical sensitivity between 10 and 100 copies DNA per amplification. Furthermore, the assay is performed with fewer hands-on steps than using the current RPA Nfo lateral flow method as dilution of amplicon is not required for lateral flow analysis. Due to the simplicity of the workflow, we believe that the lateral flow chemistry for direct detection could be readily adapted to a cost-effective single-use consumable, ideal for use in non-laboratory settings. Copyright © 2017. Published by Elsevier Inc.

  9. GENOMIC PROFILING BY MULTIPLEX LIGATION - DEPENDENT PROBE AMPLIFICATION IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS

    Directory of Open Access Journals (Sweden)

    Georgiana-Emilia Grigore

    2013-11-01

    Full Text Available The clinical management of severe pathological conditions, such as B-cell chronic lymphocytic leukemia (B-CLL, is subject to continuous optimization and re-evaluation. Patients may fully benefit from rapid, standardized laboratory tools designed to facilitate their early stratification according to disease risk, stage and prognosis. Such technologies may also aid the clinician in selecting the therapeutic option with the greatest chances of success. The presence of specific genetic abnormalities are frequently associated with the clinical outcome of oncologic patients in general, and B-CLL patients in particular. In the current study, a group of 58 B-CLL patients were evaluated for the detection of gene copy number alterations (deletions or duplication/ amplifications within 45 distinct genetic targets, by means of a novel molecular methodology, Multiplex Ligation - Dependent Probe Amplification (MLPA. Simple or complex genetic defects were identified in 67% of cases, and the most common aberrations observed were: deletion of the short arm of chromosome 13 in 33% of cases, deletion of the long arm of chromosome 11 in 16% of cases, trisomy 12 in 16% of cases, and deletion of the short arm of chromosome 17 in 7% of cases. The main conclusion of the study presented here points towards MLPA as a potential key step of clinical management protocols in B-CLL, providing that it will be fully standardised for routine diagnosis.

  10. Recurrent amplification of RTEL1 and ABCA13 and its synergistic effect associated with clinicopathological data of gastric adenocarcinoma.

    Science.gov (United States)

    Araújo, T M; Seabra, A D; Lima, E M; Assumpção, P P; Montenegro, R C; Demachki, S; Burbano, R M; Khayat, A S

    2016-01-01

    Despite progression in treatment of gastric cancer, prognosis of patients remains poor, in part due to the low rate of diagnosis during its early stages. This paradigm implies the necessity to identify molecular biomarkers for early gastric cancer diagnosis, as well as for disease monitoring, thus contributing to the development of new therapeutic approaches. In a previous study, performed by array-Comparative Genomic Hybridization, we described for the first time in literature recurrent amplification of RTEL1 and ABCA13 genes in gastric cancer. Thus, the aim of the present study was to validate recurrent amplification of RTEL1 and ABCA13 genes and associate CNV status with clinicopathological data. Results showed RTEL1 and ABCA13 amplification in 38 % of samples. Statistical analysis demonstrated that RTEL amplification is more common in older patients and more associated with intestinal type and ABCA13 amplification increases the risk of lymph node metastasis and is more common in men. Co-amplification of these genes showed a significant association with advanced staging. aCGH is a very useful tool for investigating novel genes associated with carcinogenesis and RTEL1 amplification may be important for the development of gastric cancer in older patients, besides being a probable event contributing for chromosomal instability in intestinal gastric carcinogenesis. ABCA13 amplification may have age-specific function and could be considered a useful marker for predicting lymph node metastasis in resected gastric cancer patients in early stage. Lastly, RTEL1 and ABCA13 synergistic effect may be considered as a putative marker for advanced staging in gastric cancer patients.

  11. Digital Genome-Wide ncRNA Expression, Including SnoRNAs, across 11 Human Tissues Using PolyA-Neutral Amplification

    Science.gov (United States)

    Castle, John C.; Armour, Christopher D.; Löwer, Martin; Haynor, David; Biery, Matthew; Bouzek, Heather; Chen, Ronghua; Jackson, Stuart; Johnson, Jason M.; Rohl, Carol A.; Raymond, Christopher K.

    2010-01-01

    Non-coding RNAs (ncRNAs) are an essential class of molecular species that have been difficult to monitor on high throughput platforms due to frequent lack of polyadenylation. Using a polyadenylation-neutral amplification protocol and next-generation sequencing, we explore ncRNA expression in eleven human tissues. ncRNAs 7SL, U2, 7SK, and HBII-52 are expressed at levels far exceeding mRNAs. C/D and H/ACA box snoRNAs are associated with rRNA methylation and pseudouridylation, respectively: spleen expresses both, hypothalamus expresses mainly C/D box snoRNAs, and testes show enriched expression of both H/ACA box snoRNAs and RNA telomerase TERC. Within the snoRNA 14q cluster, 14q(I-6) is expressed at much higher levels than other cluster members. More reads align to mitochondrial than nuclear tRNAs. Many lincRNAs are actively transcribed, particularly those overlapping known ncRNAs. Within the Prader-Willi syndrome loci, the snoRNA HBII-85 (group I) cluster is highly expressed in hypothalamus, greater than in other tissues and greater than group II or III. Additionally, within the disease locus we find novel transcription across a 400,000 nt span in ovaries. This genome-wide polyA-neutral expression compendium demonstrates the richness of ncRNA expression, their high expression patterns, their function-specific expression patterns, and is publicly available. PMID:20668672

  12. Digital genome-wide ncRNA expression, including SnoRNAs, across 11 human tissues using polyA-neutral amplification.

    Directory of Open Access Journals (Sweden)

    John C Castle

    Full Text Available Non-coding RNAs (ncRNAs are an essential class of molecular species that have been difficult to monitor on high throughput platforms due to frequent lack of polyadenylation. Using a polyadenylation-neutral amplification protocol and next-generation sequencing, we explore ncRNA expression in eleven human tissues. ncRNAs 7SL, U2, 7SK, and HBII-52 are expressed at levels far exceeding mRNAs. C/D and H/ACA box snoRNAs are associated with rRNA methylation and pseudouridylation, respectively: spleen expresses both, hypothalamus expresses mainly C/D box snoRNAs, and testes show enriched expression of both H/ACA box snoRNAs and RNA telomerase TERC. Within the snoRNA 14q cluster, 14q(I-6 is expressed at much higher levels than other cluster members. More reads align to mitochondrial than nuclear tRNAs. Many lincRNAs are actively transcribed, particularly those overlapping known ncRNAs. Within the Prader-Willi syndrome loci, the snoRNA HBII-85 (group I cluster is highly expressed in hypothalamus, greater than in other tissues and greater than group II or III. Additionally, within the disease locus we find novel transcription across a 400,000 nt span in ovaries. This genome-wide polyA-neutral expression compendium demonstrates the richness of ncRNA expression, their high expression patterns, their function-specific expression patterns, and is publicly available.

  13. Generation of sequence signatures from DNA amplification fingerprints with mini-hairpin and microsatellite primers.

    Science.gov (United States)

    Caetano-Anollés, G; Gresshoff, P M

    1996-06-01

    DNA amplification fingerprinting (DAF) with mini-hairpins harboring arbitrary "core" sequences at their 3' termini were used to fingerprint a variety of templates, including PCR products and whole genomes, to establish genetic relationships between plant tax at the interspecific and intraspecific level, and to identify closely related fungal isolates and plant accessions. No correlation was observed between the sequence of the arbitrary core, the stability of the mini-hairpin structure and DAF efficiency. Mini-hairpin primers with short arbitrary cores and primers complementary to simple sequence repeats present in microsatellites were also used to generate arbitrary signatures from amplification profiles (ASAP). The ASAP strategy is a dual-step amplification procedure that uses at least one primer in each fingerprinting stage. ASAP was able to reproducibly amplify DAF products (representing about 10-15 kb of sequence) following careful optimization of amplification parameters such as primer and template concentration. Avoidance of primer sequences partially complementary to DAF product termini was necessary in order to produce distinct fingerprints. This allowed the combinatorial use of oligomers in nucleic acid screening, with numerous ASAP fingerprinting reactions based on a limited number of primer sequences. Mini-hairpin primers and ASAP analysis significantly increased detection of polymorphic DNA, separating closely related bermudagrass (Cynodon) cultivars and detecting putatively linked markers in bulked segregant analysis of the soybean (Glycine max) supernodulation (nitrate-tolerant symbiosis) locus.

  14. Direct Identification of Enteroviruses in Cerebrospinal Fluid of Patients with Suspected Meningitis by Nested PCR Amplification

    Directory of Open Access Journals (Sweden)

    Alexandr Krasota

    2016-01-01

    Full Text Available Enteroviruses, the most common human viral pathogens worldwide, have been associated with serous meningitis, encephalitis, syndrome of acute flaccid paralysis, myocarditis and the onset of diabetes type 1. In the future, the rapid identification of the etiological agent would allow to adjust the therapy promptly and thereby improve the course of the disease and prognosis. We developed RT-nested PCR amplification of the genomic region coding viral structural protein VP1 for direct identification of enteroviruses in clinical specimens and compared it with the existing analogs. One-hundred-fifty-nine cerebrospinal fluids (CSF from patients with suspected meningitis were studied. The amplification of VP1 genomic region using the new method was achieved for 86 (54.1% patients compared with 75 (47.2%, 53 (33.3% and 31 (19.5% achieved with previously published methods. We identified 11 serotypes of the Enterovirus species B in 2012, including relatively rare echovirus 14 (E-14, E-15 and E-32, and eight serotypes of species B and 5 enteroviruses A71 (EV-A71 in 2013. The developed method can be useful for direct identification of enteroviruses in clinical material with the low virus loads such as CSF.

  15. Miniaturized isothermal nucleic acid amplification, a review.

    Science.gov (United States)

    Asiello, Peter J; Baeumner, Antje J

    2011-04-21

    Micro-Total Analysis Systems (µTAS) for use in on-site rapid detection of DNA or RNA are increasingly being developed. Here, amplification of the target sequence is key to increasing sensitivity, enabling single-cell and few-copy nucleic acid detection. The several advantages to miniaturizing amplification reactions and coupling them with sample preparation and detection on the same chip are well known and include fewer manual steps, preventing contamination, and significantly reducing the volume of expensive reagents. To-date, the majority of miniaturized systems for nucleic acid analysis have used the polymerase chain reaction (PCR) for amplification and those systems are covered in previous reviews. This review provides a thorough overview of miniaturized analysis systems using alternatives to PCR, specifically isothermal amplification reactions. With no need for thermal cycling, isothermal microsystems can be designed to be simple and low-energy consuming and therefore may outperform PCR in portable, battery-operated detection systems in the future. The main isothermal methods as miniaturized systems reviewed here include nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), and strand displacement amplification (SDA). Also, important design criteria for the miniaturized devices are discussed. Finally, the potential of miniaturization of some new isothermal methods such as the exponential amplification reaction (EXPAR), isothermal and chimeric primer-initiated amplification of nucleic acids (ICANs), signal-mediated amplification of RNA technology (SMART) and others is presented.

  16. The genome sequence of pepper vein yellows virus (family Luteoviridae, genus Polerovirus)

    OpenAIRE

    Murakami, Ritsuko; Nakashima, Nobuhiko; Hinomoto, Norihide; Kawano, Shinji; Toyosato, Tetsuya

    2011-01-01

    The complete genome of pepper vein yellows virus (PeVYV) was sequenced using random amplification of RNA samples isolated from vector insects (Aphis gossypii) that had been given access to PeVYV-infected plants. The PeVYV genome consisted of 6244 nucleotides and had a genomic organization characteristic of members of the genus Polerovirus. PeVYV had highest amino acid sequence identities in ORF0 to ORF3 (75.9 - 91.9%) with tobacco vein distorting polerovirus, with which it was only 25.1% iden...

  17. Voltammetric determination of attomolar levels of a sequence derived from the genom of hepatitis B virus by using molecular beacon mediated circular strand displacement and rolling circle amplification.

    Science.gov (United States)

    Huang, Shan; Feng, Mengmeng; Li, Jiawen; Liu, Yi; Xiao, Qi

    2018-03-03

    The authors describe an electrochemical method for the determination of the single-stranded DNA (ssDNA) oligonucleotide with a sequence derived from the genom of hepatitis B virus (HBV). It is making use of circular strand displacement (CSD) and rolling circle amplification (RCA) strategies mediated by a molecular beacon (MB). This ssDNA hybridizes with the loop portion of the MB immobilized on the surface of a gold electrode, while primer DNA also hybridizes with the rest of partial DNA sequences of MB. This triggers the MB-mediated CSD. The RCA is then initiated to produce a long DNA strand with multiple tandem-repeat sequences, and this results in a significant increase of the differential pulse voltammetric response of the electrochemical probe Methylene Blue at a rather low working potential of -0.24 V (vs. Ag/AgCl). Under optimal experimental conditions, the assay displays an ultrahigh sensitivity (with a 2.6 aM detection limit) and excellent selectivity. Response is linear in the 10 to 700 aM DNA concentration range. Graphical abstract Schematic of a voltammetric method for the determination of attomolar levels of target DNA. It is based on molecular beacon mediated circular strand displacement and rolling circle amplification strategies. Under optimal experimental conditions, the assay displays an ultrahigh sensitivity with a 2.6 aM detection limit and excellent selectivity.

  18. Horizontal Gene Acquisitions, Mobile Element Proliferation, and Genome Decay in the Host-Restricted Plant Pathogen Erwinia Tracheiphila

    Science.gov (United States)

    Shapiro, Lori R.; Scully, Erin D.; Straub, Timothy J.; Park, Jihye; Stephenson, Andrew G.; Beattie, Gwyn A.; Gleason, Mark L.; Kolter, Roberto; Coelho, Miguel C.; De Moraes, Consuelo M.; Mescher, Mark C.; Zhaxybayeva, Olga

    2016-01-01

    Modern industrial agriculture depends on high-density cultivation of genetically similar crop plants, creating favorable conditions for the emergence of novel pathogens with increased fitness in managed compared with ecologically intact settings. Here, we present the genome sequence of six strains of the cucurbit bacterial wilt pathogen Erwinia tracheiphila (Enterobacteriaceae) isolated from infected squash plants in New York, Pennsylvania, Kentucky, and Michigan. These genomes exhibit a high proportion of recent horizontal gene acquisitions, invasion and remarkable amplification of mobile genetic elements, and pseudogenization of approximately 20% of the coding sequences. These genome attributes indicate that E. tracheiphila recently emerged as a host-restricted pathogen. Furthermore, chromosomal rearrangements associated with phage and transposable element proliferation contribute to substantial differences in gene content and genetic architecture between the six E. tracheiphila strains and other Erwinia species. Together, these data lead us to hypothesize that E. tracheiphila has undergone recent evolution through both genome decay (pseudogenization) and genome expansion (horizontal gene transfer and mobile element amplification). Despite evidence of dramatic genomic changes, the six strains are genetically monomorphic, suggesting a recent population bottleneck and emergence into E. tracheiphila’s current ecological niche. PMID:26992913

  19. Biomaterials in light amplification

    Science.gov (United States)

    Mysliwiec, Jaroslaw; Cyprych, Konrad; Sznitko, Lech; Miniewicz, Andrzej

    2017-03-01

    Biologically produced or inspired materials can serve as optical gain media, i.e. they can exhibit the phenomenon of light amplification. Some of these materials, under suitable dye-doping and optical pumping conditions, show lasing phenomena. The emerging branch of research focused on obtaining lasing action in highly disordered and highly light scattering materials, i.e. research on random lasing, is perfectly suited for biological materials. The use of biomaterials in light amplification has been extensively reported in the literature. In this review we attempt to report on progress in the development of biologically derived systems able to show the phenomena of light amplification and random lasing together with the contribution of our group to this field. The rich world of biopolymers modified with molecular aggregates and nanocrystals, and self-organized at the nanoscale, offers a multitude of possibilities for tailoring luminescent and light scattering properties that are not easily replicated in conventional organic or inorganic materials. Of particular importance and interest are light amplification and lasing, or random lasing studies in biological cells and tissues. In this review we will describe nucleic acids and their complexes employed as gain media due to their favorable optical properties and ease of manipulation. We will report on research conducted on various biomaterials showing structural analogy to nucleic acids such as fluorescent proteins, gelatins in which the first distributed feedback laser was realized, and also amyloids or silks, which, due to their dye-doped fiber-like structure, allow for light amplification. Other materials that were investigated in that respect include polysaccharides, like starch exhibiting favorable photostability in comparison to other biomaterials, and chitosan, which forms photonic crystals or cellulose. Light amplification and random lasing was not only observed in processed biomaterials but also in living

  20. Search for Genomic Alterations in Monozygotic Twins Discordant for Cleft Lip and/or Palate

    DEFF Research Database (Denmark)

    Kimani, Jane W; Yoshiura, Koh-Ichiro; Shi, Min

    2009-01-01

    consisting of 1,536 SNPs, to scan for genomic alterations in a sample of monozygotic twin pairs with discordant cleft lip and/or palate phenotypes. Paired analysis for deletions, amplifications and loss of heterozygosity, along with sequence verification of SNPs with discordant genotype calls did not reveal...... any genomic discordance between twin pairs in lymphocyte DNA samples. Our results demonstrate that postzygotic genomic alterations are not a common cause of monozygotic twin discordance for isolated cleft lip and/or palate. However, rare or balanced genomic alterations, tissue-specific events...

  1. Implementing PGD/PGD-A in IVF clinics: considerations for the best laboratory approach and management.

    Science.gov (United States)

    Capalbo, Antonio; Romanelli, Valeria; Cimadomo, Danilo; Girardi, Laura; Stoppa, Marta; Dovere, Lisa; Dell'Edera, Domenico; Ubaldi, Filippo Maria; Rienzi, Laura

    2016-10-01

    For an IVF clinic that wishes to implement preimplantation genetic diagnosis for monogenic diseases (PGD) and for aneuploidy testing (PGD-A), a global improvement is required through all the steps of an IVF treatment and patient care. At present, CCS (Comprehensive Chromosome Screening)-based trophectoderm (TE) biopsy has been demonstrated as a safe, accurate and reproducible approach to conduct PGD-A and possibly also PGD from the same biopsy. Key challenges in PGD/PGD-A implementation cover genetic and reproductive counselling, selection of the most efficient approach for blastocyst biopsy as well as of the best performing molecular technique to conduct CCS and monogenic disease analysis. Three different approaches for TE biopsy can be compared. However, among them, the application of TE biopsy approaches, entailing the zona opening when the expanded blastocyst stage is reached, represent the only biopsy methods suited with a totally undisturbed embryo culture strategy (time lapse-based incubation in a single media). Moreover, contemporary CCS technologies show a different spectrum of capabilities and limits that potentially impact the clinical outcomes, the management and the applicability of the PGD-A itself. In general, CCS approaches that avoid the use of whole genome amplification (WGA) can provide higher reliability of results with lower costs and turnaround time of analysis. The future perspectives are focused on the scrupulous and rigorous clinical validations of novel CCS methods based on targeted approaches that avoid the use of WGA, such as targeted next-generation sequencing technology, to further improve the throughput of analysis and the overall cost-effectiveness of PGD/PGD-A.

  2. Identification and differentiation of the twenty six bluetongue virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2.

    Directory of Open Access Journals (Sweden)

    Narender S Maan

    Full Text Available Bluetongue (BT is an arthropod-borne viral disease, which primarily affects ruminants in tropical and temperate regions of the world. Twenty six bluetongue virus (BTV serotypes have been recognised worldwide, including nine from Europe and fifteen in the United States. Identification of BTV serotype is important for vaccination programmes and for BTV epidemiology studies. Traditional typing methods (virus isolation and serum or virus neutralisation tests (SNT or VNT are slow (taking weeks, depend on availability of reference virus-strains or antisera and can be inconclusive. Nucleotide sequence analyses and phylogenetic comparisons of genome segment 2 (Seg-2 encoding BTV outer-capsid protein VP2 (the primary determinant of virus serotype were completed for reference strains of BTV-1 to 26, as well as multiple additional isolates from different geographic and temporal origins. The resulting Seg-2 database has been used to develop rapid (within 24 h and reliable RT-PCR-based typing assays for each BTV type. Multiple primer-pairs (at least three designed for each serotype were widely tested, providing an initial identification of serotype by amplification of a cDNA product of the expected size. Serotype was confirmed by sequencing of the cDNA amplicons and phylogenetic comparisons to previously characterised reference strains. The results from RT-PCR and sequencing were in perfect agreement with VNT for reference strains of all 26 BTV serotypes, as well as the field isolates tested. The serotype-specific primers showed no cross-amplification with reference strains of the remaining 25 serotypes, or multiple other isolates of the more closely related heterologous BTV types. The primers and RT-PCR assays developed in this study provide a rapid, sensitive and reliable method for the identification and differentiation of the twenty-six BTV serotypes, and will be updated periodically to maintain their relevance to current BTV distribution and

  3. Screening for genomic rearrangements at BRCA1 locus in Iranian ...

    Indian Academy of Sciences (India)

    2016-08-26

    Aug 26, 2016 ... Home; Journals; Journal of Genetics; Volume 92; Issue 1. Screening for genomic rearrangements at BRCA1 locus in Iranian women with breast cancer using multiplex ligation-dependent probe amplification. Vahid R. Yassaee Babak Emamalizadeh Mir Davood Omrani. Research Note Volume 92 Issue 1 ...

  4. Genomic profiling identifies GATA6 as a candidate oncogene amplified in pancreatobiliary cancer.

    Directory of Open Access Journals (Sweden)

    Kevin A Kwei

    2008-05-01

    Full Text Available Pancreatobiliary cancers have among the highest mortality rates of any cancer type. Discovering the full spectrum of molecular genetic alterations may suggest new avenues for therapy. To catalogue genomic alterations, we carried out array-based genomic profiling of 31 exocrine pancreatic cancers and 6 distal bile duct cancers, expanded as xenografts to enrich the tumor cell fraction. We identified numerous focal DNA amplifications and deletions, including in 19% of pancreatobiliary cases gain at cytoband 18q11.2, a locus uncommonly amplified in other tumor types. The smallest shared amplification at 18q11.2 included GATA6, a transcriptional regulator previously linked to normal pancreas development. When amplified, GATA6 was overexpressed at both the mRNA and protein levels, and strong immunostaining was observed in 25 of 54 (46% primary pancreatic cancers compared to 0 of 33 normal pancreas specimens surveyed. GATA6 expression in xenografts was associated with specific microarray gene-expression patterns, enriched for GATA binding sites and mitochondrial oxidative phosphorylation activity. siRNA mediated knockdown of GATA6 in pancreatic cancer cell lines with amplification led to reduced cell proliferation, cell cycle progression, and colony formation. Our findings indicate that GATA6 amplification and overexpression contribute to the oncogenic phenotypes of pancreatic cancer cells, and identify GATA6 as a candidate lineage-specific oncogene in pancreatobiliary cancer, with implications for novel treatment strategies.

  5. A genomic library-based amplification approach (GL-PCR) for the mapping of multiple IS6110 insertion sites and strain differentiation of Mycobacterium tuberculosis.

    Science.gov (United States)

    Namouchi, Amine; Mardassi, Helmi

    2006-11-01

    Evidence suggests that insertion of the IS6110 element is not without consequence to the biology of Mycobacterium tuberculosis complex strains. Thus, mapping of multiple IS6110 insertion sites in the genome of biomedically relevant clinical isolates would result in a better understanding of the role of this mobile element, particularly with regard to transmission, adaptability and virulence. In the present paper, we describe a versatile strategy, referred to as GL-PCR, that amplifies IS6110-flanking sequences based on the construction of a genomic library. M. tuberculosis chromosomal DNA is fully digested with HincII and then ligated into a plasmid vector between T7 and T3 promoter sequences. The ligation reaction product is transformed into Escherichia coli and selective PCR amplification targeting both 5' and 3' IS6110-flanking sequences are performed on the plasmid library DNA. For this purpose, four separate PCR reactions are performed, each combining an outward primer specific for one IS6110 end with either T7 or T3 primer. Determination of the nucleotide sequence of the PCR products generated from a single ligation reaction allowed mapping of 21 out of the 24 IS6110 copies of two 12 banded M. tuberculosis strains, yielding an overall sensitivity of 87,5%. Furthermore, by simply comparing the migration pattern of GL-PCR-generated products, the strategy proved to be as valuable as IS6110 RFLP for molecular typing of M. tuberculosis complex strains. Importantly, GL-PCR was able to discriminate between strains differing by a single IS6110 band.

  6. C-MET overexpression and amplification in gliomas.

    Science.gov (United States)

    Kwak, Yoonjin; Kim, Seong-Ik; Park, Chul-Kee; Paek, Sun Ha; Lee, Soon-Tae; Park, Sung-Hye

    2015-01-01

    We investigated c-Met overexpression and MET gene amplification in gliomas to determine their incidence and prognostic significance. c-Met immunohistochemistry and MET gene fluorescence in situ hybridization were carried out on tissue microarrays from 250 patients with gliomas (137 grade IV GBMs and 113 grade II and III diffuse gliomas). Clinicopathological features of these cases were reviewed. c-Met overexpression and MET gene amplification were detected in 13.1% and 5.1% of the GBMs, respectively. All the MET-amplified cases showed c-Met overexpression, but MET amplification was not always concordant with c-Met overexpression. None of grade II and III gliomas demonstrated c-Met overexpression or MET gene amplification. Mean survival of the GBM patients with MET amplification was not significantly different from patients without MET amplification (P=0.155). However, GBM patients with c-Met overexpression survived longer than patients without c-Met overexpression (P=0.035). Although MET amplification was not related to poor GBM prognosis, it is partially associated with the aggressiveness of gliomas, as MET amplification was found only in grade IV, not in grade II and III gliomas. We suggest that MET inhibitor therapy may be beneficial in about 5% GBMs, which was the incidence of MET gene amplification found in the patients included in this study.

  7. Gene Amplification on Demand Accelerates Cellobiose Utilization in Engineered Saccharomyces cerevisiae.

    Science.gov (United States)

    Oh, Eun Joong; Skerker, Jeffrey M; Kim, Soo Rin; Wei, Na; Turner, Timothy L; Maurer, Matthew J; Arkin, Adam P; Jin, Yong-Su

    2016-06-15

    Efficient microbial utilization of cellulosic sugars is essential for the economic production of biofuels and chemicals. Although the yeast Saccharomyces cerevisiae is a robust microbial platform widely used in ethanol plants using sugar cane and corn starch in large-scale operations, glucose repression is one of the significant barriers to the efficient fermentation of cellulosic sugar mixtures. A recent study demonstrated that intracellular utilization of cellobiose by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular β-glucosidase (encoded by gh1-1) can alleviate glucose repression, resulting in the simultaneous cofermentation of cellobiose and nonglucose sugars. Here we report enhanced cellobiose fermentation by engineered yeast expressing cdt-1 and gh1-1 through laboratory evolution. When cdt-1 and gh1-1 were integrated into the genome of yeast, the single copy integrant showed a low cellobiose consumption rate. However, cellobiose fermentation rates by engineered yeast increased gradually during serial subcultures on cellobiose. Finally, an evolved strain exhibited a 15-fold-higher cellobiose fermentation rate. To identify the responsible mutations in the evolved strain, genome sequencing was performed. Interestingly, no mutations affecting cellobiose fermentation were identified, but the evolved strain contained 9 copies of cdt-1 and 23 copies of gh1-1 We also traced the copy numbers of cdt-1 and gh1-1 of mixed populations during the serial subcultures. The copy numbers of cdt-1 and gh1-1 in the cultures increased gradually with similar ratios as cellobiose fermentation rates of the cultures increased. These results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step in engineered yeast and copies of genes coding for metabolic enzymes might be amplified in yeast if there is a growth advantage. This study indicates that on-demand gene amplification might be an

  8. Deconstruction of Vulnerability to Complex Diseases: Enhanced Effect Sizes and Power of Intermediate Phenotypes

    Directory of Open Access Journals (Sweden)

    David Goldman

    2007-01-01

    Full Text Available The deconstruction of vulnerability to complex disease with the help of intermediate phenotypes, including the heritable and disease-associated endophenotypes, is a legacy of Henri Begleiter. Systematic searches for genes influencing complex disorders, including bipolar disorder, have recently been completed using whole genome association (WGA, identifying a series of validated loci. Using this information, it is possible to compare effect sizes of disease loci discovered in very large samples to the effect sizes of replicated functional loci determining intermediate phenotypes that are of essential interest in psychiatric disorders. It is shown that the genes influencing intermediate phenotypes tend to have a larger effect size. Furthermore, the WGA results reveal that the number of loci of large effect size for complex diseases is limited, and yet multiple functional loci have already been identified for intermediate phenotypes relevant to psychiatric diseases, and without the benefit of WGA.

  9. Social amplification of risk

    International Nuclear Information System (INIS)

    Kasperson, R.E.; Renn, O.; Slovic, P.; Kasperson, J.X.; Emani, S.

    1989-01-01

    The risks associated with radioactive and other hazardous waste disposal may be expected to interact with societal processes to enlarge or attenuate the consequences of risks and risk events. This article summarizes a conceptual framework that depicts the social amplification of risk. Using a data base of 128 hazard events that have occurred largely over the past ten years, the authors examine the role of physical consequences, media coverage, and public perceptions of risk in generating social and economic impacts. The analysis concludes that social amplification processes substantially shape the nature and magnitude of those impacts but also that such social amplification appears to be systematically related to characteristics of the risks and risk events

  10. Origin and evolution of SINEs in eukaryotic genomes.

    Science.gov (United States)

    Kramerov, D A; Vassetzky, N S

    2011-12-01

    Short interspersed elements (SINEs) are one of the two most prolific mobile genomic elements in most of the higher eukaryotes. Although their biology is still not thoroughly understood, unusual life cycle of these simple elements amplified as genomic parasites makes their evolution unique in many ways. In contrast to most genetic elements including other transposons, SINEs emerged de novo many times in evolution from available molecules (for example, tRNA). The involvement of reverse transcription in their amplification cycle, huge number of genomic copies and modular structure allow variation mechanisms in SINEs uncommon or rare in other genetic elements (module exchange between SINE families, dimerization, and so on.). Overall, SINE evolution includes their emergence, progressive optimization and counteraction to the cell's defense against mobile genetic elements.

  11. Loop mediated isothermal amplification: An innovative gene amplification technique for animal diseases

    Directory of Open Access Journals (Sweden)

    Pravas Ranjan Sahoo

    2016-05-01

    Full Text Available India being a developing country mainly depends on livestock sector for its economy. However, nowadays, there is emergence and reemergence of more transboundary animal diseases. The existing diagnostic techniques are not so quick and with less specificity. To reduce the economy loss, there should be a development of rapid, reliable, robust diagnostic technique, which can work with high degree of sensitivity and specificity. Loop mediated isothermal amplification assay is a rapid gene amplification technique that amplifies nucleic acid under an isothermal condition with a set of designed primers spanning eight distinct sequences of the target. This assay can be used as an emerging powerful, innovative gene amplification diagnostic tool against various pathogens of livestock diseases. This review is to highlight the basic concept and methodology of this assay in livestock disease.

  12. Loop mediated isothermal amplification: An innovative gene amplification technique for animal diseases.

    Science.gov (United States)

    Sahoo, Pravas Ranjan; Sethy, Kamadev; Mohapatra, Swagat; Panda, Debasis

    2016-05-01

    India being a developing country mainly depends on livestock sector for its economy. However, nowadays, there is emergence and reemergence of more transboundary animal diseases. The existing diagnostic techniques are not so quick and with less specificity. To reduce the economy loss, there should be a development of rapid, reliable, robust diagnostic technique, which can work with high degree of sensitivity and specificity. Loop mediated isothermal amplification assay is a rapid gene amplification technique that amplifies nucleic acid under an isothermal condition with a set of designed primers spanning eight distinct sequences of the target. This assay can be used as an emerging powerful, innovative gene amplification diagnostic tool against various pathogens of livestock diseases. This review is to highlight the basic concept and methodology of this assay in livestock disease.

  13. Loop-mediated isothermal amplification for rapid and convenient detection of Mycoplasma hyopneumoniae.

    Science.gov (United States)

    Li, Jiahe; Minion, F Chris; Petersen, Andrew C; Jiang, Fei; Yang, Sheng; Guo, Panpan; Li, Jinxiang; Wu, Wenxue

    2013-04-01

    Loop-mediated isothermal amplification (LAMP), a novel method of gene amplification, was employed in this study for detecting Mycoplasma hyopneumoniae in the respiratory tract or lungs of swine. The pathogen can be detected in LAMP reactions containing as few as 10 fg purified target DNA (10 copies of M. hyopneumoniae genome) within 30 min, which was comparable to real-time PCR. After 30-min reaction at 63 °C, the addition of a certain amount of dye (SYBR Green I and hydroxyl naphthol blue at a proper ratio) into the LAMP reaction system makes the results easily determined as positive or negative by visual inspection. In addition, the LAMP was able to distinguish between M. hyopneumoniae and other closely-related mycoplasma strains, indicating a high degree of specificity. The LAMP assay was more simple and cheap, since the reaction could be completed under isothermal conditions and less laboratorial infrastructure are required. And, it was proven reliable for M. hyopneumoniae diagnosis of nasal swab and lung samples from the field.

  14. A Rapid and Reproducible Genomic DNA Extraction Protocol for Sequence-Based Identification of Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and Green Algae

    Directory of Open Access Journals (Sweden)

    Farkhondeh Saba

    2017-01-01

    Full Text Available Background:  Sequence-based identification of various microorganisms including Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and green algae necessitates an efficient and reproducible genome extraction procedure though which a pure template DNA is yielded and it can be used in polymerase chain reactions (PCR. Considering the fact that DNA extraction from these microorganisms is time consuming and laborious, we developed and standardized a safe, rapid and inexpensive miniprep protocol. Methods:  According to our results, amplification of various genomic regions including SSU, LSU, ITS, β-tubulin, actin, RPB2, and EF-1 resulted in a reproducible and efficient DNA extraction from a wide range of microorganisms yielding adequate pure genomic material for reproducible PCR-amplifications. Results:   This method relies on a temporary shock of increased concentrations of detergent which can be applied concomitant with multiple freeze-thaws to yield sufficient amount of DNA for PCR amplification of multiple or single fragments(s of the genome. As an advantage, the recipe seems very flexible, thus, various optional steps can be included depending on the samples used.Conclusion:   Having the needed flexibility in each step, this protocol is applicable on a very wide range of samples. Hence, various steps can be included depending on the desired quantity and quality.

  15. A Rapid and Reproducible Genomic DNA Extraction Protocol for Sequence-Based Identification of Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and Green Algae

    Directory of Open Access Journals (Sweden)

    Farkhondeh Saba

    2016-09-01

    Full Text Available Background:  Sequence-based identification of various microorganisms including Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and green algae necessitates an efficient and reproducible genome extraction procedure though which a pure template DNA is yielded and it can be used in polymerase chain reactions (PCR. Considering the fact that DNA extraction from these microorganisms is time consuming and laborious, we developed and standardized a safe, rapid and inexpensive miniprep protocol. Methods:  According to our results, amplification of various genomic regions including SSU, LSU, ITS, β-tubulin, actin, RPB2, and EF-1 resulted in a reproducible and efficient DNA extraction from a wide range of microorganisms yielding adequate pure genomic material for reproducible PCR-amplifications. Results:   This method relies on a temporary shock of increased concentrations of detergent which can be applied concomitant with multiple freeze-thaws to yield sufficient amount of DNA for PCR amplification of multiple or single fragments(s of the genome. As an advantage, the recipe seems very flexible, thus, various optional steps can be included depending on the samples used.Conclusion:   Having the needed flexibility in each step, this protocol is applicable on a very wide range of samples. Hence, various steps can be included depending on the desired quantity and quality.

  16. Global DNA methylation analysis using methyl-sensitive amplification polymorphism (MSAP).

    Science.gov (United States)

    Yaish, Mahmoud W; Peng, Mingsheng; Rothstein, Steven J

    2014-01-01

    DNA methylation is a crucial epigenetic process which helps control gene transcription activity in eukaryotes. Information regarding the methylation status of a regulatory sequence of a particular gene provides important knowledge of this transcriptional control. DNA methylation can be detected using several methods, including sodium bisulfite sequencing and restriction digestion using methylation-sensitive endonucleases. Methyl-Sensitive Amplification Polymorphism (MSAP) is a technique used to study the global DNA methylation status of an organism and hence to distinguish between two individuals based on the DNA methylation status determined by the differential digestion pattern. Therefore, this technique is a useful method for DNA methylation mapping and positional cloning of differentially methylated genes. In this technique, genomic DNA is first digested with a methylation-sensitive restriction enzyme such as HpaII, and then the DNA fragments are ligated to adaptors in order to facilitate their amplification. Digestion using a methylation-insensitive isoschizomer of HpaII, MspI is used in a parallel digestion reaction as a loading control in the experiment. Subsequently, these fragments are selectively amplified by fluorescently labeled primers. PCR products from different individuals are compared, and once an interesting polymorphic locus is recognized, the desired DNA fragment can be isolated from a denaturing polyacrylamide gel, sequenced and identified based on DNA sequence similarity to other sequences available in the database. We will use analysis of met1, ddm1, and atmbd9 mutants and wild-type plants treated with a cytidine analogue, 5-azaC, or zebularine to demonstrate how to assess the genetic modulation of DNA methylation in Arabidopsis. It should be noted that despite the fact that MSAP is a reliable technique used to fish for polymorphic methylated loci, its power is limited to the restriction recognition sites of the enzymes used in the genomic

  17. Comparative genomic hybridization detects novel amplifications in fibroadenomas of the breast

    DEFF Research Database (Denmark)

    Ojopi, E P; Rogatto, S R; Caldeira, J R

    2001-01-01

    Comparative genomic hybridization analysis was performed for identification of chromosomal imbalances in 23 samples of fibroadenomas of the breast. Chromosomal gains rather than losses were a feature of these lesions. Only two cases with a familial and/or previous history of breast lesions had gain...

  18. Duplex recombinase polymerase amplification assays incorporating competitive internal controls for bacterial meningitis detection.

    Science.gov (United States)

    Higgins, Owen; Clancy, Eoin; Forrest, Matthew S; Piepenburg, Olaf; Cormican, Martin; Boo, Teck Wee; O'Sullivan, Nicola; McGuinness, Claire; Cafferty, Deirdre; Cunney, Robert; Smith, Terry J

    2018-04-01

    Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technology that provides rapid and robust infectious disease pathogen detection, ideal for point-of-care (POC) diagnostics in disease-prevalent low-resource countries. We have developed and evaluated three duplex RPA assays incorporating competitive internal controls for the detection of leading bacterial meningitis pathogens. Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae singleplex RPA assays were initially developed and evaluated, demonstrating 100% specificity with limits of detection of 4.1, 8.5 and 3.9 genome copies per reaction, respectively. Each assay was further developed into internally controlled duplex RPA assays via the incorporation of internal amplification control templates. Clinical performance of each internally controlled duplex RPA assay was evaluated by testing 64 archived PCR-positive clinical samples. Compared to real-time PCR, all duplex RPA assays demonstrated 100% diagnostic specificity, with diagnostic sensitivities of 100%, 86.3% and 100% for the S. pneumoniae, N. meningitidis and H. influenzae assays, respectively. This study details the first report of internally controlled duplex RPA assays for the detection of bacterial meningitis pathogens: S. pneumoniae, N. meningitidis and H. influenzae. We have successfully demonstrated the clinical diagnostic utility of each duplex RPA assay, introducing effective diagnostic technology for POC bacterial meningitis identification in disease-prevalent developing countries. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. FancJ regulates interstrand crosslinker induced centrosome amplification through the activation of polo-like kinase 1

    Directory of Open Access Journals (Sweden)

    Jianqiu Zou

    2013-08-01

    DNA damage response (DDR and the centrosome cycle are two of the most critical processes for maintaining a stable genome in animals. Sporadic evidence suggests a connection between these two processes. Here, we report our findings that six Fanconi Anemia (FA proteins, including FancI and FancJ, localize to the centrosome. Intriguingly, we found that the localization of FancJ to the mother centrosome is stimulated by a DNA interstrand crosslinker, Mitomycin C (MMC. We further show that, in addition to its role in interstrand crosslinking (ICL repair, FancJ also regulates the normal centrosome cycle as well as ICL induced centrosome amplification by activating the polo-like kinase 1 (PLK1. We have uncovered a novel function of FancJ in centrosome biogenesis and established centrosome amplification as an integral part of the ICL response.

  20. Genomic Heterogeneity as a Barrier to Precision Medicine in Gastroesophageal Adenocarcinoma.

    Science.gov (United States)

    Pectasides, Eirini; Stachler, Matthew D; Derks, Sarah; Liu, Yang; Maron, Steven; Islam, Mirazul; Alpert, Lindsay; Kwak, Heewon; Kindler, Hedy; Polite, Blase; Sharma, Manish R; Allen, Kenisha; O'Day, Emily; Lomnicki, Samantha; Maranto, Melissa; Kanteti, Rajani; Fitzpatrick, Carrie; Weber, Christopher; Setia, Namrata; Xiao, Shu-Yuan; Hart, John; Nagy, Rebecca J; Kim, Kyoung-Mee; Choi, Min-Gew; Min, Byung-Hoon; Nason, Katie S; O'Keefe, Lea; Watanabe, Masayuki; Baba, Hideo; Lanman, Rick; Agoston, Agoston T; Oh, David J; Dunford, Andrew; Thorner, Aaron R; Ducar, Matthew D; Wollison, Bruce M; Coleman, Haley A; Ji, Yuan; Posner, Mitchell C; Roggin, Kevin; Turaga, Kiran; Chang, Paul; Hogarth, Kyle; Siddiqui, Uzma; Gelrud, Andres; Ha, Gavin; Freeman, Samuel S; Rhoades, Justin; Reed, Sarah; Gydush, Greg; Rotem, Denisse; Davison, Jon; Imamura, Yu; Adalsteinsson, Viktor; Lee, Jeeyun; Bass, Adam J; Catenacci, Daniel V

    2018-01-01

    Gastroesophageal adenocarcinoma (GEA) is a lethal disease where targeted therapies, even when guided by genomic biomarkers, have had limited efficacy. A potential reason for the failure of such therapies is that genomic profiling results could commonly differ between the primary and metastatic tumors. To evaluate genomic heterogeneity, we sequenced paired primary GEA and synchronous metastatic lesions across multiple cohorts, finding extensive differences in genomic alterations, including discrepancies in potentially clinically relevant alterations. Multiregion sequencing showed significant discrepancy within the primary tumor (PT) and between the PT and disseminated disease, with oncogene amplification profiles commonly discordant. In addition, a pilot analysis of cell-free DNA (cfDNA) sequencing demonstrated the feasibility of detecting genomic amplifications not detected in PT sampling. Lastly, we profiled paired primary tumors, metastatic tumors, and cfDNA from patients enrolled in the personalized antibodies for GEA (PANGEA) trial of targeted therapies in GEA and found that genomic biomarkers were recurrently discrepant between the PT and untreated metastases. Divergent primary and metastatic tissue profiling led to treatment reassignment in 32% (9/28) of patients. In discordant primary and metastatic lesions, we found 87.5% concordance for targetable alterations in metastatic tissue and cfDNA, suggesting the potential for cfDNA profiling to enhance selection of therapy. Significance: We demonstrate frequent baseline heterogeneity in targetable genomic alterations in GEA, indicating that current tissue sampling practices for biomarker testing do not effectively guide precision medicine in this disease and that routine profiling of metastatic lesions and/or cfDNA should be systematically evaluated. Cancer Discov; 8(1); 37-48. ©2017 AACR. See related commentary by Sundar and Tan, p. 14 See related article by Janjigian et al., p. 49 This article is highlighted

  1. Comparative Analysis of the Genomic DNA Isolation Methods on Inula sp. (Asteraceae

    Directory of Open Access Journals (Sweden)

    Emre SEVİNDİK

    2016-12-01

    Full Text Available Simple, fast, low-cost and high throughput protocols are required for DNA isolation of plant species. In this study, phenol chloroform isoamyl alcohol and commercial (Sigma DNA isolation kit methods were applied on some Inula species that belong to Asteraceae family. Genomic DNA amounts, A260, A280, A260/A230 and purity degrees (A260/A280 that were obtained through both methods were measured through electrophoresis and spectrophotometer. Additionally, PCR amplification was realized by primer pairs specific to nrDNA ITS, cpDNA ndhF (972F-1603R and trnL-F regions. Results showed that maximum genomic DNA in nanograms obtained by phenol chloroform isoamyl alcohol method. The study also revealed that I. macrocephala had the maximum DNA and I. heterolepis had the minimum DNA amount. A260/A280 purity degrees showed that the highest and lowest purity in gDNAs obtained through phenol-choloform isoamyl alcohol method were in I.aucheriana and I. salicina, respectively. The highest and lowest purity degrees of gDNAs obtained through commercial kit was observed in I. fragilis and I. macrocephala samples, respectively. PCR amplification results showed that while band profiles of each three regions (ITS, trnL-F and ndhF did not yield positive results in PCR amplifications using phenol-choloform isoamyl alcohol method; PCR band profiles obtained through commercial kit yielded positive results. As a result, it is fair to say that the relation of genomic DNA with PCR was found to be more efficient although the maximum amount of genomic DNA was obtained through phenol chloroform isoamyl alcohol method.

  2. Earthquake acceleration amplification based on single microtremor test

    Science.gov (United States)

    Jaya Syahbana, Arifan; Kurniawan, Rahmat; Soebowo, Eko

    2018-02-01

    Understanding soil dynamics is needed to understand soil behaviour, including the parameters of earthquake acceleration amplification. Many researchers now conduct single microtremor tests to obtain amplification of velocity and natural periods of soil at test sites. However, these amplification parameters are rarely used, so a method is needed to convert the velocity amplification to acceleration amplification. This paper will discuss the proposed process of changing the value of amplification. The proposed method is to integrate the time histories of the synthetic earthquake acceleration of the soil surface under the deaggregation at that location so the time histories of the velocity earthquake will be obtained. Next is to conduct a “fitting curve” between amplification by a single microtremor test with amplification of the synthetic earthquake velocity time histories. After obtaining the fitting curve time histories of velocity, differentiation will be conducted to obtain fitting curve acceleration time histories. The final step after obtaining the fitting curve is to compare the acceleration of the “fitting curve” against the histories time of the acceleration of synthetic earthquake at bedrocks to obtain single microtremor acceleration amplification factor.

  3. Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification.

    Directory of Open Access Journals (Sweden)

    David S Boyle

    Full Text Available Improved access to effective tests for diagnosing tuberculosis (TB has been designated a public health priority by the World Health Organisation. In high burden TB countries nucleic acid based TB tests have been restricted to centralised laboratories and specialised research settings. Requirements such as a constant electrical supply, air conditioning and skilled, computer literate operators prevent implementation of such tests in many settings. Isothermal DNA amplification technologies permit the use of simpler, less energy intensive detection platforms more suited to low resource settings that allow the accurate diagnosis of a disease within a short timeframe. Recombinase Polymerase Amplification (RPA is a rapid, low temperature isothermal DNA amplification reaction. We report here RPA-based detection of Mycobacterium tuberculosis complex (MTC DNA in <20 minutes at 39 °C. Assays for two MTC specific targets were investigated, IS6110 and IS1081. When testing purified MTC genomic DNA, limits of detection of 6.25 fg (IS6110 and 20 fg (IS1081were consistently achieved. When testing a convenience sample of pulmonary specimens from suspected TB patients, RPA demonstrated superior accuracy to indirect fluorescence microscopy. Compared to culture, sensitivities for the IS1081 RPA and microscopy were 91.4% (95%CI: 85, 97.9 and 86.1% (95%CI: 78.1, 94.1 respectively (n = 71. Specificities were 100% and 88.6% (95% CI: 80.8, 96.1 respectively. For the IS6110 RPA and microscopy sensitivities of 87.5% (95%CI: 81.7, 93.2 and 70.8% (95%CI: 62.9, 78.7 were obtained (n = 90. Specificities were 95.4 (95% CI: 92.3,98.1 and 88% (95% CI: 83.6, 92.4 respectively. The superior specificity of RPA for detecting tuberculosis was due to the reduced ability of fluorescence microscopy to distinguish Mtb complex from other acid fast bacteria. The rapid nature of the RPA assay and its low energy requirement compared to other amplification technologies suggest RPA-based TB

  4. One bacterial cell, one complete genome.

    Directory of Open Access Journals (Sweden)

    Tanja Woyke

    2010-04-01

    Full Text Available While the bulk of the finished microbial genomes sequenced to date are derived from cultured bacterial and archaeal representatives, the vast majority of microorganisms elude current culturing attempts, severely limiting the ability to recover complete or even partial genomes from these environmental species. Single cell genomics is a novel culture-independent approach, which enables access to the genetic material of an individual cell. No single cell genome has to our knowledge been closed and finished to date. Here we report the completed genome from an uncultured single cell of Candidatus Sulcia muelleri DMIN. Digital PCR on single symbiont cells isolated from the bacteriome of the green sharpshooter Draeculacephala minerva bacteriome allowed us to assess that this bacteria is polyploid with genome copies ranging from approximately 200-900 per cell, making it a most suitable target for single cell finishing efforts. For single cell shotgun sequencing, an individual Sulcia cell was isolated and whole genome amplified by multiple displacement amplification (MDA. Sanger-based finishing methods allowed us to close the genome. To verify the correctness of our single cell genome and exclude MDA-derived artifacts, we independently shotgun sequenced and assembled the Sulcia genome from pooled bacteriomes using a metagenomic approach, yielding a nearly identical genome. Four variations we detected appear to be genuine biological differences between the two samples. Comparison of the single cell genome with bacteriome metagenomic sequence data detected two single nucleotide polymorphisms (SNPs, indicating extremely low genetic diversity within a Sulcia population. This study demonstrates the power of single cell genomics to generate a complete, high quality, non-composite reference genome within an environmental sample, which can be used for population genetic analyzes.

  5. One Bacterial Cell, One Complete Genome

    Energy Technology Data Exchange (ETDEWEB)

    Woyke, Tanja; Tighe, Damon; Mavrommatis, Konstantinos; Clum, Alicia; Copeland, Alex; Schackwitz, Wendy; Lapidus, Alla; Wu, Dongying; McCutcheon, John P.; McDonald, Bradon R.; Moran, Nancy A.; Bristow, James; Cheng, Jan-Fang

    2010-04-26

    While the bulk of the finished microbial genomes sequenced to date are derived from cultured bacterial and archaeal representatives, the vast majority of microorganisms elude current culturing attempts, severely limiting the ability to recover complete or even partial genomes from these environmental species. Single cell genomics is a novel culture-independent approach, which enables access to the genetic material of an individual cell. No single cell genome has to our knowledge been closed and finished to date. Here we report the completed genome from an uncultured single cell of Candidatus Sulcia muelleri DMIN. Digital PCR on single symbiont cells isolated from the bacteriome of the green sharpshooter Draeculacephala minerva bacteriome allowed us to assess that this bacteria is polyploid with genome copies ranging from approximately 200?900 per cell, making it a most suitable target for single cell finishing efforts. For single cell shotgun sequencing, an individual Sulcia cell was isolated and whole genome amplified by multiple displacement amplification (MDA). Sanger-based finishing methods allowed us to close the genome. To verify the correctness of our single cell genome and exclude MDA-derived artifacts, we independently shotgun sequenced and assembled the Sulcia genome from pooled bacteriomes using a metagenomic approach, yielding a nearly identical genome. Four variations we detected appear to be genuine biological differences between the two samples. Comparison of the single cell genome with bacteriome metagenomic sequence data detected two single nucleotide polymorphisms (SNPs), indicating extremely low genetic diversity within a Sulcia population. This study demonstrates the power of single cell genomics to generate a complete, high quality, non-composite reference genome within an environmental sample, which can be used for population genetic analyzes.

  6. Loop-mediated isothermal amplification assay for rapid and sensitive detection of sheep pox and goat pox viruses in clinical samples.

    Science.gov (United States)

    Venkatesan, G; Balamurugan, V; Bhanuprakash, V; Singh, R K; Pandey, A B

    2016-06-01

    A Loop-mediated isothermal amplification (LAMP) assay targeting the highly conserved DNA polymerase gene of capripox virus genome was developed and evaluated for rapid detection of sheep pox and goat pox viruses. The optimized LAMP assay is found specific and sensitive for amplification of target DNA with a diagnostic sensitivity and specificity of 96.6% and 100% respectively compared to quantitative PCR. The detection rate of LAMP, PCR and Q-PCR assays is found to be 81.5%, 67% and 83% respectively. This LAMP assay has the potential for rapid clinical diagnosis and surveillance of sheep pox and goat pox in field diagnostic laboratories. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Diagnostic performance of a novel loop-mediated isothermal amplification (LAMP) assay targeting the apicoplast genome for malaria diagnosis in a field setting in sub-Saharan Africa.

    Science.gov (United States)

    Oriero, Eniyou C; Okebe, Joseph; Jacobs, Jan; Van Geertruyden, Jean-Pierre; Nwakanma, Davis; D'Alessandro, Umberto

    2015-10-09

    New diagnostic tools to detect reliably and rapidly asymptomatic and low-density malaria infections are needed as their treatment could interrupt transmission. Isothermal amplification techniques are being explored for field diagnosis of malaria. In this study, a novel molecular tool (loop-mediated isothermal amplification-LAMP) targeting the apicoplast genome of Plasmodium falciparum was evaluated for the detection of asymptomatic malaria-infected individuals in a rural setting in The Gambia. A blood was collected from 341 subjects (median age 9 years, range 1-68 years) screened for malaria. On site, a rapid diagnostic test (RDT, SD Bioline Malaria Antigen P.f) was performed, thick blood films (TBF) slides for microscopy were prepared and dry blood spots (DBS) were collected on Whatman(®) 903 Specimen collection paper. The TBF and DBS were transported to the field laboratory where microscopy and LAMP testing were performed. The latter was done on DNA extracted from the DBS using a crude (methanol/heating) extraction method. A laboratory-based PCR amplification was done on all the samples using DNA extracted with the Qiagen kit and its results were taken as reference for all the other tests. Plasmodium falciparum malaria prevalence was 37 % (127/341) as detected by LAMP, 30 % (104/341) by microscopy and 37 % (126/341) by RDT. Compared to the reference PCR method, sensitivity was 92 % for LAMP, 78 % for microscopy, and 76 % for RDT; specificity was 97 % for LAMP, 99 % for microscopy, and 88 % for RDT. Area under the receiver operating characteristic (ROC) curve in comparison with the reference standard was 0.94 for LAMP, 0.88 for microscopy and 0.81 for RDT. Turn-around time for the entire LAMP assay was approximately 3 h and 30 min for an average of 27 ± 9.5 samples collected per day, compared to a minimum of 10 samples an hour per operator by RDT and over 8 h by microscopy. The LAMP assay could produce reliable results the same day of the screening. It could

  8. Genomic sequencing of Pleistocene cave bears

    Energy Technology Data Exchange (ETDEWEB)

    Noonan, James P.; Hofreiter, Michael; Smith, Doug; Priest, JamesR.; Rohland, Nadin; Rabeder, Gernot; Krause, Johannes; Detter, J. Chris; Paabo, Svante; Rubin, Edward M.

    2005-04-01

    Despite the information content of genomic DNA, ancient DNA studies to date have largely been limited to amplification of mitochondrial DNA due to technical hurdles such as contamination and degradation of ancient DNAs. In this study, we describe two metagenomic libraries constructed using unamplified DNA extracted from the bones of two 40,000-year-old extinct cave bears. Analysis of {approx}1 Mb of sequence from each library showed that, despite significant microbial contamination, 5.8 percent and 1.1 percent of clones in the libraries contain cave bear inserts, yielding 26,861 bp of cave bear genome sequence. Alignment of this sequence to the dog genome, the closest sequenced genome to cave bear in terms of evolutionary distance, revealed roughly the expected ratio of cave bear exons, repeats and conserved noncoding sequences. Only 0.04 percent of all clones sequenced were derived from contamination with modern human DNA. Comparison of cave bear with orthologous sequences from several modern bear species revealed the evolutionary relationship of these lineages. Using the metagenomic approach described here, we have recovered substantial quantities of mammalian genomic sequence more than twice as old as any previously reported, establishing the feasibility of ancient DNA genomic sequencing programs.

  9. Synthetic assembly of novel avidin-biotin-GlcNAc (ABG) complex as an attractive bio-probe and its interaction with wheat germ agglutinin (WGA).

    Science.gov (United States)

    Kumari, Amrita; Koyama, Tetsuo; Hatano, Ken; Matsuoka, Koji

    2016-10-01

    A tetravalent GlcNAc pendant glycocluster was constructed with terminal biotin through C6 linker. To acquire the multivalent carbohydrate-protein interactions, we synthesized a glycopolymer of tetrameric structure using N-acetyl-d-glucosamine (GlcNAc) as the target carbohydrate by the use of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) as coupling reagent, followed by biotin-avidin complexation leading to the formation of glycocluster of avidin-biotin-GlcNAc conjugate (ABG complex). The dynamic light scattering (DLS) system was implied for size detection and to check the binding affinity of GlcNAc conjugate with a WGA lectin we use fluorometric assay by means of specific excitation of tryptophan at λex 295nm and it was found to be very high Ka∼1.39×10(7) M(-1) in case of ABG complex as compared to GlcNAc only Ka∼1.01×10(4) M(-1) with the phenomenon proven to be due to glycocluster effect. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. In vitro evolution of terminal protein-containing genomes

    Science.gov (United States)

    Esteban, José A.; Blanco, Luis; Villar, Laurentino; Salas, Margarita

    1997-01-01

    A new self-sustained terminal protein-primed DNA amplification system has been used to describe in vitro evolutionary changes affecting maintenance of the genome size of bacteriophage φ29. These changes involve generation and efficient amplification of short palindromic molecules containing an inverted duplication of one of the original DNA ends. A template-switching mechanism is proposed to account for the appearance of these molecules. After their formation, they would replicate by means of hairpin intermediates. Relevant kinetic information about this DNA replication system has been obtained from the competition between the input full-length φ29 DNA and its derived truncated versions. The physiological relevance of these molecules and the mechanisms to control their formation are discussed. PMID:9096322

  11. Combined amplification and hybridization techniques for genome scanning in vegetatively propagated crops

    International Nuclear Information System (INIS)

    Kahl, G.; Ramser, J.; Terauchi, R.; Lopez-Peralta, C.; Asemota, H.N.; Weising, K.

    1998-01-01

    A combination of PCR- and hybridization-based genome scanning techniques and sequence comparisons between non-coding chloroplast DNA flanking tRNA genes has been employed to screen Dioscorea species for intra- and interspecific genetic diversity. This methodology detected extensive polymorphisms within Dioscorea bulbifera L., and revealed taxonomic and phylogenetic relationships among cultivated Guinea yams varieties and their potential wild progenitors. Finally, screening of yam germplasm grown in Jamaica permitted reliable discrimination between all major cultivars. Genome scanning by micro satellite-primed PCR (MP-PCR) and random amplified polymorphic DNA (RAPD) analysis in combination with the novel random amplified micro satellite polymorphisms (RAMPO) hybridization technique has shown high potential for the genetic analysis of yams, and holds promise for other vegetatively propagated orphan crops. (author)

  12. LATERAL GENE TRANSFER AND THE HISTORY OF BACTERIAL GENOMES

    Energy Technology Data Exchange (ETDEWEB)

    Howard Ochman

    2006-02-22

    The aims of this research were to elucidate the role and extent of lateral transfer in the differentiation of bacterial strains and species, and to assess the impact of gene transfer on the evolution of bacterial genomes. The ultimate goal of the project is to examine the dynamics of a core set of protein-coding genes (i.e., those that are distributed universally among Bacteria) by developing conserved primers that would allow their amplification and sequencing in any bacterial taxa. In addition, we adopted a bioinformatic approach to elucidate the extent of lateral gene transfer in sequenced genome.

  13. GMO detection in food and feed through screening by visual loop-mediated isothermal amplification assays.

    Science.gov (United States)

    Wang, Cong; Li, Rong; Quan, Sheng; Shen, Ping; Zhang, Dabing; Shi, Jianxin; Yang, Litao

    2015-06-01

    Isothermal DNA/RNA amplification techniques are the primary methodology for developing on-spot rapid nucleic acid amplification assays, and the loop-mediated isothermal amplification (LAMP) technique has been developed and applied in the detection of foodborne pathogens, plant/animal viruses, and genetically modified (GM) food/feed contents. In this study, one set of LAMP assays targeting on eight frequently used universal elements, marker genes, and exogenous target genes, such as CaMV35S promoter, FMV35S promoter, NOS, bar, cry1Ac, CP4 epsps, pat, and NptII, were developed for visual screening of GM contents in plant-derived food samples with high efficiency and accuracy. For these eight LAMP assays, their specificity was evaluated by testing commercial GM plant events and their limits of detection were also determined, which are 10 haploid genome equivalents (HGE) for FMV35S promoter, cry1Ac, and pat assays, as well as five HGE for CaMV35S promoter, bar, NOS terminator, CP4 epsps, and NptII assays. The screening applicability of these LAMP assays was further validated successfully using practical canola, soybean, and maize samples. The results suggested that the established visual LAMP assays are applicable and cost-effective for GM screening in plant-derived food samples.

  14. Amplification and high-level expression of heat shock protein 90 marks aggressive phenotypes of human epidermal growth factor receptor 2 negative breast cancer.

    Science.gov (United States)

    Cheng, Qing; Chang, Jeffrey T; Geradts, Joseph; Neckers, Leonard M; Haystead, Timothy; Spector, Neil L; Lyerly, H Kim

    2012-04-17

    Although human epidermal growth factor receptor 2 (HER2) positive or estrogen receptor (ER) positive breast cancers are treated with clinically validated anti-HER2 or anti-estrogen therapies, intrinsic and acquired resistance to these therapies appears in a substantial proportion of breast cancer patients and new therapies are needed. Identification of additional molecular factors, especially those characterized by aggressive behavior and poor prognosis, could prioritize interventional opportunities to improve the diagnosis and treatment of breast cancer. We compiled a collection of 4,010 breast tumor gene expression data derived from 23 datasets that have been posted on the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database. We performed a genome-scale survival analysis using Cox-regression survival analyses, and validated using Kaplan-Meier Estimates survival and Cox Proportional-Hazards Regression survival analyses. We conducted a genome-scale analysis of chromosome alteration using 481 breast cancer samples obtained from The Cancer Genome Atlas (TCGA), from which combined expression and copy number data were available. We assessed the correlation between somatic copy number alterations and gene expression using analysis of variance (ANOVA). Increased expression of each of the heat shock protein (HSP) 90 isoforms, as well as HSP transcriptional factor 1 (HSF1), was correlated with poor prognosis in different subtypes of breast cancer. High-level expression of HSP90AA1 and HSP90AB1, two cytoplasmic HSP90 isoforms, was driven by chromosome coding region amplifications and were independent factors that led to death from breast cancer among patients with triple-negative (TNBC) and HER2-/ER+ subtypes, respectively. Furthermore, amplification of HSF1 was correlated with higher HSP90AA1 and HSP90AB1 mRNA expression among the breast cancer cells without amplifications of these two genes. A collection of HSP90AA1, HSP90AB1 and HSF

  15. Significance of genomic instability in breast cancer in atomic bomb survivors: analysis of microarray-comparative genomic hybridization

    Directory of Open Access Journals (Sweden)

    Oikawa Masahiro

    2011-12-01

    Full Text Available Abstract Background It has been postulated that ionizing radiation induces breast cancers among atomic bomb (A-bomb survivors. We have reported a higher incidence of HER2 and C-MYC oncogene amplification in breast cancers from A-bomb survivors. The purpose of this study was to clarify the effect of A-bomb radiation exposure on genomic instability (GIN, which is an important hallmark of carcinogenesis, in archival formalin-fixed paraffin-embedded (FFPE tissues of breast cancer by using microarray-comparative genomic hybridization (aCGH. Methods Tumor DNA was extracted from FFPE tissues of invasive ductal cancers from 15 survivors who were exposed at 1.5 km or less from the hypocenter and 13 calendar year-matched non-exposed patients followed by aCGH analysis using a high-density oligonucleotide microarray. The total length of copy number aberrations (CNA was used as an indicator of GIN, and correlation with clinicopathological factors were statistically tested. Results The mean of the derivative log ratio spread (DLRSpread, which estimates the noise by calculating the spread of log ratio differences between consecutive probes for all chromosomes, was 0.54 (range, 0.26 to 1.05. The concordance of results between aCGH and fluorescence in situ hybridization (FISH for HER2 gene amplification was 88%. The incidence of HER2 amplification and histological grade was significantly higher in the A-bomb survivors than control group (P = 0.04, respectively. The total length of CNA tended to be larger in the A-bomb survivors (P = 0.15. Correlation analysis of CNA and clinicopathological factors revealed that DLRSpread was negatively correlated with that significantly (P = 0.034, r = -0.40. Multivariate analysis with covariance revealed that the exposure to A-bomb was a significant (P = 0.005 independent factor which was associated with larger total length of CNA of breast cancers. Conclusions Thus, archival FFPE tissues from A-bomb survivors are useful for

  16. Significance of genomic instability in breast cancer in atomic bomb survivors: analysis of microarray-comparative genomic hybridization

    International Nuclear Information System (INIS)

    Oikawa, Masahiro; Yoshiura, Koh-ichiro; Kondo, Hisayoshi; Miura, Shiro; Nagayasu, Takeshi; Nakashima, Masahiro

    2011-01-01

    It has been postulated that ionizing radiation induces breast cancers among atomic bomb (A-bomb) survivors. We have reported a higher incidence of HER2 and C-MYC oncogene amplification in breast cancers from A-bomb survivors. The purpose of this study was to clarify the effect of A-bomb radiation exposure on genomic instability (GIN), which is an important hallmark of carcinogenesis, in archival formalin-fixed paraffin-embedded (FFPE) tissues of breast cancer by using microarray-comparative genomic hybridization (aCGH). Tumor DNA was extracted from FFPE tissues of invasive ductal cancers from 15 survivors who were exposed at 1.5 km or less from the hypocenter and 13 calendar year-matched non-exposed patients followed by aCGH analysis using a high-density oligonucleotide microarray. The total length of copy number aberrations (CNA) was used as an indicator of GIN, and correlation with clinicopathological factors were statistically tested. The mean of the derivative log ratio spread (DLRSpread), which estimates the noise by calculating the spread of log ratio differences between consecutive probes for all chromosomes, was 0.54 (range, 0.26 to 1.05). The concordance of results between aCGH and fluorescence in situ hybridization (FISH) for HER2 gene amplification was 88%. The incidence of HER2 amplification and histological grade was significantly higher in the A-bomb survivors than control group (P = 0.04, respectively). The total length of CNA tended to be larger in the A-bomb survivors (P = 0.15). Correlation analysis of CNA and clinicopathological factors revealed that DLRSpread was negatively correlated with that significantly (P = 0.034, r = -0.40). Multivariate analysis with covariance revealed that the exposure to A-bomb was a significant (P = 0.005) independent factor which was associated with larger total length of CNA of breast cancers. Thus, archival FFPE tissues from A-bomb survivors are useful for genome-wide aCGH analysis. Our results suggested that A

  17. Significance of genomic instability in breast cancer in atomic bomb survivors: analysis of microarray-comparative genomic hybridization.

    Science.gov (United States)

    Oikawa, Masahiro; Yoshiura, Koh-ichiro; Kondo, Hisayoshi; Miura, Shiro; Nagayasu, Takeshi; Nakashima, Masahiro

    2011-12-07

    It has been postulated that ionizing radiation induces breast cancers among atomic bomb (A-bomb) survivors. We have reported a higher incidence of HER2 and C-MYC oncogene amplification in breast cancers from A-bomb survivors. The purpose of this study was to clarify the effect of A-bomb radiation exposure on genomic instability (GIN), which is an important hallmark of carcinogenesis, in archival formalin-fixed paraffin-embedded (FFPE) tissues of breast cancer by using microarray-comparative genomic hybridization (aCGH). Tumor DNA was extracted from FFPE tissues of invasive ductal cancers from 15 survivors who were exposed at 1.5 km or less from the hypocenter and 13 calendar year-matched non-exposed patients followed by aCGH analysis using a high-density oligonucleotide microarray. The total length of copy number aberrations (CNA) was used as an indicator of GIN, and correlation with clinicopathological factors were statistically tested. The mean of the derivative log ratio spread (DLRSpread), which estimates the noise by calculating the spread of log ratio differences between consecutive probes for all chromosomes, was 0.54 (range, 0.26 to 1.05). The concordance of results between aCGH and fluorescence in situ hybridization (FISH) for HER2 gene amplification was 88%. The incidence of HER2 amplification and histological grade was significantly higher in the A-bomb survivors than control group (P = 0.04, respectively). The total length of CNA tended to be larger in the A-bomb survivors (P = 0.15). Correlation analysis of CNA and clinicopathological factors revealed that DLRSpread was negatively correlated with that significantly (P = 0.034, r = -0.40). Multivariate analysis with covariance revealed that the exposure to A-bomb was a significant (P = 0.005) independent factor which was associated with larger total length of CNA of breast cancers. Thus, archival FFPE tissues from A-bomb survivors are useful for genome-wide aCGH analysis. Our results suggested that A

  18. Privacy amplification for quantum key distribution

    International Nuclear Information System (INIS)

    Watanabe, Yodai

    2007-01-01

    This paper examines classical privacy amplification using a universal family of hash functions. In quantum key distribution, the adversary's measurement can wait until the choice of hash functions is announced, and so the adversary's information may depend on the choice. Therefore the existing result on classical privacy amplification, which assumes the independence of the choice from the other random variables, is not applicable to this case. This paper provides a security proof of privacy amplification which is valid even when the adversary's information may depend on the choice of hash functions. The compression rate of the proposed privacy amplification can be taken to be the same as that of the existing one with an exponentially small loss in secrecy of a final key. (fast track communication)

  19. Proviral amplification of the Gypsy endogenous retrovirus of Drosophila melanogaster involves env-independent invasion of the female germline.

    OpenAIRE

    Chalvet, F; Teysset, L; Terzian, C; Prud'homme, N; Santamaria, P; Bucheton, A; Pélisson, A

    1999-01-01

    Gypsy is an infectious endogenous retrovirus of Drosophila melanogaster. The gypsy proviruses replicate very efficiently in the genome of the progeny of females homozygous for permissive alleles of the flamenco gene. This replicative transposition is correlated with derepression of gypsy expression, specifically in the somatic cells of the ovaries of the permissive mothers. The determinism of this amplification was studied further by making chimeric mothers containing different permissive/res...

  20. Non-Enzymatic Detection of Bacterial Genomic DNA Using the Bio-Barcode Assay

    Science.gov (United States)

    Hill, Haley D.; Vega, Rafael A.; Mirkin, Chad A.

    2011-01-01

    The detection of bacterial genomic DNA through a non-enzymatic nanomaterials based amplification method, the bio-barcode assay, is reported. The assay utilizes oligonucleotide functionalized magnetic microparticles to capture the target of interest from the sample. A critical step in the new assay involves the use of blocking oligonucleotides during heat denaturation of the double stranded DNA. These blockers bind to specific regions of the target DNA upon cooling, and prevent the duplex DNA from re-hybridizing, which allows the particle probes to bind. Following target isolation using the magnetic particles, oligonucleotide functionalized gold nanoparticles act as target recognition agents. The oligonucleotides on the nanoparticle (barcodes) act as amplification surrogates. The barcodes are then detected using the Scanometric method. The limit of detection for this assay was determined to be 2.5 femtomolar, and this is the first demonstration of a barcode type assay for the detection of double stranded, genomic DNA. PMID:17927207

  1. Combined amplification and hybridization techniques for genome scanning in vegetatively propagated crops

    Energy Technology Data Exchange (ETDEWEB)

    Kahl, G; Ramser, J; Terauchi, R [Biocentre, University of Frankfurt, Frankfurt am Main (Germany); Lopez-Peralta, C [IRGP, Colegio de Postgraduados, Montecillo, Edo. de Mexico, Texcoco (Mexico); Asemota, H N [Biotechnology Centre, University of the West Indies, Mona, Kingston (Jamaica); Weising, K [School of Biological Sciences, University of Auckland, Auckland (New Zealand)

    1998-10-01

    A combination of PCR- and hybridization-based genome scanning techniques and sequence comparisons between non-coding chloroplast DNA flanking tRNA genes has been employed to screen Dioscorea species for intra- and interspecific genetic diversity. This methodology detected extensive polymorphisms within Dioscorea bulbifera L., and revealed taxonomic and phylogenetic relationships among cultivated Guinea yams varieties and their potential wild progenitors. Finally, screening of yam germplasm grown in Jamaica permitted reliable discrimination between all major cultivars. Genome scanning by micro satellite-primed PCR (MP-PCR) and random amplified polymorphic DNA (RAPD) analysis in combination with the novel random amplified micro satellite polymorphisms (RAMPO) hybridization technique has shown high potential for the genetic analysis of yams, and holds promise for other vegetatively propagated orphan crops. (author) 46 refs, 3 figs, 3 tabs

  2. Efficient Audio Power Amplification - Challenges

    DEFF Research Database (Denmark)

    Andersen, Michael Andreas E.

    2005-01-01

    For more than a decade efficient audio power amplification has evolved and today switch-mode audio power amplification in various forms are the state-of-the-art. The technical steps that lead to this evolution are described and in addition many of the challenges still to be faced and where...

  3. Helicase-dependent amplification of nucleic acids.

    Science.gov (United States)

    Cao, Yun; Kim, Hyun-Jin; Li, Ying; Kong, Huimin; Lemieux, Bertrand

    2013-10-11

    Helicase-dependent amplification (HDA) is a novel method for the isothermal in vitro amplification of nucleic acids. The HDA reaction selectively amplifies a target sequence by extension of two oligonucleotide primers. Unlike the polymerase chain reaction (PCR), HDA uses a helicase enzyme to separate the deoxyribonucleic acid (DNA) strands, rather than heat denaturation. This allows DNA amplification without the need for thermal cycling. The helicase used in HDA is a helicase super family II protein obtained from a thermophilic organism, Thermoanaerobacter tengcongensis (TteUvrD). This thermostable helicase is capable of unwinding blunt-end nucleic acid substrates at elevated temperatures (60° to 65°C). The HDA reaction can also be coupled with reverse transcription for ribonucleic acid (RNA) amplification. The products of this reaction can be detected during the reaction using fluorescent probes when incubations are conducted in a fluorimeter. Alternatively, products can be detected after amplification using a disposable amplicon containment device that contains an embedded lateral flow strip. Copyright © 2013 John Wiley & Sons, Inc.

  4. The polydeoxyadenylate tract of Alu repetitive elements is polymorphic in the human genome

    International Nuclear Information System (INIS)

    Economou, E.P.; Bergen, A.W.; Warren, A.C.; Antonarakis, S.E.

    1990-01-01

    To identify DNA polymorphisms that are abundant in the human genome and are detectable by polymerase chain reaction amplification of genomic DNA, the authors hypothesize that the polydeoxyadenylate tract of the Alu family of repetitive elements is polymorphic among human chromosomes. Analysis of the 3' ends of three specific Alu sequences showed two occurrences, one in the adenosine deaminase gene and other in the β-globin pseudogene, were polymorphic. This novel class of polymorphism, termed AluVpA [Alu variable poly(A)] may represent one of the most useful and informative group of DNA markers in the human genome

  5. RABL6A, a Novel RAB-Like Protein, Controls Centrosome Amplification and Chromosome Instability in Primary Fibroblasts

    Science.gov (United States)

    Zhang, Xuefeng; Hagen, Jussara; Muniz, Viviane P.; Smith, Tarik; Coombs, Gary S.; Eischen, Christine M.; Mackie, Duncan I.; Roman, David L.; Van Rheeden, Richard; Darbro, Benjamin; Tompkins, Van S.; Quelle, Dawn E.

    2013-01-01

    RABL6A (RAB-like 6 isoform A) is a novel protein that was originally identified based on its association with the Alternative Reading Frame (ARF) tumor suppressor. ARF acts through multiple p53-dependent and p53-independent pathways to prevent cancer. How RABL6A functions, to what extent it depends on ARF and p53 activity, and its importance in normal cell biology are entirely unknown. We examined the biological consequences of RABL6A silencing in primary mouse embryo fibroblasts (MEFs) that express or lack ARF, p53 or both proteins. We found that RABL6A depletion caused centrosome amplification, aneuploidy and multinucleation in MEFs regardless of ARF and p53 status. The centrosome amplification in RABL6A depleted p53−/− MEFs resulted from centrosome reduplication via Cdk2-mediated hyperphosphorylation of nucleophosmin (NPM) at threonine-199. Thus, RABL6A prevents centrosome amplification through an ARF/p53-independent mechanism that restricts NPM-T199 phosphorylation. These findings demonstrate an essential role for RABL6A in centrosome regulation and maintenance of chromosome stability in non-transformed cells, key processes that ensure genomic integrity and prevent tumorigenesis. PMID:24282525

  6. A Perfect Match Genomic Landscape Provides a Unified Framework for the Precise Detection of Variation in Natural and Synthetic Haploid Genomes.

    Science.gov (United States)

    Palacios-Flores, Kim; García-Sotelo, Jair; Castillo, Alejandra; Uribe, Carina; Aguilar, Luis; Morales, Lucía; Gómez-Romero, Laura; Reyes, José; Garciarubio, Alejandro; Boege, Margareta; Dávila, Guillermo

    2018-04-01

    We present a conceptually simple, sensitive, precise, and essentially nonstatistical solution for the analysis of genome variation in haploid organisms. The generation of a Perfect Match Genomic Landscape (PMGL), which computes intergenome identity with single nucleotide resolution, reveals signatures of variation wherever a query genome differs from a reference genome. Such signatures encode the precise location of different types of variants, including single nucleotide variants, deletions, insertions, and amplifications, effectively introducing the concept of a general signature of variation. The precise nature of variants is then resolved through the generation of targeted alignments between specific sets of sequence reads and known regions of the reference genome. Thus, the perfect match logic decouples the identification of the location of variants from the characterization of their nature, providing a unified framework for the detection of genome variation. We assessed the performance of the PMGL strategy via simulation experiments. We determined the variation profiles of natural genomes and of a synthetic chromosome, both in the context of haploid yeast strains. Our approach uncovered variants that have previously escaped detection. Moreover, our strategy is ideally suited for further refining high-quality reference genomes. The source codes for the automated PMGL pipeline have been deposited in a public repository. Copyright © 2018 by the Genetics Society of America.

  7. The value of blastocyst culture on preimplantation genetic diagnosis%囊胚培养在植入前遗传学诊断中的价值

    Institute of Scientific and Technical Information of China (English)

    偶健; 王玮; 马燕琳; 周知; 丁洁; 王馥新; 段程颖; 李林江; 郑爱燕

    2015-01-01

    Objective To estimate the value of blastocyst culture for preimplantation genetic diagnosis (PGD).Methods Day 3 embryos were biopsied and analyzed with fluorescence in situ hybridization (FISH) technique.Embryos with normal FISH results were cultured into blastocysts,and the ones with better morphology scores were transferred.Fourteen embryos with abnormal FISH results were cultured into blastocysts.Part of the cells taken from the blastocysts were amplified by whole genomic amplification (WGA) and assessed by array-based comparative genomic hybridization (array-CGH) analysis.Results Six blastocysts with normal FISH results were transferred in 5 cycles.Four healthy babies of 3 cycles were delivered.Another one was a singleton pregnancy but with embryo growth arrest,whose villus karyotype was normal.Fourteen embryos with abnormal FISH results were cultured into blastocysts and analyzed by array-CGH.Six blastocysts were normal by array-CGH.Conclusion FISH combined with blastocyst culture may further ensure the accuracy of PGD result.Detection at the blastocyst stage can avoid false positive results and mosaic interferences on Day 3 stage and are therefore more authentic.%目的 探讨囊胚培养在植入前遗传学诊断(preimplantation genetic diagnosis,PGD)中的应用价值.方法 受精后第3天(Day 3)行胚胎活检,进行荧光原位杂交(fluorescent in situ hybridization,FISH).对于诊断为正常的胚胎,培养到囊胚阶段后选择形态评分优良的囊胚进行移植;对于诊断为异常的胚胎,有14个培养到囊胚阶段,各取其一部分细胞用于全基因组扩增(whole genomic amplification,WGA),将扩增后的DNA用微阵列比较基因组杂交(array-based comparative genomic hybridization,arrayCGH)进行再次检测.结果 FISH诊断为正常的6个囊胚进行了5个周期的胚胎移植,3个周期成功生育了4个健康婴儿,1个周期单胎妊娠见胚囊后流产,绒毛染色体检测为正常核型.FISH诊断为异常的14

  8. Efficient audio power amplification - challenges

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, Michael A.E.

    2005-07-01

    For more than a decade efficient audio power amplification has evolved and today switch-mode audio power amplification in various forms are the state-of-the-art. The technical steps that lead to this evolution are described and in addition many of the challenges still to be faced and where extensive research and development are needed is covered. (au)

  9. PriFi - Using a Multiple Alignment of Related Sequences to Find Primers for  Amplification of Homologs

    DEFF Research Database (Denmark)

    Fredslund, Jakob; Schauser, Leif; Madsen, Lene Heegaard

    2005-01-01

    Using a comparative approach, the web program PriFi (http://cgi-www.daimi.au.dk/cgi-chili/PriFi/main) designs pairs of primers useful for PCR amplification of genomic DNA in species where prior sequence information is not available. The program works with an alignment of DNA sequences from...... of a procedure for developing general markers serving as common anchor loci across species. To accommodate users with special preferences, configuration settings and criteria can be customized....

  10. The somatic genomic landscape of chromophobe renal cell carcinoma.

    Science.gov (United States)

    Davis, Caleb F; Ricketts, Christopher J; Wang, Min; Yang, Lixing; Cherniack, Andrew D; Shen, Hui; Buhay, Christian; Kang, Hyojin; Kim, Sang Cheol; Fahey, Catherine C; Hacker, Kathryn E; Bhanot, Gyan; Gordenin, Dmitry A; Chu, Andy; Gunaratne, Preethi H; Biehl, Michael; Seth, Sahil; Kaipparettu, Benny A; Bristow, Christopher A; Donehower, Lawrence A; Wallen, Eric M; Smith, Angela B; Tickoo, Satish K; Tamboli, Pheroze; Reuter, Victor; Schmidt, Laura S; Hsieh, James J; Choueiri, Toni K; Hakimi, A Ari; Chin, Lynda; Meyerson, Matthew; Kucherlapati, Raju; Park, Woong-Yang; Robertson, A Gordon; Laird, Peter W; Henske, Elizabeth P; Kwiatkowski, David J; Park, Peter J; Morgan, Margaret; Shuch, Brian; Muzny, Donna; Wheeler, David A; Linehan, W Marston; Gibbs, Richard A; Rathmell, W Kimryn; Creighton, Chad J

    2014-09-08

    We describe the landscape of somatic genomic alterations of 66 chromophobe renal cell carcinomas (ChRCCs) on the basis of multidimensional and comprehensive characterization, including mtDNA and whole-genome sequencing. The result is consistent that ChRCC originates from the distal nephron compared with other kidney cancers with more proximal origins. Combined mtDNA and gene expression analysis implicates changes in mitochondrial function as a component of the disease biology, while suggesting alternative roles for mtDNA mutations in cancers relying on oxidative phosphorylation. Genomic rearrangements lead to recurrent structural breakpoints within TERT promoter region, which correlates with highly elevated TERT expression and manifestation of kataegis, representing a mechanism of TERT upregulation in cancer distinct from previously observed amplifications and point mutations. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Genomic and gene expression signature of the pre-invasive testicular carcinoma in situ

    DEFF Research Database (Denmark)

    Almstrup, Kristian; Ottesen, Anne Marie; Sonne, Si Brask

    2005-01-01

    and increased transcriptional activation and/or deficiency in the epigenetic silencing of specific loci. Amplification of chromosome 12p, appears to be a good genomic marker of the transition from the pre-malignant to malignant CIS cell; this is consistent with recent findings of propagation advantages...

  12. Genetic signatures from amplification profiles characterize DNA mutation in somatic and radiation-induced sports of chrysanthemum

    International Nuclear Information System (INIS)

    Trigiano, R.N.; Scott, M.C.; Caetano-Anolles, G.

    1998-01-01

    The chrysanthemum (Dendranthema grandiflora Tzvelev.) cultivars 'Dark Charm', 'Salmon Charm', 'Coral Charm' and 'Dark Bronze Charm' are either radiation-induced mutants or spontaneous sports of 'Charm' and constitute a family or series of plants that primarily differ in flower color. These cultivars, which were difficult to differentiate genetically by DNA amplification fingerprinting (DAF), were easily identified by using arbitrary signatures from amplification profiles (ASAP). Genomic DNA was first amplified with three standard octamer arbitrary primers, all of which produced monomorphic profiles. Products from each of these DNA fingerprints were subsequently reamplified using four minihairpin decamer primers. The 12 primer combinations produced signatures containing approximately 37% polymorphic character loci, which were used to estimate genetic relationships between cultivars. Forty-six (32%) unique amplification products were associated with individual cultivars. The number of ASAP polymorphisms detected provided an estimate of the mutation rate in the mutant cultivars, ranging from 0.03% to 1.6% of nucleotide changes within an average of 18 kb of arbitrary amplified DAF sequence. The ASAP technique permits the clear genetic identification of somatic mutants and radiation-induced sports that are genetically highly homogeneous and should facilitate marker assisted breeding and protection of plant breeders rights of varieties or cultivars

  13. Characterisation of genetic markers in Mungbean using direct amplification of length polymorphisms (DALP)

    International Nuclear Information System (INIS)

    Kumar, S.V.; Tan, S.G.; Quah, S.C.

    2000-01-01

    A newly developed technique, Direct Amplification of Length Polymorphisms (DALP), developed by Desmarais and co-workers in 1998 was successfully used to identify and characterise new genetic markers in mungbean (Vigyia radiata). DALP uses an arbitrarily primed PCR (AP-PCR) to produce genomic fingerprints and is specifically designed to enable direct sequencing of polymorphic bands. In this study, an oligonucleotide pair DALP235 and DAPLR were tested on four varieties of mungbean (V3476, P4281, V5973 and V5784) and produced, through PCR, specific multibanded fingerprints which showed polymorphisms. These polymorphic bands are the result of length polymorphisms as well as absence and presence of bands. Some of the polymorphic zones may be codominantly inherited and may be potential microsatellites. The success of DALP in characterising new polymorphic loci and its ability to discover microsatellites without the use of priori knowledge of the mungbean genome is revolutionary. This would greatly facilitate the breeding and improvement of the crop. (author)

  14. Simultaneous detection of genetically modified organisms by multiplex ligation-dependent genome amplification and capillary gel electrophoresis with laser-induced fluorescence.

    Science.gov (United States)

    García-Cañas, Virginia; Mondello, Monica; Cifuentes, Alejandro

    2010-07-01

    In this work, an innovative method useful to simultaneously analyze multiple genetically modified organisms is described. The developed method consists in the combination of multiplex ligation-dependent genome dependent amplification (MLGA) with CGE and LIF detection using bare-fused silica capillaries. The MLGA process is based on oligonucleotide constructs, formed by a universal sequence (vector) and long specific oligonucleotides (selectors) that facilitate the circularization of specific DNA target regions. Subsequently, the circularized target sequences are simultaneously amplified with the same couple of primers and analyzed by CGE-LIF using a bare-fused silica capillary and a run electrolyte containing 2-hydroxyethyl cellulose acting as both sieving matrix and dynamic capillary coating. CGE-LIF is shown to be very useful and informative for optimizing MLGA parameters such as annealing temperature, number of ligation cycles, and selector probes concentration. We demonstrate the specificity of the method in detecting the presence of transgenic DNA in certified reference and raw commercial samples. The method developed is sensitive and allows the simultaneous detection in a single run of percentages of transgenic maize as low as 1% of GA21, 1% of MON863, and 1% of MON810 in maize samples with signal-to-noise ratios for the corresponding DNA peaks of 15, 12, and 26, respectively. These results demonstrate, to our knowledge for the first time, the great possibilities of MLGA techniques for genetically modified organisms analysis.

  15. EFFICIENCY OF REAL-TIME PCR FOR 18S rRNA AMPLIFICATION OF SORBUS DOMESTICA, L.

    Directory of Open Access Journals (Sweden)

    Petronela Poláčeková

    2012-10-01

    Full Text Available Normal 0 21 false false false SK X-NONE X-NONE Nowadays, the awareness is given more and more to underutilized and  unusual fruits. One of them is Sorbus domestica, L. not only as an endangered species, but as well as a promising and economically usable crop. The work was aimed for finding a total genomic DNA isolating methods from fresh plant material and confirmation of the optimized method by the detection of 18S rRNA gene using real-time PCR. Two commercial isolation kits were tested -  Invisorb® Spin Plant Mini Kit and Wizard ® Genomic DNA. Higher purity and yield of DNA isolation kit showed Invisorb kit. The effective and pure PCR amplification was confirmed for Invisorb, too when 20 ng undiluted DNA at annealing temperature of 64.5 °C.doi:10.5219/203

  16. Search for methylation-sensitive amplification polymorphisms in mutant figs.

    Science.gov (United States)

    Rodrigues, M G F; Martins, A B G; Bertoni, B W; Figueira, A; Giuliatti, S

    2013-07-08

    Fig (Ficus carica) breeding programs that use conventional approaches to develop new cultivars are rare, owing to limited genetic variability and the difficulty in obtaining plants via gamete fusion. Cytosine methylation in plants leads to gene repression, thereby affecting transcription without changing the DNA sequence. Previous studies using random amplification of polymorphic DNA and amplified fragment length polymorphism markers revealed no polymorphisms among select fig mutants that originated from gamma-irradiated buds. Therefore, we conducted methylation-sensitive amplified polymorphism analysis to verify the existence of variability due to epigenetic DNA methylation among these mutant selections compared to the main cultivar 'Roxo-de-Valinhos'. Samples of genomic DNA were double-digested with either HpaII (methylation sensitive) or MspI (methylation insensitive) and with EcoRI. Fourteen primer combinations were tested, and on an average, non-methylated CCGG, symmetrically methylated CmCGG, and hemimethylated hmCCGG sites accounted for 87.9, 10.1, and 2.0%, respectively. MSAP analysis was effective in detecting differentially methylated sites in the genomic DNA of fig mutants, and methylation may be responsible for the phenotypic variation between treatments. Further analyses such as polymorphic DNA sequencing are necessary to validate these differences, standardize the regions of methylation, and analyze reads using bioinformatic tools.

  17. Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing.

    Science.gov (United States)

    Castle, John; Garrett-Engele, Phil; Armour, Christopher D; Duenwald, Sven J; Loerch, Patrick M; Meyer, Michael R; Schadt, Eric E; Stoughton, Roland; Parrish, Mark L; Shoemaker, Daniel D; Johnson, Jason M

    2003-01-01

    Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing.

  18. Comprehensive Genomic Profiling of Esthesioneuroblastoma Reveals Additional Treatment Options.

    Science.gov (United States)

    Gay, Laurie M; Kim, Sungeun; Fedorchak, Kyle; Kundranda, Madappa; Odia, Yazmin; Nangia, Chaitali; Battiste, James; Colon-Otero, Gerardo; Powell, Steven; Russell, Jeffery; Elvin, Julia A; Vergilio, Jo-Anne; Suh, James; Ali, Siraj M; Stephens, Philip J; Miller, Vincent A; Ross, Jeffrey S

    2017-07-01

    Esthesioneuroblastoma (ENB), also known as olfactory neuroblastoma, is a rare malignant neoplasm of the olfactory mucosa. Despite surgical resection combined with radiotherapy and adjuvant chemotherapy, ENB often relapses with rapid progression. Current multimodality, nontargeted therapy for relapsed ENB is of limited clinical benefit. We queried whether comprehensive genomic profiling (CGP) of relapsed or refractory ENB can uncover genomic alterations (GA) that could identify potential targeted therapies for these patients. CGP was performed on formalin-fixed, paraffin-embedded sections from 41 consecutive clinical cases of ENBs using a hybrid-capture, adaptor ligation based next-generation sequencing assay to a mean coverage depth of 593X. The results were analyzed for base substitutions, insertions and deletions, select rearrangements, and copy number changes (amplifications and homozygous deletions). Clinically relevant GA (CRGA) were defined as GA linked to drugs on the market or under evaluation in clinical trials. A total of 28 ENBs harbored GA, with a mean of 1.5 GA per sample. Approximately half of the ENBs (21, 51%) featured at least one CRGA, with an average of 1 CRGA per sample. The most commonly altered gene was TP53 (17%), with GA in PIK3CA , NF1 , CDKN2A , and CDKN2C occurring in 7% of samples. We report comprehensive genomic profiles for 41 ENB tumors. CGP revealed potential new therapeutic targets, including targetable GA in the mTOR, CDK and growth factor signaling pathways, highlighting the clinical value of genomic profiling in ENB. Comprehensive genomic profiling of 41 relapsed or refractory ENBs reveals recurrent alterations or classes of mutation, including amplification of tyrosine kinases encoded on chromosome 5q and mutations affecting genes in the mTOR/PI3K pathway. Approximately half of the ENBs (21, 51%) featured at least one clinically relevant genomic alteration (CRGA), with an average of 1 CRGA per sample. The most commonly altered

  19. The detection of hTERC amplification using fluorescence in situ hybridization in the diagnosis and prognosis of cervical intraepithelial neoplasia: a case control study

    Directory of Open Access Journals (Sweden)

    Yin Geping

    2012-08-01

    Full Text Available Abstract Background Currently the routine non-invasive screening methods for cervical intraepithelial neoplasia (CIN and cervical cancer are Thinprep cytology test (TCT and human papillomavirus testing. However, both methods are limited by the high false positive and false negative rates and lack of association with patients’ prognosis, especially for the early detection of pro-malignant CIN. The aim of the study was to investigate the role of genomic amplification of human telomerase gene (hTERC in the diagnosis and prognosis of CIN. Methods The study group consisted of specimens of exfoliated cervical cells from 151 patients, including 27 with CIN I, 54 with CIN II/III, 17 with carcinoma in situ, and 28 with invasive squamous carcinoma, as well as 25 patients who were at 2-year follow-up after either Loop Electrosurgical Excision treatment (n = 11 or radical surgery (n = 14. hTERC amplification was detected by dual-color interphase fluorescence in situ hybridization (FISH, and the results were compared with TCT and histologic examination. The final diagnosis was determined by the pathological examination. The control group consisted of specimens of exfoliated cervical cells from 40 normal women. Results The percentage of cervical exfoliated cells with positive hTERC amplification and incidence rates of hTERC amplification were 9.2% ± 4.6% and 44.4% (12/27 respectively in patients with CIN I; 16.0% ± 14.4% and 85.1% (46/54 in patients with CIN II/III; 19.7% ± 13.3% and 88.3% (15 /17 in patients with carcinoma in situ; 47.0% ± 25.2% and 100% (28/28in patients with invasive squamous carcinoma. There was statistically significant difference between the control and study group (P Conclusion The detection of genomic amplification of hTERC using FISH is a non-invasive and effective approach for CIN.

  20. Comparing Whole-Genome Sequencing with Sanger Sequencing for spa Typing of Methicillin-Resistant Staphylococcus aureus

    DEFF Research Database (Denmark)

    Bartels, Mette Damkjaer; Petersen, Andreas; Worning, Peder

    2014-01-01

    spa typing of methicillin-resistant Staphylococcus aureus (MRSA) has traditionally been done by PCR amplification and Sanger sequencing of the spa repeat region. At Hvidovre Hospital, Denmark, whole-genome sequencing (WGS) of all MRSA isolates has been performed routinely since January 2013, and ...

  1. Droplet digital polymerase chain reaction detection of HER2 amplification in formalin fixed paraffin embedded breast and gastric carcinoma samples.

    Science.gov (United States)

    Zhu, Yazhen; Lu, Dan; Lira, Maruja E; Xu, Qing; Du, Yunzhi; Xiong, Jianghong; Mao, Mao; Chung, Hyun Cheol; Zheng, Guangjuan

    2016-04-01

    Human epidermal growth factor receptor 2 (HER2) is a key driver of tumorigenesis, and over-expression as a result of HER2 gene amplification has been observed in a number of solid tumors. Recently HER2 has become an important biomarker for the monoclonal antibody treatment of HER2-positive metastatic breast and advanced gastric cancer. The HER2 targeting antibody trastuzumab treatment requires accurate measurement of HER2 levels for proper diagnosis. Droplet digital PCR (ddPCR) with highly direct, precise and absolute nucleic acid quantification could be used to detect HER2 amplification levels. Our objective was to evaluate a robust, accurate and less subjective application of ddPCR for HER2 amplification levels and test the assay performance in clinical formalin-fixed paraffin-embedded (FFPE) breast and gastric carcinoma samples. Genomic DNA from HER2 amplified cell line SK-BR-3 was used to set up the ddPCR assays. The copy number of HER2 was compared to the chromosome 17 centromere reference gene (CEP17), expressed as HER2:CEP17 ratio. Genomic DNAs of FFPE specimens from 145 Asian patients with breast and gastric carcinomas were assayed using both standard methods, immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH), and ddPCR. Based on 145 clinical breast and gastric carcinoma cases, our study demonstrated a high concordance of ddPCR results to FISH and IHC. In breast cancer specimens, the ddPCR results had high concordance with FISH and IHC defined HER2 status with a sensitivity of 90.9% (30/33) and a specificity of 100% (77/77). In gastric cancer specimens that were concordant in both FISH and IHC, our assay was 95.5% concordant with FISH and IHC (21/22). ddPCR has the advantage of automation and also allows levels of HER2 amplification to be easily evaluated in large numbers of samples, and presents a potential option to define HER2 status. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Rapid and reliable extraction of genomic DNA from various wild-type and transgenic plants

    Directory of Open Access Journals (Sweden)

    Yang Moon-Sik

    2004-09-01

    Full Text Available Abstract Background DNA extraction methods for PCR-quality DNA from calluses and plants are not time efficient, since they require that the tissues be ground in liquid nitrogen, followed by precipitation of the DNA pellet in ethanol, washing and drying the pellet, etc. The need for a rapid and simple procedure is urgent, especially when hundreds of samples need to be analyzed. Here, we describe a simple and efficient method of isolating high-quality genomic DNA for PCR amplification and enzyme digestion from calluses, various wild-type and transgenic plants. Results We developed new rapid and reliable genomic DNA extraction method. With our developed method, plant genomic DNA extraction could be performed within 30 min. The method was as follows. Plant tissue was homogenized with salt DNA extraction buffer using hand-operated homogenizer and extracted by phenol:chloroform:isoamyl alcohol (25:24:1. After centrifugation, the supernatant was directly used for DNA template for PCR, resulting in successful amplification for RAPD from various sources of plants and specific foreign genes from transgenic plants. After precipitating the supernatant, the DNA was completely digested by restriction enzymes. Conclusion This DNA extraction procedure promises simplicity, speed, and efficiency, both in terms of time and the amount of plant sample required. In addition, this method does not require expensive facilities for plant genomic DNA extraction.

  3. Comparative genomic and proteomic analysis of high grade glioma primary cultures and matched tumor in situ.

    LENUS (Irish Health Repository)

    Howley, R

    2012-10-15

    Developing targeted therapies for high grade gliomas (HGG), the most common primary brain tumor in adults, relies largely on glioma cultures. However, it is unclear if HGG tumorigenic signaling pathways are retained under in-vitro conditions. Using array comparative genomic hybridization and immunohistochemical profiling, we contrasted the epidermal and platelet-derived growth factor receptor (EGFR\\/PDGFR) in-vitro pathway status of twenty-six primary HGG cultures with the pathway status of their original HGG biopsies. Genomic gains or amplifications were lost during culturing while genomic losses were more likely to be retained. Loss of EGFR amplification was further verified immunohistochemically when EGFR over expression was decreased in the majority of cultures. Conversely, PDGFRα and PDGFRβ were more abundantly expressed in primary cultures than in the original tumor (p<0.05). Despite these genomic and proteomic differences, primary HGG cultures retained key aspects of dysregulated tumorigenic signaling. Both in-vivo and in-vitro the presence of EGFR resulted in downstream activation of P70s6K while reduced downstream activation was associated with the presence of PDGFR and the tumor suppressor, PTEN. The preserved pathway dysregulation make this glioma model suitable for further studies of glioma tumorigenesis, however individual culture related differences must be taken into consideration when testing responsiveness to chemotherapeutic agents.

  4. Improved Method for Direct Detection of Environmental Microorganisms Using an Amplification of 16S rDNA Region

    Science.gov (United States)

    Tsujimura, M.; Akutsu, J.; Zhang, Z.; Sasaki, M.; Tajima, H.; Kawarabayasi, Y.

    2004-12-01

    The thermostable proteins or enzymes were expected to be capable to be utilized in many areas of industries. Many thermophilic microorganisms, which possess the thermostable proteins or enzymes, were identified from the extreme environment. However, many unidentified and uncultivable microorganisms are still remaining in the environment on the earth. It is generally said that the cultivable microorganisms are less than 1% of entire microorganisms living in the earth, remaining over 99% are still uncultivable. As an approach to the uncultivable microorganisms, the PCR amplification of 16S rDNA region using primer sets designed from the conserved region has been generally utilized for detection and community analysis of microorganism in the environment. However, the facts, that PCR amplification introduces the mutation in the amplified DNA fragment and efficiency of PCR amplification is depend on the sequences of primer sets, indicated that the improving of PCR analysis was necessary for more correct detection of microorganisms. As the result of evaluation for the quality of DNA polymerases, sequences of primers used for amplification and conditions of PCR amplification, the DNA polymerase, the primer set and the conditions for amplification, which did not amplify the DNA fragment from the DNA contaminated within the DNA polymerase itself, were successfully selected. Also the rate of mutation in the DNA fragment amplified was evaluated using this conditions and the genomic DNA from cultivable microbes as a template. The result indicated the rate of mutation introduced by PCR was approximately 0.1% to 0.125%. The improved method using these conditions and error rate calculated was applied for the analysis of microorganisms in the geothermal environment. The result indicated that four kinds of dominant microorganisms, including both of bacteria and archaea, were alive within soil in the hot spring in Tohoku Area. We would like to apply this improved method to detection

  5. The genome sequence of pepper vein yellows virus (family Luteoviridae, genus Polerovirus).

    Science.gov (United States)

    Murakami, Ritsuko; Nakashima, Nobuhiko; Hinomoto, Norihide; Kawano, Shinji; Toyosato, Tetsuya

    2011-05-01

    The complete genome of pepper vein yellows virus (PeVYV) was sequenced using random amplification of RNA samples isolated from vector insects (Aphis gossypii) that had been given access to PeVYV-infected plants. The PeVYV genome consisted of 6244 nucleotides and had a genomic organization characteristic of members of the genus Polerovirus. PeVYV had highest amino acid sequence identities in ORF0 to ORF3 (75.9 - 91.9%) with tobacco vein distorting polerovirus, with which it was only 25.1% identical in ORF5. These sequence comparisons and previously studied biological properties indicate that PeVYV is a distinctly different virus and belongs to a new species of the genus Polerovirus.

  6. Simultaneous Aurora-A/STK15 overexpression and centrosome amplification induce chromosomal instability in tumour cells with a MIN phenotype

    International Nuclear Information System (INIS)

    Lentini, Laura; Amato, Angela; Schillaci, Tiziana; Di Leonardo, Aldo

    2007-01-01

    Genetic instability is a hallmark of tumours and preneoplastic lesions. The predominant form of genome instability in human cancer is chromosome instability (CIN). CIN is characterized by chromosomal aberrations, gains or losses of whole chromosomes (aneuploidy), and it is often associated with centrosome amplification. Centrosomes control cell division by forming a bipolar mitotic spindle and play an essential role in the maintenance of chromosomal stability. However, whether centrosome amplification could directly cause aneuploidy is not fully established. Also, alterations in genes required for mitotic progression could be involved in CIN. A major candidate is represented by Aurora-A/STK15 that associates with centrosomes and is overexpressed in several types of human tumour. Centrosome amplification were induced by hydroxyurea treatment and visualized by immunofluorescence microscopy. Aurora-A/STK15 ectopic expression was achieved by retroviral infection and puromycin selection in HCT116 tumour cells. Effects of Aurora-A/STK15 depletion on centrosome status and ploidy were determined by Aurora-A/STK15 transcriptional silencing by RNA interference. Changes in the expression levels of some mitotic genes were determined by Real time RT-PCR. We investigated whether amplification of centrosomes and overexpression of Aurora-A/STK15 induce CIN using as a model system a colon carcinoma cell line (HCT116). We found that in HCT116 cells, chromosomally stable and near diploid cells harbouring a MIN phenotype, centrosome amplification induced by hydroxyurea treatment is neither maintained nor induces aneuploidy. On the contrary, ectopic overexpression of Aurora-A/STK15 induced supernumerary centrosomes and aneuploidy. Aurora-A/STK15 transcriptional silencing by RNA interference in cells ectopically overexpressing this kinase promptly decreased cell numbers with supernumerary centrosomes and aneuploidy. Our results show that centrosome amplification alone is not sufficient

  7. Simultaneous Aurora-A/STK15 overexpression and centrosome amplification induce chromosomal instability in tumour cells with a MIN phenotype

    Directory of Open Access Journals (Sweden)

    Schillaci Tiziana

    2007-11-01

    Full Text Available Abstract Background Genetic instability is a hallmark of tumours and preneoplastic lesions. The predominant form of genome instability in human cancer is chromosome instability (CIN. CIN is characterized by chromosomal aberrations, gains or losses of whole chromosomes (aneuploidy, and it is often associated with centrosome amplification. Centrosomes control cell division by forming a bipolar mitotic spindle and play an essential role in the maintenance of chromosomal stability. However, whether centrosome amplification could directly cause aneuploidy is not fully established. Also, alterations in genes required for mitotic progression could be involved in CIN. A major candidate is represented by Aurora-A/STK15 that associates with centrosomes and is overexpressed in several types of human tumour. Methods Centrosome amplification were induced by hydroxyurea treatment and visualized by immunofluorescence microscopy. Aurora-A/STK15 ectopic expression was achieved by retroviral infection and puromycin selection in HCT116 tumour cells. Effects of Aurora-A/STK15 depletion on centrosome status and ploidy were determined by Aurora-A/STK15 transcriptional silencing by RNA interference. Changes in the expression levels of some mitotic genes were determined by Real time RT-PCR. Results We investigated whether amplification of centrosomes and overexpression of Aurora-A/STK15 induce CIN using as a model system a colon carcinoma cell line (HCT116. We found that in HCT116 cells, chromosomally stable and near diploid cells harbouring a MIN phenotype, centrosome amplification induced by hydroxyurea treatment is neither maintained nor induces aneuploidy. On the contrary, ectopic overexpression of Aurora-A/STK15 induced supernumerary centrosomes and aneuploidy. Aurora-A/STK15 transcriptional silencing by RNA interference in cells ectopically overexpressing this kinase promptly decreased cell numbers with supernumerary centrosomes and aneuploidy. Conclusion Our

  8. Rapid and sensitive detection of canine parvovirus type 2 by recombinase polymerase amplification.

    Science.gov (United States)

    Wang, Jianchang; Liu, Libing; Li, Ruiwen; Wang, Jinfeng; Fu, Qi; Yuan, Wanzhe

    2016-04-01

    A novel recombinase polymerase amplification (RPA)-based method for detection of canine parvovirus type 2 (CPV-2) was developed. Sensitivity analysis showed that the detection limit of RPA was 10 copies of CPV-2 genomic DNA. RPA amplified both CPV-2a and -2b DNA but did not amplify the template of other important dog viruses (CCoV, PRV or CDV), demonstrating high specificity. The method was further validated with 57 canine fecal samples. An outstanding advantage of RPA is that it is an isothermal reaction and can be performed in a water bath, making RPA a potential alternative method for CPV-2 detection in resource-limited settings.

  9. HER-2 amplification in tubular carcinoma of the breast.

    Science.gov (United States)

    Oakley, Gerard J; Tubbs, Raymond R; Crowe, Joseph; Sebek, Bruce; Budd, G Thomas; Patrick, Rebecca J; Procop, Gary W

    2006-07-01

    The prognostic and therapeutic implications of HER-2 gene amplification and estrogen and progesterone receptor status in breast cancer are well described. To address the relative paucity of information concerning HER-2 amplification for tubular carcinomas, we assessed the frequency of gene amplification in 55 tubular carcinomas of the breast from 54 patients, 5 of which had axillary node metastases. The HER-2 gene copy number was assessed by fluorescence in situ hybridization for the majority of tumors analyzed, whereas estrogen and progesterone receptor status was achieved by immunohistochemical analysis. HER-2 gene amplification was not observed in any of the tumors examined, and most were estrogen receptor-positive. This HER-2 gene amplification frequency was significantly lower than the frequency of gene amplification previously reported for all invasive ductal carcinoma of no special type (P < .01). HER-2 gene amplification likely occurs infrequently, or not at all, in tubular carcinomas of the breast, whereas most express estrogen receptors.

  10. Clinical application of somatosensory amplification in psychosomatic medicine

    Directory of Open Access Journals (Sweden)

    Nakao Mutsuhiro

    2007-10-01

    Full Text Available Abstract Many patients with somatoform disorders are frequently encountered in psychosomatic clinics as well as in primary care clinics. To assess such patients objectively, the concept of somatosensory amplification may be useful. Somatosensory amplification refers to the tendency to experience a somatic sensation as intense, noxious, and disturbing. It may have a role in a variety of medical conditions characterized by somatic symptoms that are disproportionate to demonstrable organ pathology. It may also explain some of the variability in somatic symptomatology found among different patients with the same serious medical disorder. It has been assessed with a self-report questionnaire, the Somatosensory Amplification Scale. This instrument was developed in a clinical setting in the U.S., and the reliability and validity of the Japanese and Turkish versions have been confirmed as well. Many studies have attempted to clarify the specific role of somatosensory amplification as a pathogenic mechanism in somatization. It has been reported that somatosensory amplification does not correlate with heightened sensitivity to bodily sensations and that emotional reactivity exerts its influence on somatization via a negatively biased reporting style. According to our recent electroencephalographic study, somatosensory amplification appears to reflect some aspects of long-latency cognitive processing rather than short-latency interoceptive sensitivity. The concept of somatosensory amplification can be useful as an indicator of somatization in the therapy of a broad range of disorders, from impaired self-awareness to various psychiatric disorders. It also provides useful information for choosing appropriate pharmacological or psychological therapy. While somatosensory amplification has a role in the presentation of somatic symptoms, it is closely associated with other factors, namely, anxiety, depression, and alexithymia that may also influence the same

  11. Amplification of pico-scale DNA mediated by bacterial carrier DNA for small-cell-number transcription factor ChIP-seq

    DEFF Research Database (Denmark)

    Jakobsen, Janus S; Bagger, Frederik O; Hasemann, Marie S

    2015-01-01

    BACKGROUND: Chromatin-Immunoprecipitation coupled with deep sequencing (ChIP-seq) is used to map transcription factor occupancy and generate epigenetic profiles genome-wide. The requirement of nano-scale ChIP DNA for generation of sequencing libraries has impeded ChIP-seq on in vivo tissues of low...... transcription factor (CEBPA) and histone mark (H3K4me3) ChIP. We further demonstrate that genomic profiles are highly resilient to changes in carrier DNA to ChIP DNA ratios. CONCLUSIONS: This represents a significant advance compared to existing technologies, which involve either complex steps of pre...... cell numbers. RESULTS: We describe a robust, simple and scalable methodology for ChIP-seq of low-abundant cell populations, verified down to 10,000 cells. By employing non-mammalian genome mapping bacterial carrier DNA during amplification, we reliably amplify down to 50 pg of ChIP DNA from...

  12. Comparison of esterase gene amplification, gene expression and esterase activity in insecticide susceptible and resistant strains of the brown planthopper, Nilaparvata lugens (Stål).

    Science.gov (United States)

    Vontas, J G; Small, G J; Hemingway, J

    2000-12-01

    Organophosphorus and carbamate insecticide resistance in Nilaparvata lugens is based on amplification of a carboxylesterase gene, Nl-EST1. An identical gene occurs in susceptible insects. Quantitative real-time PCR was used to demonstrate that Nl-EST1 is amplified 3-7-fold in the genome of resistant compared to susceptible planthoppers. Expression levels were similar to amplification levels, with 1-15-fold more Nl-EST1 mRNA in individual insects and 5-11-fold more Nl-EST1 mRNA in mass whole body homogenates of resistant females compared to susceptibles. These values corresponded to an 8-10-fold increase in esterase activity in the head and thorax of individual resistant insects. Although amplification, expression and activity levels of Nl-EST1 in resistant N. lugens were similar, the correlation between esterase activity and Nl-EST1 mRNA levels in resistant individuals was not linear.

  13. Genomic and Epigenomic Alterations in Cancer.

    Science.gov (United States)

    Chakravarthi, Balabhadrapatruni V S K; Nepal, Saroj; Varambally, Sooryanarayana

    2016-07-01

    Multiple genetic and epigenetic events characterize tumor progression and define the identity of the tumors. Advances in high-throughput technologies, like gene expression profiling, next-generation sequencing, proteomics, and metabolomics, have enabled detailed molecular characterization of various tumors. The integration and analyses of these high-throughput data have unraveled many novel molecular aberrations and network alterations in tumors. These molecular alterations include multiple cancer-driving mutations, gene fusions, amplification, deletion, and post-translational modifications, among others. Many of these genomic events are being used in cancer diagnosis, whereas others are therapeutically targeted with small-molecule inhibitors. Multiple genes/enzymes that play a role in DNA and histone modifications are also altered in various cancers, changing the epigenomic landscape during cancer initiation and progression. Apart from protein-coding genes, studies are uncovering the critical regulatory roles played by noncoding RNAs and noncoding regions of the genome during cancer progression. Many of these genomic and epigenetic events function in tandem to drive tumor development and metastasis. Concurrent advances in genome-modulating technologies, like gene silencing and genome editing, are providing ability to understand in detail the process of cancer initiation, progression, and signaling as well as opening up avenues for therapeutic targeting. In this review, we discuss some of the recent advances in cancer genomic and epigenomic research. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  14. Cloning-free genome alterations in Saccharomyces cerevisiae using adaptamer-mediated PCR

    DEFF Research Database (Denmark)

    Reid, Robert J D; Lisby, Michael; Rothstein, Rodney

    2002-01-01

    . Furthermore, many of the techniques described here rely on preexisting and commercially available adaptamer sets that can be obtained inexpensively rather than designing new primers for every experiment. Although a cost is incurred when performing multiple PCR amplifications, the increase in recombination...... efficiency is dramatic. Finally, the adaptamer-mediated PCR fusion methodology is versatile and can be applied to varied genome manipulations....

  15. Whole Genome Analyses of a Well-Differentiated Liposarcoma Reveals Novel SYT1 and DDR2 Rearrangements

    Science.gov (United States)

    Egan, Jan B.; Barrett, Michael T.; Champion, Mia D.; Middha, Sumit; Lenkiewicz, Elizabeth; Evers, Lisa; Francis, Princy; Schmidt, Jessica; Shi, Chang-Xin; Van Wier, Scott; Badar, Sandra; Ahmann, Gregory; Kortuem, K. Martin; Boczek, Nicole J.; Fonseca, Rafael; Craig, David W.; Carpten, John D.; Borad, Mitesh J.; Stewart, A. Keith

    2014-01-01

    Liposarcoma is the most common soft tissue sarcoma, but little is known about the genomic basis of this disease. Given the low cell content of this tumor type, we utilized flow cytometry to isolate the diploid normal and aneuploid tumor populations from a well-differentiated liposarcoma prior to array comparative genomic hybridization and whole genome sequencing. This work revealed massive highly focal amplifications throughout the aneuploid tumor genome including MDM2, a gene that has previously been found to be amplified in well-differentiated liposarcoma. Structural analysis revealed massive rearrangement of chromosome 12 and 11 gene fusions, some of which may be part of double minute chromosomes commonly present in well-differentiated liposarcoma. We identified a hotspot of genomic instability localized to a region of chromosome 12 that includes a highly conserved, putative L1 retrotransposon element, LOC100507498 which resides within a gene cluster (NAV3, SYT1, PAWR) where 6 of the 11 fusion events occurred. Interestingly, a potential gene fusion was also identified in amplified DDR2, which is a potential therapeutic target of kinase inhibitors such as dastinib, that are not routinely used in the treatment of patients with liposarcoma. Furthermore, 7 somatic, damaging single nucleotide variants have also been identified, including D125N in the PTPRQ protein. In conclusion, this work is the first to report the entire genome of a well-differentiated liposarcoma with novel chromosomal rearrangements associated with amplification of therapeutically targetable genes such as MDM2 and DDR2. PMID:24505276

  16. Whole genome analyses of a well-differentiated liposarcoma reveals novel SYT1 and DDR2 rearrangements.

    Directory of Open Access Journals (Sweden)

    Jan B Egan

    Full Text Available Liposarcoma is the most common soft tissue sarcoma, but little is known about the genomic basis of this disease. Given the low cell content of this tumor type, we utilized flow cytometry to isolate the diploid normal and aneuploid tumor populations from a well-differentiated liposarcoma prior to array comparative genomic hybridization and whole genome sequencing. This work revealed massive highly focal amplifications throughout the aneuploid tumor genome including MDM2, a gene that has previously been found to be amplified in well-differentiated liposarcoma. Structural analysis revealed massive rearrangement of chromosome 12 and 11 gene fusions, some of which may be part of double minute chromosomes commonly present in well-differentiated liposarcoma. We identified a hotspot of genomic instability localized to a region of chromosome 12 that includes a highly conserved, putative L1 retrotransposon element, LOC100507498 which resides within a gene cluster (NAV3, SYT1, PAWR where 6 of the 11 fusion events occurred. Interestingly, a potential gene fusion was also identified in amplified DDR2, which is a potential therapeutic target of kinase inhibitors such as dastinib, that are not routinely used in the treatment of patients with liposarcoma. Furthermore, 7 somatic, damaging single nucleotide variants have also been identified, including D125N in the PTPRQ protein. In conclusion, this work is the first to report the entire genome of a well-differentiated liposarcoma with novel chromosomal rearrangements associated with amplification of therapeutically targetable genes such as MDM2 and DDR2.

  17. Centrosome clustering and cyclin D1 gene amplification in double minutes are common events in chromosomal unstable bladder tumors

    International Nuclear Information System (INIS)

    Rey, Javier del; Prat, Esther; Ponsa, Immaculada; Lloreta, Josep; Gelabert, Antoni; Algaba, Ferran; Camps, Jordi; Miró, Rosa

    2010-01-01

    Aneuploidy, centrosome abnormalities and gene amplification are hallmarks of chromosome instability (CIN) in cancer. Yet there are no studies of the in vivo behavior of these phenomena within the same bladder tumor. Twenty-one paraffin-embedded bladder tumors were analyzed by conventional comparative genome hybridization and fluorescence in situ hybridization (FISH) with a cyclin D1 gene (CCND1)/centromere 11 dual-color probe. Immunofluorescent staining of α, β and γ tubulin was also performed. Based on the CIN index, defined as the percentage of cells not displaying the modal number for chromosome 11, tumors were classified as CIN-negative and CIN-positive. Fourteen out of 21 tumors were considered CIN-positive. All T1G3 tumors were included in the CIN-positive group whereas the majority of Ta samples were classified as CIN-negative tumors. Centrosome clustering was observed in six out of 12 CIN-positive tumors analyzed. CCND1 amplification in homogeneously staining regions was present in six out of 14 CIN-positive tumors; three of them also showed amplification of this gene in double minutes. Complex in vivo behavior of CCND1 amplicon in bladder tumor cells has been demonstrated by accurate FISH analysis on paraffin-embedded tumors. Positive correlation between high heterogeneity, centrosome abnormalities and CCND1 amplification was found in T1G3 bladder carcinomas. This is the first study to provide insights into the coexistence of CCND1 amplification in homogeneously staining regions and double minutes in primary bladder tumors. It is noteworthy that those patients whose tumors showed double minutes had a significantly shorter overall survival rate (p < 0.001)

  18. Involvement of Disperse Repetitive Sequences in Wheat/Rye Genome Adjustment

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    Manuela Silva

    2012-07-01

    Full Text Available The union of different genomes in the same nucleus frequently results in hybrid genotypes with improved genome plasticity related to both genome remodeling events and changes in gene expression. Most modern cereal crops are polyploid species. Triticale, synthesized by the cross between wheat and rye, constitutes an excellent model to study polyploidization functional implications. We intend to attain a deeper knowledge of dispersed repetitive sequence involvement in parental genome reshuffle in triticale and in wheat-rye addition lines that have the entire wheat genome plus each rye chromosome pair. Through Random Amplified Polymorphic DNA (RAPD analysis with OPH20 10-mer primer we unraveled clear alterations corresponding to the loss of specific bands from both parental genomes. Moreover, the sequential nature of those events was revealed by the increased absence of rye-origin bands in wheat-rye addition lines in comparison with triticale. Remodeled band sequencing revealed that both repetitive and coding genome domains are affected in wheat-rye hybrid genotypes. Additionally, the amplification and sequencing of pSc20H internal segments showed that the disappearance of parental bands may result from restricted sequence alterations and unraveled the involvement of wheat/rye related repetitive sequences in genome adjustment needed for hybrid plant stabilization.

  19. GMO detection using a bioluminescent real time reporter (BART of loop mediated isothermal amplification (LAMP suitable for field use

    Directory of Open Access Journals (Sweden)

    Kiddle Guy

    2012-04-01

    Full Text Available Abstract Background There is an increasing need for quantitative technologies suitable for molecular detection in a variety of settings for applications including food traceability and monitoring of genetically modified (GM crops and their products through the food processing chain. Conventional molecular diagnostics utilising real-time polymerase chain reaction (RT-PCR and fluorescence-based determination of amplification require temperature cycling and relatively complex optics. In contrast, isothermal amplification coupled to a bioluminescent output produced in real-time (BART occurs at a constant temperature and only requires a simple light detection and integration device. Results Loop mediated isothermal amplification (LAMP shows robustness to sample-derived inhibitors. Here we show the applicability of coupled LAMP and BART reactions (LAMP-BART for determination of genetically modified (GM maize target DNA at low levels of contamination (0.1-5.0% GM using certified reference material, and compare this to RT-PCR. Results show that conventional DNA extraction methods developed for PCR may not be optimal for LAMP-BART quantification. Additionally, we demonstrate that LAMP is more tolerant to plant sample-derived inhibitors, and show this can be exploited to develop rapid extraction techniques suitable for simple field-based qualitative tests for GM status determination. We also assess the effect of total DNA assay load on LAMP-BART quantitation. Conclusions LAMP-BART is an effective and sensitive technique for GM detection with significant potential for quantification even at low levels of contamination and in samples derived from crops such as maize with a large genome size. The resilience of LAMP-BART to acidic polysaccharides makes it well suited to rapid sample preparation techniques and hence to both high throughput laboratory settings and to portable GM detection applications. The impact of the plant sample matrix and genome loading

  20. Twin target self-amplification-based DNA machine for highly sensitive detection of cancer-related gene.

    Science.gov (United States)

    Xu, Huo; Jiang, Yifan; Liu, Dengyou; Liu, Kai; Zhang, Yafeng; Yu, Suhong; Shen, Zhifa; Wu, Zai-Sheng

    2018-06-29

    The sensitive detection of cancer-related genes is of great significance for early diagnosis and treatment of human cancers, and previous isothermal amplification sensing systems were often based on the reuse of target DNA, the amplification of enzymatic products and the accumulation of reporting probes. However, no reporting probes are able to be transformed into target species and in turn initiate the signal of other probes. Herein we reported a simple, isothermal and highly sensitive homogeneous assay system for tumor suppressor p53 gene detection based on a new autonomous DNA machine, where the signaling probe, molecular beacon (MB), was able to execute the function similar to target DNA besides providing the common signal. In the presence of target p53 gene, the operation of DNA machine can be initiated, and cyclical nucleic acid strand-displacement polymerization (CNDP) and nicking/polymerization cyclical amplification (NPCA) occur, during which the MB was opened by target species and cleaved by restriction endonuclease. In turn, the cleaved fragments could activate the next signaling process as target DNA did. According to the functional similarity, the cleaved fragment was called twin target, and the corresponding fashion to amplify the signal was named twin target self-amplification. Utilizing this newly-proposed DNA machine, the target DNA could be detected down to 0.1 pM with a wide dynamic range (6 orders of magnitude) and single-base mismatched targets were discriminated, indicating a very high assay sensitivity and good specificity. In addition, the DNA machine was not only used to screen the p53 gene in complex biological matrix but also was capable of practically detecting genomic DNA p53 extracted from A549 cell line. This indicates that the proposed DNA machine holds the potential application in biomedical research and early clinical diagnosis. Copyright © 2018 Elsevier B.V. All rights reserved.

  1. Real-Time Fluorescence Loop Mediated Isothermal Amplification for the Detection of Acinetobacter baumannii

    Science.gov (United States)

    Wang, Qinqin; Zhou, Yanbin; Li, Shaoli; Zhuo, Chao; Xu, Siqi; Huang, Lixia; Yang, Ling; Liao, Kang

    2013-01-01

    Background Detection of Acinetobacter baumannii has been relying primarily on bacterial culture that often fails to return useful results in time. Although DNA-based assays are more sensitive than bacterial culture in detecting the pathogen, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. In addition, these molecular tools require expensive laboratory instruments. Therefore, establishing molecular tools for field use require simpler molecular platforms. The loop-mediated isothermal amplification method is relatively simple and can be improved for better use in a routine clinical bacteriology laboratory. A simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in the same platform has been developed in recent years. This method is referred to as real-time loop-mediated isothermal amplification. In this study, we attempted to utilize this method for rapid detection of A. baumannii. Methodology and Significant Findings Species-specific primers were designed to test the utility of this method. Clinical samples of A. baumannii were used to determine the sensitivity and specificity of this system compared to bacterial culture and a polymerase chain reaction method. All positive samples isolated from sputum were confirmed to be the species of Acinetobacter by 16S rRNA gene sequencing. The RealAmp method was found to be simpler and allowed real-time detection of DNA amplification, and could distinguish A. baumannii from Acinetobacter calcoaceticus and Acinetobacter genomic species 3. DNA was extracted by simple boiling method. Compared to bacterial culture, the sensitivity and specificity of RealAmp in detecting A. baumannii was 98.9% and 75.0%, respectively. Conclusion The RealAmp assay only requires a single unit, and the assay positivity can be verified by visual inspection. Therefore, this assay has

  2. Targeting helicase-dependent amplification products with an electrochemical genosensor for reliable and sensitive screening of genetically modified organisms.

    Science.gov (United States)

    Moura-Melo, Suely; Miranda-Castro, Rebeca; de-Los-Santos-Álvarez, Noemí; Miranda-Ordieres, Arturo J; Dos Santos Junior, J Ribeiro; da Silva Fonseca, Rosana A; Lobo-Castañón, Maria Jesús

    2015-08-18

    Cultivation of genetically modified organisms (GMOs) and their use in food and feed is constantly expanding; thus, the question of informing consumers about their presence in food has proven of significant interest. The development of sensitive, rapid, robust, and reliable methods for the detection of GMOs is crucial for proper food labeling. In response, we have experimentally characterized the helicase-dependent isothermal amplification (HDA) and sequence-specific detection of a transgene from the Cauliflower Mosaic Virus 35S Promoter (CaMV35S), inserted into most transgenic plants. HDA is one of the simplest approaches for DNA amplification, emulating the bacterial replication machinery, and resembling PCR but under isothermal conditions. However, it usually suffers from a lack of selectivity, which is due to the accumulation of spurious amplification products. To improve the selectivity of HDA, which makes the detection of amplification products more reliable, we have developed an electrochemical platform targeting the central sequence of HDA copies of the transgene. A binary monolayer architecture is built onto a thin gold film where, upon the formation of perfect nucleic acid duplexes with the amplification products, these are enzyme-labeled and electrochemically transduced. The resulting combined system increases genosensor detectability up to 10(6)-fold, allowing Yes/No detection of GMOs with a limit of detection of ∼30 copies of the CaMV35S genomic DNA. A set of general utility rules in the design of genosensors for detection of HDA amplicons, which may assist in the development of point-of-care tests, is also included. The method provides a versatile tool for detecting nucleic acids with extremely low abundance not only for food safety control but also in the diagnostics and environmental control areas.

  3. Amplification of 9q34 in childhood adrenocortical tumors: a specific feature unrelated to ethnic origin or living conditions

    Directory of Open Access Journals (Sweden)

    Figueiredo B.C.

    2000-01-01

    Full Text Available Adrenocortical tumors (ACT in children under 15 years of age exhibit some clinical and biological features distinct from ACT in adults. Cell proliferation, hypertrophy and cell death in adrenal cortex during the last months of gestation and the immediate postnatal period seem to be critical for the origin of ACT in children. Studies with large numbers of patients with childhood ACT have indicated a median age at diagnosis of about 4 years. In our institution, the median age was 3 years and 5 months, while the median age for first signs and symptoms was 2 years and 5 months (N = 72. Using the comparative genomic hybridization technique, we have reported a high frequency of 9q34 amplification in adenomas and carcinomas. This finding has been confirmed more recently by investigators in England. The lower socioeconomic status, the distinctive ethnic groups and all the regional differences in Southern Brazil in relation to patients in England indicate that these differences are not important to determine 9q34 amplification. Candidate amplified genes mapped to this locus are currently being investigated and Southern blot results obtained so far have discarded amplification of the abl oncogene. Amplification of 9q34 has not been found to be related to tumor size, staging, or malignant histopathological features, nor does it seem to be responsible for the higher incidence of ACT observed in Southern Brazil, but could be related to an ACT from embryonic origin.

  4. Real-time sequence-validated loop-mediated isothermal amplification assays for detection of Middle East respiratory syndrome coronavirus (MERS-CoV.

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    Sanchita Bhadra

    Full Text Available The Middle East respiratory syndrome coronavirus (MERS-CoV, an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported infections we have developed asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF1a and ORF1b genes and upstream of the E gene. Additionally we applied one-step strand displacement probes (OSD for real-time sequence-specific verification of LAMP amplicons. Asymmetric amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU (5 to 50 PFU/ml of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens.

  5. Chromosomal amplifications, 3q gain and deletions of 2q33-q37 are the frequent genetic changes in cervical carcinoma

    International Nuclear Information System (INIS)

    Rao, Pulivarthi H; Murty, Vundavalli VVS; Arias-Pulido, Hugo; Lu, Xin-Yan; Harris, Charles P; Vargas, Hernan; Zhang, Fang F; Narayan, Gopeshwar; Schneider, Achim; Terry, Mary Beth

    2004-01-01

    Carcinoma of uterine cervix is the second most common cancers among women worldwide. Combined radiation and chemotherapy is the choice of treatment for advanced stages of the disease. The prognosis is poor, with a five-year survival rate ranging from about 20–65%, depending on stage of the disease. Therefore, genetic characterization is essential for understanding the biology and clinical heterogeneity in cervical cancer (CC). We used a genome-wide screening method – comparative genomic hybridization (CGH) to identify DNA copy number changes in 77 patients with cervical cancer. We applied categorical and survival analyses to analyze whether chromosomal changes were related to clinico-pathologic characteristics and patients survival. The CGH analysis revealed a loss of 2q33-q37 (57.1%), gain of 3q (54.5%) and chromosomal amplifications (20.77%) as frequent genetic changes. A total of 15 amplified chromosomal sites were detected in 16 cases that include 1p31, 2q32, 7q22, 8q21.2-q24, 9p22, 10q21, 10q24, 11q13, 11q21, 12q15, 14q12, 17p11.2, 17q22, 18p11.2, and 19q13.1. Recurrent amplified sites were noted at 11q13, 11q21, and 19q13.1. The genomic alterations were further evaluated for prognostic significance in CC patients, and we did not find any correlation with a number of clinical or histological parameters. The tumors harboring HPV18 exhibited higher genomic instability compared to tumors with HPV 16. This study demonstrated that 2q33-q37 deletions, 3q gains and chromosomal amplifications as characteristic changes in invasive CC. These genetic alterations will aid in the identification of novel tumor suppressor gene(s) at 2q33-q37 and oncogenes at amplified chromosomal sites. Molecular characterization of these chromosomal changes utilizing the current genomic technologies will provide new insights into the biology and clinical behavior of CC

  6. The B73 maize genome: complexity, diversity, and dynamics.

    Science.gov (United States)

    Schnable, Patrick S; Ware, Doreen; Fulton, Robert S; Stein, Joshua C; Wei, Fusheng; Pasternak, Shiran; Liang, Chengzhi; Zhang, Jianwei; Fulton, Lucinda; Graves, Tina A; Minx, Patrick; Reily, Amy Denise; Courtney, Laura; Kruchowski, Scott S; Tomlinson, Chad; Strong, Cindy; Delehaunty, Kim; Fronick, Catrina; Courtney, Bill; Rock, Susan M; Belter, Eddie; Du, Feiyu; Kim, Kyung; Abbott, Rachel M; Cotton, Marc; Levy, Andy; Marchetto, Pamela; Ochoa, Kerri; Jackson, Stephanie M; Gillam, Barbara; Chen, Weizu; Yan, Le; Higginbotham, Jamey; Cardenas, Marco; Waligorski, Jason; Applebaum, Elizabeth; Phelps, Lindsey; Falcone, Jason; Kanchi, Krishna; Thane, Thynn; Scimone, Adam; Thane, Nay; Henke, Jessica; Wang, Tom; Ruppert, Jessica; Shah, Neha; Rotter, Kelsi; Hodges, Jennifer; Ingenthron, Elizabeth; Cordes, Matt; Kohlberg, Sara; Sgro, Jennifer; Delgado, Brandon; Mead, Kelly; Chinwalla, Asif; Leonard, Shawn; Crouse, Kevin; Collura, Kristi; Kudrna, Dave; Currie, Jennifer; He, Ruifeng; Angelova, Angelina; Rajasekar, Shanmugam; Mueller, Teri; Lomeli, Rene; Scara, Gabriel; Ko, Ara; Delaney, Krista; Wissotski, Marina; Lopez, Georgina; Campos, David; Braidotti, Michele; Ashley, Elizabeth; Golser, Wolfgang; Kim, HyeRan; Lee, Seunghee; Lin, Jinke; Dujmic, Zeljko; Kim, Woojin; Talag, Jayson; Zuccolo, Andrea; Fan, Chuanzhu; Sebastian, Aswathy; Kramer, Melissa; Spiegel, Lori; Nascimento, Lidia; Zutavern, Theresa; Miller, Beth; Ambroise, Claude; Muller, Stephanie; Spooner, Will; Narechania, Apurva; Ren, Liya; Wei, Sharon; Kumari, Sunita; Faga, Ben; Levy, Michael J; McMahan, Linda; Van Buren, Peter; Vaughn, Matthew W; Ying, Kai; Yeh, Cheng-Ting; Emrich, Scott J; Jia, Yi; Kalyanaraman, Ananth; Hsia, An-Ping; Barbazuk, W Brad; Baucom, Regina S; Brutnell, Thomas P; Carpita, Nicholas C; Chaparro, Cristian; Chia, Jer-Ming; Deragon, Jean-Marc; Estill, James C; Fu, Yan; Jeddeloh, Jeffrey A; Han, Yujun; Lee, Hyeran; Li, Pinghua; Lisch, Damon R; Liu, Sanzhen; Liu, Zhijie; Nagel, Dawn Holligan; McCann, Maureen C; SanMiguel, Phillip; Myers, Alan M; Nettleton, Dan; Nguyen, John; Penning, Bryan W; Ponnala, Lalit; Schneider, Kevin L; Schwartz, David C; Sharma, Anupma; Soderlund, Carol; Springer, Nathan M; Sun, Qi; Wang, Hao; Waterman, Michael; Westerman, Richard; Wolfgruber, Thomas K; Yang, Lixing; Yu, Yeisoo; Zhang, Lifang; Zhou, Shiguo; Zhu, Qihui; Bennetzen, Jeffrey L; Dawe, R Kelly; Jiang, Jiming; Jiang, Ning; Presting, Gernot G; Wessler, Susan R; Aluru, Srinivas; Martienssen, Robert A; Clifton, Sandra W; McCombie, W Richard; Wing, Rod A; Wilson, Richard K

    2009-11-20

    We report an improved draft nucleotide sequence of the 2.3-gigabase genome of maize, an important crop plant and model for biological research. Over 32,000 genes were predicted, of which 99.8% were placed on reference chromosomes. Nearly 85% of the genome is composed of hundreds of families of transposable elements, dispersed nonuniformly across the genome. These were responsible for the capture and amplification of numerous gene fragments and affect the composition, sizes, and positions of centromeres. We also report on the correlation of methylation-poor regions with Mu transposon insertions and recombination, and copy number variants with insertions and/or deletions, as well as how uneven gene losses between duplicated regions were involved in returning an ancient allotetraploid to a genetically diploid state. These analyses inform and set the stage for further investigations to improve our understanding of the domestication and agricultural improvements of maize.

  7. Realization of the conceptual ideal for x-ray amplification

    Energy Technology Data Exchange (ETDEWEB)

    Borisov, Alex B; Racz, Ervin; Zhang Ping; McCorkindale, John C; Khan, Shahab F; Poopalasingam, Sankar; Zhao Ji; Rhodes, Charles K [Laboratory for X-Ray Microimaging and Bioinformatics, Department of Physics, University of Illinois at Chicago, Chicago, IL 60607-7059 (United States)

    2008-05-28

    The Xe(L) system is an amplifier with fundamentally different dynamic characteristics from all previously developed laser amplifiers; it represents the conceptual ideal through full utilization of the Kramers-Kronig relations that fundamentally couple the dispersive and absorptive components. The dispersive response of the system, through optimal governance of the power compression, rules the amplification and establishes a minimum gain for the amplifier. Accordingly, the amplification requires a minimum value of the dispersion to be surpassed; the corresponding gain follows automatically. As a leading consequence, since this minimum gain is sufficiently high, the key experimental observation is the uniform presence of saturated amplification signaled by strong spectral hole burning on all transitions exhibiting amplification, including double-vacancy lines. This cardinal signature demonstrates that the amplification is legislated by the saturated gain g{sub s}, not the corresponding small signal value g{sub 0}. The chief outcome is that explosive dispersion yields perforce explosive amplification and the efficient generation of maximally bright coherent power.

  8. An aptasensor for staphylococcus aureus based on nicking enzyme amplification reaction and rolling circle amplification.

    Science.gov (United States)

    Xu, Jingguo; Guo, Jia; Maina, Sarah Wanjiku; Yang, Yumeng; Hu, Yimin; Li, Xuanxuan; Qiu, Jiarong; Xin, Zhihong

    2018-05-15

    An ultra-sensitive aptamer-based biosensor for the detection of staphylococcus aureus was established by adopting the nicking enzyme amplification reaction (NEAR) and the rolling circle amplification (RCA) technologies. Aptamer-probe (AP), containing an aptamer and a probe sequence, was developed to act as the recognition unit of the biosensor, which was specifically bound to S. aureus. The probe was released from AP and initiated into the subsequent DNA amplification reactions where S. aureus was present, converting the detection of S. aureus to the investigation of probe oligonucleotide. The RCA amplification products contained a G-quadruplex motif and formed a three dimensional structure in presence of hemin. The G4/hemin complex showed horseradish peroxidase (HRP)-mimic activity and catalyzed the chemiluminescence reaction of luminol mediated by H 2 O 2 . The results showed that the established biosensor could detect S. aureus specifically with a good linear correlation at 5-10 4  CFU/mL. The signal values based on NEAR-RCA two-step cycle were boosted acutely, much higher than that relied on one-cycle magnification. The limit of detection (LoD) was determined to be as low as 5 CFU/mL. The established aptasensor exhibited a good discrimination of living against dead S. aureus, and can be applied to detect S. aureus in the food industry. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. Identifying genes that extend life span using a high-throughput screening system.

    Science.gov (United States)

    Chen, Cuiying; Contreras, Roland

    2007-01-01

    We developed a high-throughput functional genomic screening system that allows identification of genes prolonging lifespan in the baker's yeast Saccharomyces cerevisiae. The method is based on isolating yeast mother cells with a higher than average number of cell divisions as indicated by the number of bud scars on their surface. Fluorescently labeled wheat germ agglutinin (WGA) was used for specific staining of chitin, a major component of bud scars. The critical new steps in our bud-scar-sorting system are the use of small microbeads, which allows successive rounds of purification and regrowth of the mother cells (M-cell), and utilization of flow cytometry to sort and isolate cells with a longer lifespan based on the number of bud scars specifically labeled with WGA.

  10. Spinal cord projections of the rat main forelimb nerves, studied by transganglionic transport of WGA-HRP and by the disappearance of acid phosphatase.

    Science.gov (United States)

    Castro-Lopes, J M; Coimbra, A

    1991-03-01

    The spinal cord projections of the 3 main forelimb nerves-median, radial and ulnar, were studied in the rat dorsal horn with transganglionic transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP), or using the disappearance of fluoride resistant acid phosphatase (FRAP) after nerve section. The projection patterns in lamina II were similar following the two procedures. The median and the radial nerve fibers projected to the medial and the intermediate thirds, respectively, of the dorsal horn lamina II in spinal cord segments C4-C8. The ulnar nerve projected to segments C6-C8 between the areas occupied by the other two nerves. The FRAP method also showed that the lateral part of lamina II, which was not filled by radial nerve fibers, received the projections from the dorsal cutaneous branches of cervical spinal nerves. In addition, FRAP disappeared from the medial end of segment T1 after skin incisions extending from the medial brachium to the axilla, which seemed due to severance of the cutaneous branchlets of the lateral anterior thoracic nerve. The FRAP procedure is thus sensitive enough to detect fibers in lamina II arising from small peripheral nerves, and may be used as an alternative to the anterograde tracing methods whenever there are no overlapping projections.

  11. Rapid genome detection of Schmallenberg virus and bovine viral diarrhea virus by use of isothermal amplification methods and high-speed real-time reverse transcriptase PCR.

    Science.gov (United States)

    Aebischer, Andrea; Wernike, Kerstin; Hoffmann, Bernd; Beer, Martin

    2014-06-01

    Over the past few years, there has been an increasing demand for rapid and simple diagnostic tools that can be applied outside centralized laboratories by using transportable devices. In veterinary medicine, such mobile test systems would circumvent barriers associated with the transportation of samples and significantly reduce the time to diagnose important infectious animal diseases. Among a wide range of available technologies, high-speed real-time reverse transcriptase quantitative PCR (RT-qPCR) and the two isothermal amplification techniques loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) represent three promising candidates for integration into mobile pen-side tests. The aim of this study was to investigate the performance of these amplification strategies and to evaluate their suitability for field application. In order to enable a valid comparison, novel pathogen-specific assays have been developed for the detection of Schmallenberg virus and bovine viral diarrhea virus. The newly developed assays were evaluated in comparison with established standard RT-qPCR using samples from experimentally or field-infected animals. Even though all assays allowed detection of the target virus in less than 30 min, major differences were revealed concerning sensitivity, specificity, robustness, testing time, and complexity of assay design. These findings indicated that the success of an assay will depend on the integrated amplification technology. Therefore, the application-specific pros and cons of each method that were identified during this study provide very valuable insights for future development and optimization of pen-side tests. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  12. Targeting MET Amplification as a New Oncogenic Driver

    International Nuclear Information System (INIS)

    Kawakami, Hisato; Okamoto, Isamu; Okamoto, Wataru; Tanizaki, Junko; Nakagawa, Kazuhiko; Nishio, Kazuto

    2014-01-01

    Certain genetically defined cancers are dependent on a single overactive oncogene for their proliferation and survival, a phenomenon known as “oncogene addiction”. A new generation of drugs that selectively target such “driver oncogenes” manifests a clinical efficacy greater than that of conventional chemotherapy in appropriate genetically defined patients. MET is a proto-oncogene that encodes a receptor tyrosine kinase, and aberrant activation of MET signaling occurs in a subset of advanced cancers as result of various genetic alterations including gene amplification, polysomy, and gene mutation. Our preclinical studies have shown that inhibition of MET signaling either with the small-molecule MET inhibitor crizotinib or by RNA interference targeted to MET mRNA resulted in marked antitumor effects in cancer cell lines with MET amplification both in vitro and in vivo. Furthermore, patients with non-small cell lung cancer or gastric cancer positive for MET amplification have shown a pronounced clinical response to crizotinib. Accumulating preclinical and clinical evidence thus suggests that MET amplification is an “oncogenic driver” and therefore a valid target for treatment. However, the prevalence of MET amplification has not been fully determined, possibly in part because of the difficulty in evaluating gene amplification. In this review, we provide a rationale for targeting this genetic alteration in cancer therapy

  13. Targeting MET Amplification as a New Oncogenic Driver

    Energy Technology Data Exchange (ETDEWEB)

    Kawakami, Hisato [Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan); Okamoto, Isamu, E-mail: okamotoi@kokyu.med.kyushu-u.ac.jp [Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan); Center for Clinical and Translational Research, Kyushu University Hospital, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582 (Japan); Okamoto, Wataru [Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan); Division of Transrlational Research, Exploratory Oncology Research & Clinical Trial Center, National Cancer Center, 6-5-1 Kashiwanoha, Kashiwa, Chiba 277-8577 (Japan); Tanizaki, Junko [Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan); Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, HIM223, 450 Brookline Avenue, Boston, MA 02215 (United States); Nakagawa, Kazuhiko [Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan); Nishio, Kazuto [Department of Genome Biology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan)

    2014-07-22

    Certain genetically defined cancers are dependent on a single overactive oncogene for their proliferation and survival, a phenomenon known as “oncogene addiction”. A new generation of drugs that selectively target such “driver oncogenes” manifests a clinical efficacy greater than that of conventional chemotherapy in appropriate genetically defined patients. MET is a proto-oncogene that encodes a receptor tyrosine kinase, and aberrant activation of MET signaling occurs in a subset of advanced cancers as result of various genetic alterations including gene amplification, polysomy, and gene mutation. Our preclinical studies have shown that inhibition of MET signaling either with the small-molecule MET inhibitor crizotinib or by RNA interference targeted to MET mRNA resulted in marked antitumor effects in cancer cell lines with MET amplification both in vitro and in vivo. Furthermore, patients with non-small cell lung cancer or gastric cancer positive for MET amplification have shown a pronounced clinical response to crizotinib. Accumulating preclinical and clinical evidence thus suggests that MET amplification is an “oncogenic driver” and therefore a valid target for treatment. However, the prevalence of MET amplification has not been fully determined, possibly in part because of the difficulty in evaluating gene amplification. In this review, we provide a rationale for targeting this genetic alteration in cancer therapy.

  14. biomvRhsmm: Genomic Segmentation with Hidden Semi-Markov Model

    Directory of Open Access Journals (Sweden)

    Yang Du

    2014-01-01

    Full Text Available High-throughput technologies like tiling array and next-generation sequencing (NGS generate continuous homogeneous segments or signal peaks in the genome that represent transcripts and transcript variants (transcript mapping and quantification, regions of deletion and amplification (copy number variation, or regions characterized by particular common features like chromatin state or DNA methylation ratio (epigenetic modifications. However, the volume and output of data produced by these technologies present challenges in analysis. Here, a hidden semi-Markov model (HSMM is implemented and tailored to handle multiple genomic profile, to better facilitate genome annotation by assisting in the detection of transcripts, regulatory regions, and copy number variation by holistic microarray or NGS. With support for various data distributions, instead of limiting itself to one specific application, the proposed hidden semi-Markov model is designed to allow modeling options to accommodate different types of genomic data and to serve as a general segmentation engine. By incorporating genomic positions into the sojourn distribution of HSMM, with optional prior learning using annotation or previous studies, the modeling output is more biologically sensible. The proposed model has been compared with several other state-of-the-art segmentation models through simulation benchmarking, which shows that our efficient implementation achieves comparable or better sensitivity and specificity in genomic segmentation.

  15. The draft genome of the transgenic tropical fruit tree papaya (Carica papaya Linnaeus).

    Science.gov (United States)

    Ming, Ray; Hou, Shaobin; Feng, Yun; Yu, Qingyi; Dionne-Laporte, Alexandre; Saw, Jimmy H; Senin, Pavel; Wang, Wei; Ly, Benjamin V; Lewis, Kanako L T; Salzberg, Steven L; Feng, Lu; Jones, Meghan R; Skelton, Rachel L; Murray, Jan E; Chen, Cuixia; Qian, Wubin; Shen, Junguo; Du, Peng; Eustice, Moriah; Tong, Eric; Tang, Haibao; Lyons, Eric; Paull, Robert E; Michael, Todd P; Wall, Kerr; Rice, Danny W; Albert, Henrik; Wang, Ming-Li; Zhu, Yun J; Schatz, Michael; Nagarajan, Niranjan; Acob, Ricelle A; Guan, Peizhu; Blas, Andrea; Wai, Ching Man; Ackerman, Christine M; Ren, Yan; Liu, Chao; Wang, Jianmei; Wang, Jianping; Na, Jong-Kuk; Shakirov, Eugene V; Haas, Brian; Thimmapuram, Jyothi; Nelson, David; Wang, Xiyin; Bowers, John E; Gschwend, Andrea R; Delcher, Arthur L; Singh, Ratnesh; Suzuki, Jon Y; Tripathi, Savarni; Neupane, Kabi; Wei, Hairong; Irikura, Beth; Paidi, Maya; Jiang, Ning; Zhang, Wenli; Presting, Gernot; Windsor, Aaron; Navajas-Pérez, Rafael; Torres, Manuel J; Feltus, F Alex; Porter, Brad; Li, Yingjun; Burroughs, A Max; Luo, Ming-Cheng; Liu, Lei; Christopher, David A; Mount, Stephen M; Moore, Paul H; Sugimura, Tak; Jiang, Jiming; Schuler, Mary A; Friedman, Vikki; Mitchell-Olds, Thomas; Shippen, Dorothy E; dePamphilis, Claude W; Palmer, Jeffrey D; Freeling, Michael; Paterson, Andrew H; Gonsalves, Dennis; Wang, Lei; Alam, Maqsudul

    2008-04-24

    Papaya, a fruit crop cultivated in tropical and subtropical regions, is known for its nutritional benefits and medicinal applications. Here we report a 3x draft genome sequence of 'SunUp' papaya, the first commercial virus-resistant transgenic fruit tree to be sequenced. The papaya genome is three times the size of the Arabidopsis genome, but contains fewer genes, including significantly fewer disease-resistance gene analogues. Comparison of the five sequenced genomes suggests a minimal angiosperm gene set of 13,311. A lack of recent genome duplication, atypical of other angiosperm genomes sequenced so far, may account for the smaller papaya gene number in most functional groups. Nonetheless, striking amplifications in gene number within particular functional groups suggest roles in the evolution of tree-like habit, deposition and remobilization of starch reserves, attraction of seed dispersal agents, and adaptation to tropical daylengths. Transgenesis at three locations is closely associated with chloroplast insertions into the nuclear genome, and with topoisomerase I recognition sites. Papaya offers numerous advantages as a system for fruit-tree functional genomics, and this draft genome sequence provides the foundation for revealing the basis of Carica's distinguishing morpho-physiological, medicinal and nutritional properties.

  16. Rapid aneuploidy diagnosis by multiplex ligation-dependent probe amplification and array comparative genomic hybridization in pregnancy with major congenital malformations

    Directory of Open Access Journals (Sweden)

    Chih-Ping Chen

    2011-03-01

    Conclusions: Prenatal diagnosis of major congenital malformations should alert one to the possibility of chromosomal abnormalities. Multiplex ligation-dependent probe amplification and aCGH have the advantage of rapid aneuploidy diagnosis of common aneuploidies in cases with major congenital malformations.

  17. Alternative Chemical Amplification Methods for Peroxy Radical Detection

    Science.gov (United States)

    Wood, E. C. D.

    2014-12-01

    Peroxy radicals (HO2, CH3O2, etc.) are commonly detected by the chemical amplification technique, in which ambient air is mixed with high concentrations of CO and NO, initiating a chain reaction that produces 30 - 200 NO2 molecules per sampled peroxy radical. The NO2 is then measured by one of several techniques. With the exception of CIMS-based techniques, the chemical amplification method has undergone only incremental improvements since it was first introduced in 1982. The disadvantages of the technique include the need to use high concentrations of CO and the greatly reduced sensitivity of the amplification chain length in the presence of water vapor. We present a new chemical amplification scheme in which either ethane or acetaldehyde is used in place of CO, with the NO2 product detected using Cavity Attenuated Phase Shift spectroscopy (CAPS). Under dry conditions, the amplification factor of the alternative amplifiers are approximately six times lower than the CO-based amplifier. The relative humidity "penalty" is not as severe, however, such that at typical ambient relative humidity (RH) values the amplification factor is within a factor of three of the CO-based amplifier. Combined with the NO2 sensitivity of CAPS and a dual-channel design, the detection limit of the ethane amplifier is less than 2 ppt (1 minute average, signal-to-noise ratio 2). The advantages of these alternative chemical amplification schemes are improved safety, a reduced RH correction, and increased sensitivity to organic peroxy radicals relative to HO2.

  18. Genomic Analysis of the Chicken Infectious Anemia Virus in a Specific Pathogen-Free Chicken Population in China.

    Science.gov (United States)

    Li, Yang; Wang, Yixin; Fang, Lichun; Fu, Jiayuan; Cui, Shuai; Zhao, Yingjie; Cui, Zhizhong; Chang, Shuang; Zhao, Peng

    2016-01-01

    The antibody to chicken infectious anemia virus (CIAV) was positive in a specific pathogen-free (SPF) chicken population by ELISA test in our previous inspection, indicating a possible infection with CIAV. In this study, blood samples collected from the SPF chickens were used to isolate CIAV by inoculating into MSB1 cells and PCR amplification. A CIAV strain (SD1403) was isolated and successfully identified. Three overlapping genomic fragments were obtained by PCR amplification and sequencing. The full genome sequence of the SD1403 strain was obtained by aligning the sequences. The genome of the SD1403 strain was 2293 bp with a nucleotide identity of 94.8% to 98.5% when compared with 30 referred CIAV strains. The viral proteins VP2 and VP3 were highly conserved, but VP1 was not relatively conserved. Both amino acids 139 and 144 of VP1 were glutamine, which was in accord with the low pathogenic characteristics. In this study, we first reported that CIAV exists in Chinese SPF chicken populations and may be an important reason why attenuated vaccine can be contaminated with CIAV.

  19. Genomic Analysis of the Chicken Infectious Anemia Virus in a Specific Pathogen-Free Chicken Population in China

    Directory of Open Access Journals (Sweden)

    Yang Li

    2016-01-01

    Full Text Available The antibody to chicken infectious anemia virus (CIAV was positive in a specific pathogen-free (SPF chicken population by ELISA test in our previous inspection, indicating a possible infection with CIAV. In this study, blood samples collected from the SPF chickens were used to isolate CIAV by inoculating into MSB1 cells and PCR amplification. A CIAV strain (SD1403 was isolated and successfully identified. Three overlapping genomic fragments were obtained by PCR amplification and sequencing. The full genome sequence of the SD1403 strain was obtained by aligning the sequences. The genome of the SD1403 strain was 2293 bp with a nucleotide identity of 94.8% to 98.5% when compared with 30 referred CIAV strains. The viral proteins VP2 and VP3 were highly conserved, but VP1 was not relatively conserved. Both amino acids 139 and 144 of VP1 were glutamine, which was in accord with the low pathogenic characteristics. In this study, we first reported that CIAV exists in Chinese SPF chicken populations and may be an important reason why attenuated vaccine can be contaminated with CIAV.

  20. Analysis of transposons and repeat composition of the sunflower (Helianthus annuus L.) genome.

    Science.gov (United States)

    Cavallini, Andrea; Natali, Lucia; Zuccolo, Andrea; Giordani, Tommaso; Jurman, Irena; Ferrillo, Veronica; Vitacolonna, Nicola; Sarri, Vania; Cattonaro, Federica; Ceccarelli, Marilena; Cionini, Pier Giorgio; Morgante, Michele

    2010-02-01

    A sample-sequencing strategy combined with slot-blot hybridization and FISH was used to study the composition of the repetitive component of the sunflower genome. One thousand six hundred thirty-eight sequences for a total of 954,517 bp were analyzed. The fraction of sequences that can be classified as repetitive using computational and hybridization approaches amounts to 62% in total. Almost two thirds remain as yet uncharacterized in nature. Of those characterized, most belong to the gypsy superfamily of LTR-retrotransposons. Unlike in other species, where single families can account for large fractions of the genome, it appears that no transposon family has been amplified to very high levels in sunflower. All other known classes of transposable elements were also found. One family of unknown nature (contig 61) was the most repeated in the sunflower genome. The evolution of the repetitive component in the Helianthus genus and in other Asteraceae was studied by comparative analysis of the hybridization of total genomic DNAs from these species to the sunflower small-insert library and compared to gene-based phylogeny. Very little similarity is observed between Helianthus species and two related Asteraceae species outside of the genus. Most repetitive elements are similar in annual and perennial Helianthus species indicating that sequence amplification largely predates such divergence. Gypsy-like elements are more represented in the annuals than in the perennials, while copia-like elements are similarly represented, attesting a different amplification history of the two superfamilies of LTR-retrotransposons in the Helianthus genus.

  1. A Parvovirus B19 synthetic genome: sequence features and functional competence.

    Science.gov (United States)

    Manaresi, Elisabetta; Conti, Ilaria; Bua, Gloria; Bonvicini, Francesca; Gallinella, Giorgio

    2017-08-01

    Central to genetic studies for Parvovirus B19 (B19V) is the availability of genomic clones that may possess functional competence and ability to generate infectious virus. In our study, we established a new model genetic system for Parvovirus B19. A synthetic approach was followed, by design of a reference genome sequence, by generation of a corresponding artificial construct and its molecular cloning in a complete and functional form, and by setup of an efficient strategy to generate infectious virus, via transfection in UT7/EpoS1 cells and amplification in erythroid progenitor cells. The synthetic genome was able to generate virus with biological properties paralleling those of native virus, its infectious activity being dependent on the preservation of self-complementarity and sequence heterogeneity within the terminal regions. A virus of defined genome sequence, obtained from controlled cell culture conditions, can constitute a reference tool for investigation of the structural and functional characteristics of the virus. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Development and characterization of genomic SSR markers for Anneslea fragrans (Pentaphylacaceae).

    Science.gov (United States)

    Sun, Lijing; Meng, Kaikai; Liao, Boyong; Li, Chunmei; Zhang, Yue; Liao, Wenbo; Chen, Sufang

    2017-10-01

    The genus Anneslea (Pentaphylacaceae) contains four species and six varieties, most of which are locally endemic. Here, simple sequence repeat (SSR) markers were developed for the conservation of these species. The genome of A. fragrans was sequenced and de novo assembled into 445,162 contigs, of which 30,409 SSR loci were detected. Primers for 100 SSR loci were validated with PCR amplification in three populations of A. fragrans . Seventy-nine loci successfully amplified, and 30 were polymorphic. The mean number of alleles, observed heterozygosity, and expected heterozygosity were 7.01 ± 1.60, 0.817 ± 0.241, and 0.796 ± 0.145, respectively. Most primers could be amplified in Ternstroemia gymnanthera , T. kwangtungensis , and Cleyera pachyphylla . Our study demonstrated that shotgun genome sequencing is an efficient way to develop genomic SSR markers for nonmodel species. These genomic SSR loci will be valuable in population genetic studies in Anneslea and its relatives.

  3. Accurate quantification of mouse mitochondrial DNA without co-amplification of nuclear mitochondrial insertion sequences.

    Science.gov (United States)

    Malik, Afshan N; Czajka, Anna; Cunningham, Phil

    2016-07-01

    Mitochondria contain an extra-nuclear genome in the form of mitochondrial DNA (MtDNA), damage to which can lead to inflammation and bioenergetic deficit. Changes in MtDNA levels are increasingly used as a biomarker of mitochondrial dysfunction. We previously reported that in humans, fragments in the nuclear genome known as nuclear mitochondrial insertion sequences (NumtS) affect accurate quantification of MtDNA. In the current paper our aim was to determine whether mouse NumtS affect the quantification of MtDNA and to establish a method designed to avoid this. The existence of NumtS in the mouse genome was confirmed using blast N, unique MtDNA regions were identified using FASTA, and MtDNA primers which do not co-amplify NumtS were designed and tested. MtDNA copy numbers were determined in a range of mouse tissues as the ratio of the mitochondrial and nuclear genome using real time qPCR and absolute quantification. Approximately 95% of mouse MtDNA was duplicated in the nuclear genome as NumtS which were located in 15 out of 21 chromosomes. A unique region was identified and primers flanking this region were used. MtDNA levels differed significantly in mouse tissues being the highest in the heart, with levels in descending order (highest to lowest) in kidney, liver, blood, brain, islets and lung. The presence of NumtS in the nuclear genome of mouse could lead to erroneous data when studying MtDNA content or mutation. The unique primers described here will allow accurate quantification of MtDNA content in mouse models without co-amplification of NumtS. Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

  4. Multiplex, rapid and sensitive isothermal detection of nucleic-acid sequence by endonuclease restriction-mediated real-time multiple cross displacement amplification

    Directory of Open Access Journals (Sweden)

    Yi eWang

    2016-05-01

    Full Text Available We have devised a novel isothermal amplification technology, termed endonuclease restriction-mediated real-time multiple cross displacement amplification (ET-MCDA, which facilitated multiplex, rapid, specific and sensitive detection of nucleic-acid sequences at a constant temperature. The ET-MCDA integrated multiple cross displacement amplification strategy, restriction endonuclease cleavage and real-time fluorescence detection technique. In the ET-MCDA system, the functional cross primer E-CP1 or E-CP2 was constructed by adding a short sequence at the 5’ end of CP1 or CP2, respectively, and the new E-CP1 or E-CP2 primer was labelled at the 5’ end with a fluorophore and in the middle with a dark quencher. The restriction endonuclease Nb.BsrDI specifically recognized the short sequence and digested the newly synthesized double-stranded terminal sequences (5’ end short sequences and their complementary sequences, which released the quenching, resulting on a gain of fluorescence signal. Thus, the ET-MCDA allowed real-time detection of single or multiple targets in only a single reaction, and the positive results were observed in as short as 12 minutes, detecting down to 3.125 fg of genomic DNA per tube. Moreover, the analytical specificity and the practical application of the ET-MCDA were also successfully evaluated in this study. Here we provided the details on the novel ET-MCDA technique and expounded the basic ET-MCDA amplification mechanism.

  5. Multiplex, Rapid, and Sensitive Isothermal Detection of Nucleic-Acid Sequence by Endonuclease Restriction-Mediated Real-Time Multiple Cross Displacement Amplification.

    Science.gov (United States)

    Wang, Yi; Wang, Yan; Zhang, Lu; Liu, Dongxin; Luo, Lijuan; Li, Hua; Cao, Xiaolong; Liu, Kai; Xu, Jianguo; Ye, Changyun

    2016-01-01

    We have devised a novel isothermal amplification technology, termed endonuclease restriction-mediated real-time multiple cross displacement amplification (ET-MCDA), which facilitated multiplex, rapid, specific and sensitive detection of nucleic-acid sequences at a constant temperature. The ET-MCDA integrated multiple cross displacement amplification strategy, restriction endonuclease cleavage and real-time fluorescence detection technique. In the ET-MCDA system, the functional cross primer E-CP1 or E-CP2 was constructed by adding a short sequence at the 5' end of CP1 or CP2, respectively, and the new E-CP1 or E-CP2 primer was labeled at the 5' end with a fluorophore and in the middle with a dark quencher. The restriction endonuclease Nb.BsrDI specifically recognized the short sequence and digested the newly synthesized double-stranded terminal sequences (5' end short sequences and their complementary sequences), which released the quenching, resulting on a gain of fluorescence signal. Thus, the ET-MCDA allowed real-time detection of single or multiple targets in only a single reaction, and the positive results were observed in as short as 12 min, detecting down to 3.125 fg of genomic DNA per tube. Moreover, the analytical specificity and the practical application of the ET-MCDA were also successfully evaluated in this study. Here, we provided the details on the novel ET-MCDA technique and expounded the basic ET-MCDA amplification mechanism.

  6. Amplification of a transcriptionally active DNA sequence in the human brain

    International Nuclear Information System (INIS)

    Yakovlev, A.G.; Sazonov, A.E.; Spunde, A.Ya.; Gindilis, V.M.

    1986-01-01

    The authors present their findings of tissue-specific amplification of a DNA fragment actively transcribed in the human brain. This genome fragment was found in the library complement of cDNA of the human brain and evidently belongs to a new class of moderate repetitions of DNA with an unstable copying capacity in the human genome. The authors isolated total cell RNA from various human tissues (brain, placenta), and rat tissues (brain, liver), by the method of hot phenol extraction with guanidine thiocynate. The poly(A + ) RNA fraction was isolated by chromatography. Synthesis of cDNA was done on a matrix of poly(A + ) RNA of human brain. The cDNA obtained was cloned in plasmid pBR322 for the PstI site using (dC/dG) sequences synthesized on the 3' ends of the vector molecule and cDNA respectively. In cloning 75 ng cDNA, the authors obtained approximately 10 5 recombinant. This library was analyzed by the hybridization method on columns with two radioactive ( 32 P) probes: the total cDNA preparation and the total nuclear DNA from the human brain. The number of copies of the cloned DNA fragment in the genome was determined by dot hybridization. Restricting fragments of human and rat DNA genomes homologous to the cloned cDNA were identified on radio-autographs. In each case, 10 micrograms of EcoRI DNA hydrolyzate was fractionated in 1% agarose gel. The probe was also readied with RNA samples fractionated in agarose gel with formaldehyde and transferred to a nitrocellulose filter under weak vacuum. The filter was hybridized with 0.1 micrograms DNA pAG 02, labeled with ( 32 P) to a specific activity of 0.5-1 x 10 9 counts/min x microgram. The autograph was exposed with amplifying screens at -70 0 C for 2 days

  7. Lidar using the backscatter amplification effect

    Science.gov (United States)

    Razenkov, Igor A.; Banakh, Victor A.

    2018-04-01

    Experimental data proving the possibility of lidar measurement of the refractive turbulence strength based on the effect of backscatter amplification (BSA) are reported. It is shown that the values of the amplification factor correlate with the variance of random jitter of optical image of an incoherent light source depending on the value of the structure constant of the air refractive index turbulent fluctuations averaged over the probing path. This paper presents the results of measurements of the BSA factor in comparison with the simultaneous measurements of the BSA peak, which is very narrow and only occurs on the laser beam axis. It is constructed the range-time images of the derivative of the amplification factor gives a comprehensive picture of the location of turbulent zones and their temporal dynamics.

  8. Novel mutations underlying argininosuccinic aciduria in Saudi Arabia

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    Rashed Mohamed S

    2010-03-01

    Full Text Available Abstract Background Argininosuccinic aciduria (ASAuria is an autosomal recessive disorder of the urea cycle relatively common in Saudi Arabia as a consequence of extensive consanguinity. It is the most common urea cycle disorder identified in the Saudi population, which therefore prioritizes the need to delineate the underlying molecular defects leading to disease. Findings We utilized Whole Genome Amplification (WGA, PCR and direct sequencing to identify mutations underlying ASAuria cases diagnosed by our institution. A missense mutation that accounts for 50% of Saudi ASAuria patients was recently reported by our laboratory. In this study we report a further six novel mutations (and one previously reported found in Saudi patients with ASAuria. The novel four missense, one nonsense and one splice-site mutation were confirmed by their absence in >300 chromosomes from the normal population. Pathogenicity of the novel splice-site mutation was also confirmed using reverse transcriptase-PCR analysis. Cross species amino acid conservation at the substituted residues described were observed in some but not all instances. Conclusions Together, the eight mutations described by our laboratory, encompass >90% of ASAuria patients in Saudi Arabia and add to about 45 other ASAuria mutations reported worldwide.

  9. Molecular identification of Clonorchis sinensis and discrimination with other opisthorchid liver fluke species using multiple Ligation-depended Probe Amplification (MLPA

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    Liang Chi

    2011-06-01

    Full Text Available Abstract Background Infections with the opisthorchid liver flukes Clonorchis sinensis, Opisthorchis viverrini, and O. felineus cause severe health problems globally, particularly in Southeast Asia. Early identification of the infection is essential to provide timely and appropriate chemotherapy to patients. Results In this study we evaluate a PCR-based molecular identification method, Multiplex Ligation-dependent Probe Amplification (MLPA, which allows rapid and specific detection of single nucleotide acid differences between Clonorchis sinensis, Opisthorchis viverrini and O. felineus. Three probe pairs were derived from the Internally Transcribed Spacer 1 (ITS1 of three opisthorchid liver flukes using a systematic phylogenetic analysis. Specific loci were detected in all three species, yielding three amplicons with 198,172 and 152 bp, respectively, while no cross reactions were observed. A panel of 66 C. sinensis isolates was screened using MLPA. All species were positively identified, and no inhibition was observed. The detection limit was 103 copies of the ITS gene for the three liver flukes, or about 60 pg genomic DNA for Clonorchis sinensis. Amplification products can be detected by electrophoresis on agarose gel or in a capillary sequencer. In addition, genomic DNA of Clonorchis sinensis in fecal samples of infected rats was positively amplified by MLPA. Conclusion The flexibility and specificity make MLPA a potential tool for specific identification of infections by opisthorchid liver flukes in endemic areas.

  10. Establishment of recombinase polymerase amplification assay for five hemorrhagic fever-related viruses

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    Xue-feng CAO

    2017-08-01

    Full Text Available Objective To establish a one-step recombinase polymerase amplification (RPA method for pathogen screening and rapid detection in the field targeting for five hemorrhagic fever related viruses (Zaire ebola virus, Sudan ebola virus, Marburg virus, Lassa virus and Yellow fever virus. Methods The specific nucleic acid (NA fragments of each virus were selected as target genes by genome sequence analysis, and the primers and probes for RPA assays were designed according to the sequence. A series of diluted template genes were used for RPA detection to determine the sensitivity. The hemorrhagic fever-related viral nucleic acids were used for RPA detection to determine the specificity. The amplification experiments were carried out at different temperature ranging from 37℃ to 42℃ to validate the reaction temperature range. Results The RPA reaction systems of the five hemorrhagic fever viruses could effectively amplify the target genes, the sensitivities were between 1.5×102 and 1.5×103 copies. No cross reactions existed with the other hemorrhagic fever-related viral genes. Meanwhile, RPA assay could effectively amplify the target genes at 37-42℃. Conclusion The isothermal RPA assays of five hemorrhagic fever viruses are established, which may amply target genes fast and react at a wide temperature range, and be potentially useful for in field pathogens detection. DOI: 10.11855/j.issn.0577-7402.2017.06.09

  11. Spheromak Impedance and Current Amplification

    International Nuclear Information System (INIS)

    Fowler, T K; Hua, D D; Stallard, B W

    2002-01-01

    It is shown that high current amplification can be achieved only by injecting helicity on the timescale for reconnection, τ REC , which determines the effective impedance of the spheromak. An approximate equation for current amplification is: dI TOR 2 /dt ∼ I 2 /τ REC - I TOR 2 /τ closed where I is the gun current, I TOR is the spheromak toroidal current and τ CLOSED is the ohmic decay time of the spheromak. Achieving high current amplification, I TOR >> I, requires τ REC CLOSED . For resistive reconnection, this requires reconnection in a cold zone feeding helicity into a hot zone. Here we propose an impedance model based on these ideas in a form that can be implemented in the Corsica-based helicity transport code. The most important feature of the model is the possibility that τ REC actually increases as the spheromak temperature increases, perhaps accounting for the ''voltage sag'' observed in some experiments, and a tendency toward a constant ratio of field to current, B ∝ I, or I TOR ∼ I. Program implications are discussed

  12. Direct amplification of casework bloodstains using the Promega PowerPlex(®) 21 PCR amplification system.

    Science.gov (United States)

    Gray, Kerryn; Crowle, Damian; Scott, Pam

    2014-09-01

    A significant number of evidence items submitted to Forensic Science Service Tasmania (FSST) are blood swabs or bloodstained items. Samples from these items routinely undergo phenol:chloroform:isoamyl alcohol organic extraction and quantitative Polymerase Chain Reaction (qPCR) testing prior to PowerPlex(®) 21 amplification. This multi-step process has significant cost and timeframe implications in a fiscal climate of tightening government budgets, pressure towards improved operating efficiencies, and an increasing emphasis on rapid techniques better supporting intelligence-led policing. Direct amplification of blood and buccal cells on cloth and Whatman FTA™ card with PowerPlex(®) 21 has already been successfully implemented for reference samples, eliminating the requirement for sample pre-treatment. Scope for expanding this method to include less pristine casework blood swabs and samples from bloodstained items was explored in an endeavour to eliminate lengthy DNA extraction, purification and qPCR steps for a wider subset of samples. Blood was deposited onto a range of substrates including those historically found to inhibit STR amplification. Samples were collected with micro-punch, micro-swab, or both. The potential for further fiscal savings via reduced volume amplifications was assessed by amplifying all samples at full and reduced volume (25 and 13μL). Overall success rate data showed 80% of samples yielded a complete profile at reduced volume, compared to 78% at full volume. Particularly high success rates were observed for the blood on fabric/textile category with 100% of micro-punch samples yielding complete profiles at reduced volume and 85% at full volume. Following the success of this trial, direct amplification of suitable casework blood samples has been implemented at reduced volume. Significant benefits have been experienced, most noticeably where results from crucial items have been provided to police investigators prior to interview of

  13. Genomic Alterations in Primary Gastric Adenocarcinomas Correlate with Clinicopathological Characteristics and Survival

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    Marjan M. Weiss

    2004-01-01

    Full Text Available Background & aims: Pathogenesis of gastric cancer is driven by an accumulation of genetic changes that to a large extent occur at the chromosomal level. In order to investigate the patterns of chromosomal aberrations in gastric carcinomas, we performed genome‐wide microarray based comparative genomic hybridisation (microarray CGH. With this recently developed technique chromosomal aberrations can be studied with high resolution and sensitivity. Methods: Array CGH was applied to a series of 35 gastric adenocarcinomas using a genome‐wide scanning array with 2275 BAC and P1 clones spotted in triplicate. Each clone contains at least one STS for linkage to the sequence of the human genome. These arrays provide an average resolution of 1.4 Mb across the genome. DNA copy number changes were correlated with clinicopathological tumour characteristics as well as survival. Results: All thirty‐five cancers showed chromosomal aberrations and 16 of the 35 tumours showed one or more amplifications. The most frequent aberrations are gains of 8q24.2, 8q24.1, 20q13.12, 20q13.2, 7p11.2, 1q32.3, 8p23.1–p23.3, losses of 5q14.1, 18q22.1, 19p13.12–p13.3, 9p21.3–p24.3, 17p13.1–p13.3, 13q31.1, 16q22.1, 21q21.3, and amplifications of 7q21–q22, and 12q14.1–q21.1. These aberrations were correlated to clinicopathological characteristics and survival. Gain of 1q32.3 was significantly correlated with lymph node status (p=0.007. Tumours with loss of 18q22.1, as well as tumours with amplifications were associated with poor survival (p=0.02, both. Conclusions: Microarray CGH has revealed several chromosomal regions that have not been described before in gastric cancer at this frequency and resolution, such as amplification of at 7q21–q22 and 12q14.1–q21.1, as well gains at 1q32.3, 7p11.2, and losses at 13q13.1. Interestingly, gain of 1q32.3 and loss of 18q22.1 are associated with a bad prognosis indicating that these regions could harbour gene(s that may

  14. Sequencing and annotation of mitochondrial genomes from individual parasitic helminths.

    Science.gov (United States)

    Jex, Aaron R; Littlewood, D Timothy; Gasser, Robin B

    2015-01-01

    Mitochondrial (mt) genomics has significant implications in a range of fundamental areas of parasitology, including evolution, systematics, and population genetics as well as explorations of mt biochemistry, physiology, and function. Mt genomes also provide a rich source of markers to aid molecular epidemiological and ecological studies of key parasites. However, there is still a paucity of information on mt genomes for many metazoan organisms, particularly parasitic helminths, which has often related to challenges linked to sequencing from tiny amounts of material. The advent of next-generation sequencing (NGS) technologies has paved the way for low cost, high-throughput mt genomic research, but there have been obstacles, particularly in relation to post-sequencing assembly and analyses of large datasets. In this chapter, we describe protocols for the efficient amplification and sequencing of mt genomes from small portions of individual helminths, and highlight the utility of NGS platforms to expedite mt genomics. In addition, we recommend approaches for manual or semi-automated bioinformatic annotation and analyses to overcome the bioinformatic "bottleneck" to research in this area. Taken together, these approaches have demonstrated applicability to a range of parasites and provide prospects for using complete mt genomic sequence datasets for large-scale molecular systematic and epidemiological studies. In addition, these methods have broader utility and might be readily adapted to a range of other medium-sized molecular regions (i.e., 10-100 kb), including large genomic operons, and other organellar (e.g., plastid) and viral genomes.

  15. Added Value of HER-2 Amplification Testing by Multiplex Ligation-Dependent Probe Amplification in Invasive Breast Cancer

    Science.gov (United States)

    Kuijpers, Chantal C. H. J.; Moelans, Cathy B.; van Slooten, Henk-Jan; Horstman, Anja; Hinrichs, John W. J.; Al-Janabi, Shaimaa; van Diest, Paul J.; Jiwa, Mehdi

    2013-01-01

    Background HER-2 is a prognostic and predictive marker, but as yet no technique is perfectly able to identify patients likely to benefit from HER-2 targeted therapies. We aimed to prospectively assess the added value of first-line co-testing by IHC, and multiplex ligation-dependent probe amplification (MLPA) and chromogenic in situ hybridization (CISH). Methods As local validation, HER-2 MLPA and CISH were compared in 99 breast cancers. Next, we reviewed 937 invasive breast cancers, from 4 Dutch pathology laboratories, that were prospectively assessed for HER-2 by IHC and MLPA (and CISH in selected cases). Results The validation study demonstrated 100% concordance between CISH and MLPA, if both methods were assessable and conclusive (81.8% of cases). Significant variation regarding percentages IHC 0/1+ and 2+ cases was observed between the laboratories (pCISH was 98.1% (575/586) (Kappa = 0.94). Of the IHC 3+ cases, 6.7% failed to reveal gene amplification, whereas 0.8% of the IHC 0/1+ cases demonstrated gene amplification. Results remained discordant after retrospective review in 3/11 discordant cases. In the remaining 8 cases the original IHC score was incorrect or adapted after repeated IHC staining. Conclusions MLPA is a low-cost and quantitative high-throughput technique with near perfect concordance with CISH. The use of MLPA in routinely co-testing all breast cancers may reduce HER-2 testing variation between laboratories, may serve as quality control for IHC, will reveal IHC 0/1+ patients with gene amplification, likely responsive to trastuzumab, and identify IHC 3+ cases without gene amplification that may respond less well. PMID:24324739

  16. Added value of HER-2 amplification testing by multiplex ligation-dependent probe amplification in invasive breast cancer.

    Directory of Open Access Journals (Sweden)

    Chantal C H J Kuijpers

    Full Text Available BACKGROUND: HER-2 is a prognostic and predictive marker, but as yet no technique is perfectly able to identify patients likely to benefit from HER-2 targeted therapies. We aimed to prospectively assess the added value of first-line co-testing by IHC, and multiplex ligation-dependent probe amplification (MLPA and chromogenic in situ hybridization (CISH. METHODS: As local validation, HER-2 MLPA and CISH were compared in 99 breast cancers. Next, we reviewed 937 invasive breast cancers, from 4 Dutch pathology laboratories, that were prospectively assessed for HER-2 by IHC and MLPA (and CISH in selected cases. RESULTS: The validation study demonstrated 100% concordance between CISH and MLPA, if both methods were assessable and conclusive (81.8% of cases. Significant variation regarding percentages IHC 0/1+ and 2+ cases was observed between the laboratories (p<0.0001. Overall concordance between IHC and MLPA/CISH was 98.1% (575/586 (Kappa = 0.94. Of the IHC 3+ cases, 6.7% failed to reveal gene amplification, whereas 0.8% of the IHC 0/1+ cases demonstrated gene amplification. Results remained discordant after retrospective review in 3/11 discordant cases. In the remaining 8 cases the original IHC score was incorrect or adapted after repeated IHC staining. CONCLUSIONS: MLPA is a low-cost and quantitative high-throughput technique with near perfect concordance with CISH. The use of MLPA in routinely co-testing all breast cancers may reduce HER-2 testing variation between laboratories, may serve as quality control for IHC, will reveal IHC 0/1+ patients with gene amplification, likely responsive to trastuzumab, and identify IHC 3+ cases without gene amplification that may respond less well.

  17. Strand Invasion Based Amplification (SIBA®): a novel isothermal DNA amplification technology demonstrating high specificity and sensitivity for a single molecule of target analyte.

    Science.gov (United States)

    Hoser, Mark J; Mansukoski, Hannu K; Morrical, Scott W; Eboigbodin, Kevin E

    2014-01-01

    Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR) in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA). SIBA technology is resistant to non-specific amplification, is able to detect a single molecule of target analyte, and does not require target-specific probes. The technology relies on the recombinase-dependent insertion of an invasion oligonucleotide (IO) into the double-stranded target nucleic acid. The duplex regions peripheral to the IO insertion site dissociate, thereby enabling target-specific primers to bind. A polymerase then extends the primers onto the target nucleic acid leading to exponential amplification of the target. The primers are not substrates for the recombinase and are, therefore unable to extend the target template in the absence of the IO. The inclusion of 2'-O-methyl RNA to the IO ensures that it is not extendible and that it does not take part in the extension of the target template. These characteristics ensure that the technology is resistant to non-specific amplification since primer dimers or mis-priming are unable to exponentially amplify. Consequently, SIBA is highly specific and able to distinguish closely-related species with single molecule sensitivity in the absence of complex probes or sophisticated laboratory equipment. Here, we describe this technology in detail and demonstrate its use for the detection of Salmonella.

  18. Strand Invasion Based Amplification (SIBA®: a novel isothermal DNA amplification technology demonstrating high specificity and sensitivity for a single molecule of target analyte.

    Directory of Open Access Journals (Sweden)

    Mark J Hoser

    Full Text Available Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA. SIBA technology is resistant to non-specific amplification, is able to detect a single molecule of target analyte, and does not require target-specific probes. The technology relies on the recombinase-dependent insertion of an invasion oligonucleotide (IO into the double-stranded target nucleic acid. The duplex regions peripheral to the IO insertion site dissociate, thereby enabling target-specific primers to bind. A polymerase then extends the primers onto the target nucleic acid leading to exponential amplification of the target. The primers are not substrates for the recombinase and are, therefore unable to extend the target template in the absence of the IO. The inclusion of 2'-O-methyl RNA to the IO ensures that it is not extendible and that it does not take part in the extension of the target template. These characteristics ensure that the technology is resistant to non-specific amplification since primer dimers or mis-priming are unable to exponentially amplify. Consequently, SIBA is highly specific and able to distinguish closely-related species with single molecule sensitivity in the absence of complex probes or sophisticated laboratory equipment. Here, we describe this technology in detail and demonstrate its use for the detection of Salmonella.

  19. Magnetic field amplification in interstellar collisionless shock waves

    International Nuclear Information System (INIS)

    Chevalier, R.A.

    1977-01-01

    It is stated that it is commonly assumed that a simple compression of the magnetic field occurs in interstellar shock waves. Recent space observations of the Earth's bow shock have shown that turbulent amplification of the magnetic field can occur in a collisionless shock. It is shown here that radio observations of Tycho's supernova remnant indicate the presence of a shock wave with such magnetic field amplification. There is at present no theory for the microinstabilities that give rise to turbulent amplification of the magnetic field. Despite the lack of theoretical understanding the possibility of field amplification in interstellar shock waves is here considered. In Tycho's supernova remnant there is evidence for the presence of a collisionless shock, and this is discussed. On the basis of observations of the Earth's bow shock, it is expected that turbulent magnetic field amplification occurs in the shock wave of this remnant, and this is supported by radio observations of the remnant. Consideration is given as to what extent the magnetic field is amplified in the shock wave on the basis of the non-thermal radio flux. (U.K.)

  20. Renal Cell Carcinoma With Chromosome 6p Amplification Including the TFEB Gene: A Novel Mechanism of Tumor Pathogenesis?

    Science.gov (United States)

    Williamson, Sean R; Grignon, David J; Cheng, Liang; Favazza, Laura; Gondim, Dibson D; Carskadon, Shannon; Gupta, Nilesh S; Chitale, Dhananjay A; Kalyana-Sundaram, Shanker; Palanisamy, Nallasivam

    2017-03-01

    Amplification of chromosome 6p has been implicated in aggressive behavior in several cancers, but has not been characterized in renal cell carcinoma (RCC). We identified 9 renal tumors with amplification of chromosome 6p including the TFEB gene, 3 by fluorescence in situ hybridization, and 6 from the Cancer Genome Atlas (TCGA) databases. Patients' ages were 28 to 78 years (median, 61 y). Most tumors were high stage (7/9 pT3a, 2/9 pN1). Using immunohistochemistry, 2/4 were positive for melanocytic markers and cathepsin K. Novel TFEB fusions were reported by TCGA in 2; however, due to a small composition of fusion transcripts compared with full-length transcripts (0.5/174 and 3.3/132 FPKM), we hypothesize that these represent secondary fusions due to amplification. Five specimens (4 TCGA, 1 fluorescence in situ hybridization) had concurrent chromosome 3p copy number loss or VHL deletion. However, these did not resemble clear cell RCC, had negative carbonic anhydrase IX labeling, lacked VHL mutation, and had papillary or unclassified histology (2/4 had gain of chromosome 7 or 17). One tumor each had somatic FH mutation and SMARCB1 mutation. Chromosome 6p amplification including TFEB is a previously unrecognized cytogenetic alteration in RCC, associated with heterogenous tubulopapillary eosinophilic and clear cell histology. The combined constellation of features does not fit cleanly into an existing tumor category (unclassified), most closely resembling papillary or translocation RCC. The tendency for high tumor stage, varied tubulopapillary morphology, and a subset with melanocytic marker positivity suggests the possibility of a unique tumor type, despite some variation in appearance and genetics.

  1. Museum genomics: low-cost and high-accuracy genetic data from historical specimens.

    Science.gov (United States)

    Rowe, Kevin C; Singhal, Sonal; Macmanes, Matthew D; Ayroles, Julien F; Morelli, Toni Lyn; Rubidge, Emily M; Bi, Ke; Moritz, Craig C

    2011-11-01

    Natural history collections are unparalleled repositories of geographical and temporal variation in faunal conditions. Molecular studies offer an opportunity to uncover much of this variation; however, genetic studies of historical museum specimens typically rely on extracting highly degraded and chemically modified DNA samples from skins, skulls or other dried samples. Despite this limitation, obtaining short fragments of DNA sequences using traditional PCR amplification of DNA has been the primary method for genetic study of historical specimens. Few laboratories have succeeded in obtaining genome-scale sequences from historical specimens and then only with considerable effort and cost. Here, we describe a low-cost approach using high-throughput next-generation sequencing to obtain reliable genome-scale sequence data from a traditionally preserved mammal skin and skull using a simple extraction protocol. We show that single-nucleotide polymorphisms (SNPs) from the genome sequences obtained independently from the skin and from the skull are highly repeatable compared to a reference genome. © 2011 Blackwell Publishing Ltd.

  2. PCR amplification on microarrays of gel immobilized oligonucleotides

    Science.gov (United States)

    Strizhkov, Boris; Tillib, Sergei; Mikhailovich, Vladimir; Mirzabekov, Andrei

    2003-11-04

    The invention relates two general methods for performing PCR amplification, combined with the detection and analysis of the PCR products on a microchip. In the first method, the amplification occurs both outside and within a plurality of gel pads on a microchip, with at least one oligonucleotide primer immobilized in a gel pad. In the second method, PCR amplification also takes place within gel pads on a microchip, but the pads are surrounded by a hydrophobic liquid such as that which separates the individual gel pads into environments which resemble micro-miniaturized test tubes.

  3. [Genetic system for maintaining the mitochondrial human genome in yeast Yarrowia lipolytica].

    Science.gov (United States)

    Isakova, E P; Deryabina, Yu I; Velyakova, A V; Biryukova, J K; Teplova, V V; Shevelev, A B

    2016-01-01

    For the first time, the possibility of maintaining an intact human mitochondrial genome in a heterologous system in the mitochondria of yeast Yarrowia lipolytica is shown. A method for introducing directional changes into the structure of the mitochondrial human genome replicating in Y. lipolytica by an artificially induced ability of yeast mitochondria for homologous recombination is proposed. A method of introducing and using phenotypic selection markers for the presence or absence of defects in genes tRNA-Lys and tRNA-Leu of the mitochondrial genome is developed. The proposed system can be used to correct harmful mutations of the human mitochondrial genome associated with mitochondrial diseases and for preparative amplification of intact mitochondrial DNA with an adjusted sequence in yeast cells. The applicability of the new system for the correction of mutations in the genes of Lys- and Leu-specific tRNAs of the human mitochondrial genome associated with serious and widespread human mitochondrial diseases such as myoclonic epilepsy with lactic acidosis (MELAS) and myoclonic epilepsy with ragged-red fibers (MERRF) is shown.

  4. Plasticity of the Leishmania genome leading to gene copy number variations and drug resistance [version 1; referees: 5 approved

    Directory of Open Access Journals (Sweden)

    Marie-Claude N. Laffitte

    2016-09-01

    Full Text Available Leishmania has a plastic genome, and drug pressure can select for gene copy number variation (CNV. CNVs can apply either to whole chromosomes, leading to aneuploidy, or to specific genomic regions. For the latter, the amplification of chromosomal regions occurs at the level of homologous direct or inverted repeated sequences leading to extrachromosomal circular or linear amplified DNAs. This ability of Leishmania to respond to drug pressure by CNVs has led to the development of genomic screens such as Cos-Seq, which has the potential of expediting the discovery of drug targets for novel promising drug candidates.

  5. Study on high gain broadband optical parametric chirped pulse amplification

    International Nuclear Information System (INIS)

    Zhang, S.K.; Fujita, M.; Yamanaka, C.; Yoshida, H.; Kodama, R.; Fujita, H.; Nakatsuka, M.; Izawa, Y.

    2000-01-01

    Optical parametric chirped pulse amplification has apparent advantages over the current schemes for high energy ultrashort pulse amplification. High gain in a single pass amplification, small B-integral, low heat deposition, high contrast ratio and, especially the extremely broad gain bandwidth with large-size crystals available bring people new hope for over multi-PW level at which the existing Nd:glass systems suffered difficulties. In this paper we present simulation and experimental studies for a high gain optical parametric chirped pulse amplification system which may be used as a preamplifier to replace the current complicated regenerative system or multi-pass Ti:sapphire amplifiers. Investigations on the amplification bandwidth and gain with BBO are performed. Analysis and discussions are also given. (author)

  6. HER2 amplification, overexpression and score criteria in esophageal adenocarcinoma

    Science.gov (United States)

    Hu, Yingchuan; Bandla, Santhoshi; Godfrey, Tony E.; Tan, Dongfeng; Luketich, James D.; Pennathur, Arjun; Qiu, Xing; Hicks, David G.; Peters, Jeffrey; Zhou, Zhongren

    2011-01-01

    The HER2 oncogene was recently reported to be amplified and overexpressed in esophageal adenocarcinoma. However, the relationship of HER2 amplification in esophageal adenocarcinoma with prognosis has not been well defined. The scoring systems for clinically evaluating HER2 in esophageal adenocarcinoma are not established. The aims of the study were to establish a HER2 scoring system and comprehensively investigate HER2 amplification and overexpression in esophageal adenocarcinoma and its precursor lesion. Using a tissue microarray, containing 116 cases of esophageal adenocarcinoma, 34 cases of BE, 18 cases of low grade dysplasia and 15 cases of high grade dysplasia, HER2 amplification and overexpression were analyzed by HercepTest and CISH methods. The amplification frequency in an independent series of 116 esophageal adenocarcinoma samples was also analyzed using Affymetrix SNP 6.0 microarrays. In our studies, we have found that HER2 amplification does not associate with poor prognosis in total 232 esophageal adenocarcinoma patients by CISH and high density microarrays. We further confirm the similar frequency of HER2 amplification by CISH (18.10%; 21/116) and SNP 6.0 microarrays (16.4%, 19/116) in esophageal adenocarcinoma. HER2 protein overexpression was observed in 12.1 % (14/116) of esophageal adenocarcinoma and 6.67% (1/15) of HGD. No HER2 amplification or overexpression was identified in BE or LGD. All HER2 protein overexpression cases showed HER2 gene amplification. Gene amplification was found to be more frequent by CISH than protein overexpression in esophageal adenocarcinoma (18.10% vs 12.9%). A modified two-step model for esophageal adenocarcinoma HER-2 testing is recommend for clinical esophageal adenocarcinoma HER-2 trial. PMID:21460800

  7. Amplification of hofmeister effect by alcohols.

    Science.gov (United States)

    Xu, Yun; Liu, Guangming

    2014-07-03

    We have demonstrated that Hofmeister effect can be amplified by adding alcohols to aqueous solutions. The lower critical solution temperature behavior of poly(N-isopropylacrylamide) has been employed as the model system to study the amplification of Hofmeister effect. The alcohols can more effectively amplify the Hofmeister effect following the series methanol alcohols and following the series d-sorbitol ≈ xylitol ≈ meso-erythritol alcohols. Our study reveals that the relative extent of amplification of Hofmeister effect is determined by the stability of the water/alcohol complex, which is strongly dependent on the chemical structure of alcohols. The more stable solvent complex formed via stronger hydrogen bonds can more effectively differentiate the anions through the anion-solvent complex interactions, resulting in a stronger amplification of Hofmeister effect. This study provides an alternative method to tune the relative strength of Hofmeister effect besides salt concentration.

  8. Expression microarray reproducibility is improved by optimising purification steps in RNA amplification and labelling

    Directory of Open Access Journals (Sweden)

    Brenton James D

    2004-01-01

    Full Text Available Abstract Background Expression microarrays have evolved into a powerful tool with great potential for clinical application and therefore reliability of data is essential. RNA amplification is used when the amount of starting material is scarce, as is frequently the case with clinical samples. Purification steps are critical in RNA amplification and labelling protocols, and there is a lack of sufficient data to validate and optimise the process. Results Here the purification steps involved in the protocol for indirect labelling of amplified RNA are evaluated and the experimentally determined best method for each step with respect to yield, purity, size distribution of the transcripts, and dye coupling is used to generate targets tested in replicate hybridisations. DNase treatment of diluted total RNA samples followed by phenol extraction is the optimal way to remove genomic DNA contamination. Purification of double-stranded cDNA is best achieved by phenol extraction followed by isopropanol precipitation at room temperature. Extraction with guanidinium-phenol and Lithium Chloride precipitation are the optimal methods for purification of amplified RNA and labelled aRNA respectively. Conclusion This protocol provides targets that generate highly reproducible microarray data with good representation of transcripts across the size spectrum and a coefficient of repeatability significantly better than that reported previously.

  9. Integrative Genome Comparison of Primary and Metastatic Melanomas

    Science.gov (United States)

    Feng, Bin; Nazarian, Rosalynn M.; Bosenberg, Marcus; Wu, Min; Scott, Kenneth L.; Kwong, Lawrence N.; Xiao, Yonghong; Cordon-Cardo, Carlos; Granter, Scott R.; Ramaswamy, Sridhar; Golub, Todd; Duncan, Lyn M.; Wagner, Stephan N.; Brennan, Cameron; Chin, Lynda

    2010-01-01

    A cardinal feature of malignant melanoma is its metastatic propensity. An incomplete view of the genetic events driving metastatic progression has been a major barrier to rational development of effective therapeutics and prognostic diagnostics for melanoma patients. In this study, we conducted global genomic characterization of primary and metastatic melanomas to examine the genomic landscape associated with metastatic progression. In addition to uncovering three genomic subclasses of metastastic melanomas, we delineated 39 focal and recurrent regions of amplification and deletions, many of which encompassed resident genes that have not been implicated in cancer or metastasis. To identify progression-associated metastasis gene candidates, we applied a statistical approach, Integrative Genome Comparison (IGC), to define 32 genomic regions of interest that were significantly altered in metastatic relative to primary melanomas, encompassing 30 resident genes with statistically significant expression deregulation. Functional assays on a subset of these candidates, including MET, ASPM, AKAP9, IMP3, PRKCA, RPA3, and SCAP2, validated their pro-invasion activities in human melanoma cells. Validity of the IGC approach was further reinforced by tissue microarray analysis of Survivin showing significant increased protein expression in thick versus thin primary cutaneous melanomas, and a progression correlation with lymph node metastases. Together, these functional validation results and correlative analysis of human tissues support the thesis that integrated genomic and pathological analyses of staged melanomas provide a productive entry point for discovery of melanoma metastases genes. PMID:20520718

  10. A Whole Genome Association Study on Meat Quality Traits Using High Density SNP Chips in a Cross between Korean Native Pig and Landrace

    Directory of Open Access Journals (Sweden)

    K.-T Lee

    2012-11-01

    Full Text Available A whole genome association (WGA study was performed to detect significant polymorphisms for meat quality traits in an F2 cross population (N = 478 that were generated with Korean native pig sires and Landrace dams in National Livestock Research Institute, Songwhan, Korea. The animals were genotyped using Illumina porcine 60k SNP beadchips, in which a set of 46,865 SNPs were available for the WGA analyses on ten carcass quality traits; live weight, crude protein, crude lipids, crude ash, water holding capacity, drip loss, shear force, CIE L, CIE a and CIE b. Phenotypes were regressed on additive and dominance effects for each SNP using a simple linear regression model, after adjusting for sex, sire and slaughter stage as fixed effects. With the significant SNPs for each trait (p<0.001, a stepwise regression procedure was applied to determine the best set of SNPs with the additive and/or dominance effects. A total of 106 SNPs, or quantitative trait loci (QTL were detected, and about 32 to 66% of the total phenotypic variation was explained by the significant SNPs for each trait. The QTL were identified in most porcine chromosomes (SSCs, in which majority of the QTL were detected in SSCs 1, 2, 12, 13, 14 and 16. Several QTL clusters were identified on SSCs 12, 16 and 17, and a cluster of QTL influencing crude protein, crude lipid, drip loss, shear force, CIE a and CIE b were located between 20 and 29 Mb of SSC12. A pleiotropic QTL for drip loss, CIE L and CIE b was also detected on SSC16. These QTL need to be validated in commercial pig populations for genetic improvement in meat quality via marker-assisted selection.

  11. Distinct evolutionary mechanisms for genomic imbalances in high-risk and low-risk neuroblastomas

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    Gisselsson David

    2007-09-01

    Full Text Available Abstract Background Neuroblastoma (NB is the most common extracranial solid tumour of childhood. Several genomic imbalances correlate to prognosis in NB, with structural rearrangements, including gene amplification, in a near-diploid setting typically signifying high-risk tumours and numerical changes in a near-triploid setting signifying low-risk tumours. Little is known about the temporal sequence in which these imbalances occur during the carcinogenic process. Methods We have reconstructed the appearance of cytogenetic imbalances in 270 NBs by first grouping tumours and imbalances through principal component analysis and then using the number of imbalances in each tumour as an indicator of evolutionary progression. Results Tumours clustered in four sub-groups, dominated respectively by (1 gene amplification in double minute chromosomes and few other aberrations, (2 gene amplification and loss of 1p sequences, (3 loss of 1p and other structural aberrations including gain of 17q, and (4 whole-chromosome gains and losses. Temporal analysis showed that the structural changes in groups 1–3 were acquired in a step-wise fashion, with loss of 1p sequences and the emergence of double minute chromosomes as the earliest cytogenetic events. In contrast, the gains and losses of whole chromosomes in group 4 occurred through multiple simultaneous events leading to a near-triploid chromosome number. Conclusion The finding of different temporal patterns for the acquisition of genomic imbalances in high-risk and low-risk NBs lends strong support to the hypothesis that these tumours are biologically diverse entities, evolving through distinct genetic mechanisms.

  12. Amplification of Chirality in Hydrogen-Bonded Tetrarosette Helices

    NARCIS (Netherlands)

    Mateos timoneda, Miguel; Crego Calama, Mercedes; Reinhoudt, David

    2006-01-01

    The amplification of chirality in hydrogen-bonded tetrarosette assemblies under thermodynamic equilibrium is described. The extent of the chiral amplification obtained by means of “sergeants-and-soldiers” experiments depends only on the structure of the assembly and it is independent of the

  13. Leishmania naiffi and Leishmania guyanensis reference genomes highlight genome structure and gene evolution in the Viannia subgenus.

    Science.gov (United States)

    Coughlan, Simone; Taylor, Ali Shirley; Feane, Eoghan; Sanders, Mandy; Schonian, Gabriele; Cotton, James A; Downing, Tim

    2018-04-01

    The unicellular protozoan parasite Leishmania causes the neglected tropical disease leishmaniasis, affecting 12 million people in 98 countries. In South America, where the Viannia subgenus predominates, so far only L. ( Viannia ) braziliensis and L. ( V. ) panamensis have been sequenced, assembled and annotated as reference genomes. Addressing this deficit in molecular information can inform species typing, epidemiological monitoring and clinical treatment. Here, L. ( V. ) naiffi and L. ( V. ) guyanensis genomic DNA was sequenced to assemble these two genomes as draft references from short sequence reads. The methods used were tested using short sequence reads for L. braziliensis M2904 against its published reference as a comparison. This assembly and annotation pipeline identified 70 additional genes not annotated on the original M2904 reference. Phylogenetic and evolutionary comparisons of L. guyanensis and L. naiffi with 10 other Viannia genomes revealed four traits common to all Viannia : aneuploidy, 22 orthologous groups of genes absent in other Leishmania subgenera, elevated TATE transposon copies and a high NADH-dependent fumarate reductase gene copy number. Within the Viannia , there were limited structural changes in genome architecture specific to individual species: a 45 Kb amplification on chromosome 34 was present in all bar L. lainsoni , L. naiffi had a higher copy number of the virulence factor leishmanolysin, and laboratory isolate L. shawi M8408 had a possible minichromosome derived from the 3' end of chromosome 34 . This combination of genome assembly, phylogenetics and comparative analysis across an extended panel of diverse Viannia has uncovered new insights into the origin and evolution of this subgenus and can help improve diagnostics for leishmaniasis surveillance.

  14. Birth of a healthy infant following preimplantation PKHD1 haplotyping for autosomal recessive polycystic kidney disease using multiple displacement amplification

    Science.gov (United States)

    Janson, Marleen M.; Roesler, Mark R.; Avner, Ellis D.; Strawn, Estil Y.; Bick, David P.

    2010-01-01

    Purpose To develop a reliable preimplantation genetic diagnosis protocol for couples who both carry a mutant PKHD1 gene wishing to conceive children unaffected with autosomal recessive polycystic kidney disease (ARPKD). Methods Development of a unique protocol for preimplantation genetic testing using whole genome amplification of single blastomeres by multiple displacement amplification (MDA), and haplotype analysis with novel short tandem repeat (STR) markers from the PKHD1 gene and flanking sequences, and a case report of successful utilization of the protocol followed by successful IVF resulting in the birth of an infant unaffected with ARPKD. Results We have developed 20 polymorphic STR markers suitable for linkage analysis of ARPKD. These linked STR markers have enabled unambiguous identification of the PKHD1 haplotypes of embryos produced by at-risk couples. Conclusions We have developed a reliable protocol for preimplantation genetic diagnosis of ARPKD using single-cell MDA products for PKHD1 haplotyping. PMID:20490649

  15. THE USE OF MULTIPLE DISPLACEMENT AMPLIFICATION TO INCREASE THE DETECTION AND GENOTYPING OF TRYPANOSOMA SPECIES SAMPLES IMMOBILISED ON FTA FILTERS

    Science.gov (United States)

    MORRISON, LIAM J.; McCORMACK, GILLIAN; SWEENEY, LINDSAY; LIKEUFACK, ANNE C. L.; TRUC, PHILIPPE; TURNER, C. MICHAEL; TAIT, ANDY; MacLEOD, ANNETTE

    2007-01-01

    Whole genome amplification methods are a recently developed tool for amplifying DNA from limited template. We report its application in trypanosome infections, characterised by low parasitaemias. Multiple Displacement Amplification (MDA) amplifies DNA with a simple in vitro step, and was evaluated on mouse blood samples on FTA filter cards with known numbers of Trypanosoma brucei parasites. The data showed a twenty-fold increase in the number of PCRs possible per sample, using primers diagnostic for the multi-copy ribosomal ITS region or 177 bp repeats, and a twenty-fold increase in sensitivity over nested PCR against a single copy microsatellite. Using MDA for microsatellite genotyping caused allele dropout at low DNA concentrations, which was overcome by pooling multiple MDA reactions. The validity of using MDA was established with samples from Human African Trypanosomiasis patients. The use of MDA allows maximal use of finite DNA samples and may prove a valuable tool in studies where multiple reactions are necessary, such as population genetic analyses. PMID:17556624

  16. PDGFRa amplification in multiple skin lesions of undifferentiated pleomorphic sarcoma: A clue for intimal sarcoma metastases.

    Science.gov (United States)

    Osio, Amélie; Vignon-Pennamen, Marie-Dominique; Pedeutour, Florence; Le Maignan, Christine; Koskas, Fabien; Lebbé, Célèste; Janin, Anne; Battistella, Maxime

    2017-05-01

    A 62-year-old human immunodeficiency virus-positive man was admitted for multiple cutaneous and subcutaneous nodules on his lower limbs, corresponding to an undifferentiated proliferation of spindle and pleomorphic cells, with irregular nuclei and numerous mitoses. The tumor cells were negative for a large panel of immunohistochemical markers, except CD10. MDM2 immunohistochemical staining was also negative, leading to the diagnosis of Fédération Nationale des Centres de Lutte contre le Cancer grade III undifferentiated pleomorphic sarcoma (UPS). Array-comparative genomic hybridization showed a highly complex karyotype, with amplification of the 4q12 region, an area that contains only the platelet-derived growth factor receptor α (PDGFRa) gene. This amplification of PDFGRa, molecular hallmark of intimal sarcoma (IS), led to the diagnosis of skin IS metastasis. A positron emission tomography showed a hypermetabolic mass protruding in the preaortic area, consistent with the diagnosis of aortic IS. Our study shows that a rare differential diagnosis in peripheral UPS can be IS skin metastasis, and underlines the importance of molecular analyses in UPS. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. High resolution array-based comparative genomic hybridisation of medulloblastomas and supra-tentorial primitive neuroectodermal tumours

    Science.gov (United States)

    McCabe, Martin Gerard; Ichimura, Koichi; Liu, Lu; Plant, Karen; Bäcklund, L Magnus; Pearson, Danita M; Collins, Vincent Peter

    2010-01-01

    Medulloblastomas and supratentorial primitive neuroectodermal tumours are aggressive childhood tumours. We report our findings using array comparative genomic hybridisation (CGH) on a whole-genome BAC/PAC/cosmid array with a median clone separation of 0.97Mb to study 34 medulloblastomas and 7 supratentorial primitive neuroectodermal tumours. Array CGH allowed identification and mapping of numerous novel small regions of copy number change to genomic sequence, in addition to the large regions already known from previous studies. Novel amplifications were identified, some encompassing oncogenes, MYCL1, PDGFRA, KIT and MYB, not previously reported to show amplification in these tumours. In addition, one supratentorial primitive neuroectodermal tumour had lost both copies of the tumour suppressor genes CDKN2A & CDKN2B. Ten medulloblastomas had findings suggestive of isochromosome 17q. In contrast to previous reports using conventional CGH, array CGH identified three distinct breakpoints in these cases: Ch 17: 17940393-19251679 (17p11.2, n=6), Ch 17: 20111990-23308272 (17p11.2-17q11.2, n=4) and Ch 17: 38425359-39091575 (17q21.31, n=1). Significant differences were found in the patterns of copy number change between medulloblastomas and supratentorial primitive neuroectodermal tumours, providing further evidence that these tumours are genetically distinct despite their morphological and behavioural similarities. PMID:16783165

  18. A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification

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    Xia Li

    2016-10-01

    Full Text Available Bisphenol A (BPA detection in drinking water and food packaging materials has attracted much attention since the discovery that BPA can interfere with normal physiological processes and cause adverse health effects. Here, we constructed a label-free aptamer fluorescent assay for selective and sensitive detection of BPA based on the rolling circle amplification (RCA/Exonuclease III (Exo III-combined cascade amplification strategy. First, the duplex DNA probe (RP with anti-BPA aptamer and trigger sequence was designed for BPA recognition and signal amplification. Next, under the action of BPA, the trigger probe was liberated from RP to initiate RCA reaction as primary amplification. Subsequently, the RCA products were used to trigger Exo III assisted secondary amplification with the help of hairpin probes, producing plenty of “G-quadruplex” in lantern-like structures. Finally, the continuously enriched “G-quadruplex lanterns” were lightened by zinc(II-protoporphyrin IX (ZnPPIX generating enhanced fluorescence signals. By integrating the primary RCA and secondary Exo III mediated cascade amplification strategy, this method displayed an excellent sensitivity with the detection limits of 5.4 × 10−17 M. In addition, the anti-BPA aptamer exhibits high recognition ability with BPA, guaranteeing the specificity of detection. The reporter signal probe (G-quadruplex with ZnPPIX provides a label-free fluorescence signals readout without complicated labeling procedures, making the method simple in design and cost-effective in operation. Moreover, environmental samples analysis was also performed, suggesting that our strategy was reliable and had a great potential application in environmental monitoring.

  19. A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification.

    Science.gov (United States)

    Li, Xia; Song, Juan; Xue, Qing-Wang; You, Fu-Heng; Lu, Xia; Kong, Yan-Cong; Ma, Shu-Yi; Jiang, Wei; Li, Chen-Zhong

    2016-10-21

    Bisphenol A (BPA) detection in drinking water and food packaging materials has attracted much attention since the discovery that BPA can interfere with normal physiological processes and cause adverse health effects. Here, we constructed a label-free aptamer fluorescent assay for selective and sensitive detection of BPA based on the rolling circle amplification (RCA)/Exonuclease III (Exo III)-combined cascade amplification strategy. First, the duplex DNA probe (RP) with anti-BPA aptamer and trigger sequence was designed for BPA recognition and signal amplification. Next, under the action of BPA, the trigger probe was liberated from RP to initiate RCA reaction as primary amplification. Subsequently, the RCA products were used to trigger Exo III assisted secondary amplification with the help of hairpin probes, producing plenty of "G-quadruplex" in lantern-like structures. Finally, the continuously enriched "G-quadruplex lanterns" were lightened by zinc(II)-protoporphyrin IX (ZnPPIX) generating enhanced fluorescence signals. By integrating the primary RCA and secondary Exo III mediated cascade amplification strategy, this method displayed an excellent sensitivity with the detection limits of 5.4 × 10 -17 M. In addition, the anti-BPA aptamer exhibits high recognition ability with BPA, guaranteeing the specificity of detection. The reporter signal probe (G-quadruplex with ZnPPIX) provides a label-free fluorescence signals readout without complicated labeling procedures, making the method simple in design and cost-effective in operation. Moreover, environmental samples analysis was also performed, suggesting that our strategy was reliable and had a great potential application in environmental monitoring.

  20. Social amplification of risk: a conceptual framework

    International Nuclear Information System (INIS)

    Kasperson, R.E.; Renn, O.; Slovic, P.; Brown, H.S.; Emel, J.; Goble, R.; Kasperson, J.X.; Ratick, S.

    1988-01-01

    One of the most perplexing problems in risk analysis is why some relatively minor risks or risk events, as assessed by technical experts, often elicit strong public concerns and result in substantial impacts upon society and economy. This article sets forth a conceptual framework that seeks to link systematically the technical assessment of risk with psychological, sociological, and cultural perspectives of risk perception and risk-related behavior. The main thesis is that hazards interact with psychological, social, institutional, and cultural processes in ways that may amplify or attenuate public responses to the risk or risk event. A structural description of the social amplification of risk is now possible. Amplification occurs at two stages: in the transfer of information about the risk, and in the response mechanisms of society. Signals about risk are processed by individual and social amplification stations, including the scientist who communicates the risk assessment, the news media, cultural groups, interpersonal networks, and others. Key steps of amplifications can be identified at each stage. The amplified risk leads to behavioral responses, which, in turn, result in secondary impacts. Models are presented that portray the elements and linkages in the proposed conceptual framework

  1. Initiation of genome instability and preneoplastic processes through loss of Fhit expression.

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    Joshua C Saldivar

    Full Text Available Genomic instability drives tumorigenesis, but how it is initiated in sporadic neoplasias is unknown. In early preneoplasias, alterations at chromosome fragile sites arise due to DNA replication stress. A frequent, perhaps earliest, genetic alteration in preneoplasias is deletion within the fragile FRA3B/FHIT locus, leading to loss of Fhit protein expression. Because common chromosome fragile sites are exquisitely sensitive to replication stress, it has been proposed that their clonal alterations in cancer cells are due to stress sensitivity rather than to a selective advantage imparted by loss of expression of fragile gene products. Here, we show in normal, transformed, and cancer-derived cell lines that Fhit-depletion causes replication stress-induced DNA double-strand breaks. Using DNA combing, we observed a defect in replication fork progression in Fhit-deficient cells that stemmed primarily from fork stalling and collapse. The likely mechanism for the role of Fhit in replication fork progression is through regulation of Thymidine kinase 1 expression and thymidine triphosphate pool levels; notably, restoration of nucleotide balance rescued DNA replication defects and suppressed DNA breakage in Fhit-deficient cells. Depletion of Fhit did not activate the DNA damage response nor cause cell cycle arrest, allowing continued cell proliferation and ongoing chromosomal instability. This finding was in accord with in vivo studies, as Fhit knockout mouse tissue showed no evidence of cell cycle arrest or senescence yet exhibited numerous somatic DNA copy number aberrations at replication stress-sensitive loci. Furthermore, cells established from Fhit knockout tissue showed rapid immortalization and selection of DNA deletions and amplifications, including amplification of the Mdm2 gene, suggesting that Fhit loss-induced genome instability facilitates transformation. We propose that loss of Fhit expression in precancerous lesions is the first step in the

  2. DNA amplification is rare in normal human cells

    International Nuclear Information System (INIS)

    Wright, J.A.; Watt, F.M.; Hudson, D.L.; Stark, G.R.; Smith, H.S.; Hancock, M.C.

    1990-01-01

    Three types of normal human cells were selected in tissue culture with three drugs without observing a single amplification event from a total of 5 x 10 8 cells. No drug-resistant colonies were observed when normal foreskin keratinocytes were selected with N-(phosphonacetyl)-L-aspartate or with hydroxyurea or when normal mammary epithelial cells were selected with methotrexate. Some slightly resistant colonies with limited potential for growth were obtained when normal diploid fibroblast cells derived from fetal lung were selected with methotrexate or hydroxyurea but careful copy-number analysis of the dihydrofolate reductase and ribonucleotide reductase genes revealed no evidence of amplification. The rarity of DNA amplification in normal human cells contrasts strongly with the situation in tumors and in established cell lines, where amplification of onogenes and of genes mediating drug resistance is frequent. The results suggest that tumors and cell lines have acquired the abnormal ability to amplify DNA with high frequency

  3. Highly parallel and short-acting amplification with locus-specific primers to detect single nucleotide polymorphisms by the DigiTag2 assay.

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    Nao Nishida

    Full Text Available The DigiTag2 assay enables analysis of a set of 96 SNPs using Kapa 2GFast HotStart DNA polymerase with a new protocol that has a total running time of about 7 hours, which is 6 hours shorter than the previous protocol. Quality parameters (conversion rate, call rate, reproducibility and concordance were at the same levels as when genotype calls were acquired using the previous protocol. Multiplex PCR with 192 pairs of locus-specific primers was available for target preparation in the DigiTag2 assay without the optimization of reaction conditions, and quality parameters had the same levels as those acquired with 96-plex PCR. The locus-specific primers were able to achieve sufficient (concentration of target amplicon ≥5 nM and specific (concentration of unexpected amplicons <2 nM amplification within 2 hours, were also able to achieve detectable amplifications even when working in a 96-plex or 192-plex form. The improved DigiTag2 assay will be an efficient platform for screening an intermediate number of SNPs (tens to hundreds of sites in the replication analysis after genome-wide association study. Moreover, highly parallel and short-acting amplification with locus-specific primers may thus facilitate widespread application to other PCR-based assays.

  4. Amplification biases: possible differences among deviating gene expressions

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    Piumi Francois

    2008-01-01

    Full Text Available Abstract Background Gene expression profiling has become a tool of choice to study pathological or developmental questions but in most cases the material is scarce and requires sample amplification. Two main procedures have been used: in vitro transcription (IVT and polymerase chain reaction (PCR, the former known as linear and the latter as exponential. Previous reports identified enzymatic pitfalls in PCR and IVT protocols; however the possible differences between the sequences affected by these amplification defaults were only rarely explored. Results Screening a bovine cDNA array dedicated to embryonic stages with embryonic (n = 3 and somatic tissues (n = 2, we proceeded to moderate amplifications starting from 1 μg of total RNA (global PCR or IVT one round. Whatever the tissue, 16% of the probes were involved in deviating gene expressions due to amplification defaults. These distortions were likely due to the molecular features of the affected sequences (position within a gene, GC content, hairpin number but also to the relative abundance of these transcripts within the tissues. These deviating genes mainly encoded housekeeping genes from physiological or cellular processes (70% and constituted 2 subsets which did not overlap (molecular features, signal intensities, gene ID. However, the differential expressions identified between embryonic stages were both reliable (minor intersect with biased expressions and relevant (biologically validated. In addition, the relative expression levels of those genes were biologically similar between amplified and unamplified samples. Conclusion Conversely to the most recent reports which challenged the use of intense amplification procedures on minute amounts of RNA, we chose moderate PCR and IVT amplifications for our gene profiling study. Conclusively, it appeared that systematic biases arose even with moderate amplification procedures, independently of (i the sample used: brain, ovary or embryos, (ii

  5. Widespread hypomethylation occurs early and synergizes with gene amplification during esophageal carcinogenesis.

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    Hector Alvarez

    2011-03-01

    Full Text Available Although a combination of genomic and epigenetic alterations are implicated in the multistep transformation of normal squamous esophageal epithelium to Barrett esophagus, dysplasia, and adenocarcinoma, the combinatorial effect of these changes is unknown. By integrating genome-wide DNA methylation, copy number, and transcriptomic datasets obtained from endoscopic biopsies of neoplastic progression within the same individual, we are uniquely able to define the molecular events associated progression of Barrett esophagus. We find that the previously reported global hypomethylation phenomenon in cancer has its origins at the earliest stages of epithelial carcinogenesis. Promoter hypomethylation synergizes with gene amplification and leads to significant upregulation of a chr4q21 chemokine cluster and other transcripts during Barrett neoplasia. In contrast, gene-specific hypermethylation is observed at a restricted number of loci and, in combination with hemi-allelic deletions, leads to downregulatation of selected transcripts during multistep progression. We also observe that epigenetic regulation during epithelial carcinogenesis is not restricted to traditionally defined "CpG islands," but may also occur through a mechanism of differential methylation outside of these regions. Finally, validation of novel upregulated targets (CXCL1 and 3, GATA6, and DMBT1 in a larger independent panel of samples confirms the utility of integrative analysis in cancer biomarker discovery.

  6. Analysis of high-throughput sequencing and annotation strategies for phage genomes.

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    Matthew R Henn

    Full Text Available BACKGROUND: Bacterial viruses (phages play a critical role in shaping microbial populations as they influence both host mortality and horizontal gene transfer. As such, they have a significant impact on local and global ecosystem function and human health. Despite their importance, little is known about the genomic diversity harbored in phages, as methods to capture complete phage genomes have been hampered by the lack of knowledge about the target genomes, and difficulties in generating sufficient quantities of genomic DNA for sequencing. Of the approximately 550 phage genomes currently available in the public domain, fewer than 5% are marine phage. METHODOLOGY/PRINCIPAL FINDINGS: To advance the study of phage biology through comparative genomic approaches we used marine cyanophage as a model system. We compared DNA preparation methodologies (DNA extraction directly from either phage lysates or CsCl purified phage particles, and sequencing strategies that utilize either Sanger sequencing of a linker amplification shotgun library (LASL or of a whole genome shotgun library (WGSL, or 454 pyrosequencing methods. We demonstrate that genomic DNA sample preparation directly from a phage lysate, combined with 454 pyrosequencing, is best suited for phage genome sequencing at scale, as this method is capable of capturing complete continuous genomes with high accuracy. In addition, we describe an automated annotation informatics pipeline that delivers high-quality annotation and yields few false positives and negatives in ORF calling. CONCLUSIONS/SIGNIFICANCE: These DNA preparation, sequencing and annotation strategies enable a high-throughput approach to the burgeoning field of phage genomics.

  7. Evaluation of the AGCU Expressmarker 16 and 22 PCR Amplification Kits Using Biological Samples Applied to FTA Micro Cards in Reduced Volume Direct PCR Amplification Reactions

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    Samantha J Ogden

    2015-01-01

    Full Text Available This study evaluated the performance of the  Wuxi AGCU ScienTech Incorporation (HuiShan, Wuxi, China AGCU Expressmarker 16 (EX 16 and 22 (EX22 short tandem repeat (STR amplification kits in reduced reaction volumes using direct polymerase chain reaction (PCR amplification workflows. The commercially available PowerPlex® 21 (PP21 System (Promega, Wisconsin, USA, which follows similar direct workflows, was used as a reference. Anticoagulate blood applied to chemically impregnated  FTA TM Micro Cards (GE Healthcare UK Limited, Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK was used to represent a complex biological sample. Allelic concordance, first-pass success rate, average peak heights, heterozygous peak height ratios (HPHRs, and intracolor and intercolor peak height balance were determined. In reduced volume PCR reactions, the performances of both the EX16 and EX22 STR amplification kits were comparable to that of the PP21 System. The level of performance was maintained at PCR reaction volumes, which are 40% of that recommended. The EX22 and PP21 System kits possess comparable overlapping genome coverage. This study evaluated the performance of the AGCU EX16 and EX22 STR amplification kits in reduced PCR reaction volumes using direct workflows in combination with whole blood applied to FTA TM Micro Cards. Allelic concordance, first-pass success rate, average peak heights, HPHRs, and intracolor and intercolor peak height balance were determined. A concordance analysis was completed that compared the performance of the EX16 and EX22 kits using human blood applied to FTA Micro Cards in combination with full, half, and reduced PCR reaction volumes. The PP21 System (Promega was used as a reference kit. Where appropriate, the distributions of data were assessed using the Shapiro-Wilk test. For normally-distributed data, statistics were calculated using analysis of variance (ANOVA and for nonparametric data the Wilcoxon

  8. [Prognostic significance of MYCN amplification in children neuroblastic tumors].

    Science.gov (United States)

    Niu, Huilin; Xu, Tao; Wang, Fenghua; Chen, Zhengrong; Gao, Qiu; Yi, Peng; Xia, Jianqing

    2015-02-01

    To summarize the clinicopathologic features of neuroblastic tumors (NT), and to explore the prognostic significance of MYCN amplification in NT. The clinicopathologic data of 267 NT were reviewed. MYCN gene amplification was detected by fluorescence in situ hybridization (FISH) in 119 cases and the relationship with pathological characteristics and prognostic significance were analyzed. The study included 267 cases of children NT from patients aged from 1 day to 13 years (median 27 months). The male to female ratio was 1.43. There were 38 cases (14.2%), 43 cases (16.1%), 71 cases (26.6%), and 115 cases (43.1%) of INSS stages I, II, III and IV respectively.Favorable histology group had 157 cases (59.9%); unfavorable histology group had 110 cases (40.1%).Of the 119 NT cases with MYCN FISH performed, 18 cases (15.1%) showed amplification and the signal ratio of MYCN to CEP2 was 4.08-43.29. One hundred and one cases of non-amplified MYCN included MYCN gain in 79 cases (66.3%) and MYCN negative in 22 cases (18.5%). MYCN expression showed significant difference (P = 0.000) between ages, gender, NT type and MKI, but not INPC and clinical stage (P > 0.05).Of the 18 cases with MYCN amplification, 3 were undifferentiated, and 15 poorly differentiated; 17 had high MKI and one moderate MKI. All 18 cases were in unfavorable histology group; the overall survival rate was 3/18, with an average survival time of (17.9 ± 2.4) months.Of the 101 MYCN non-amplification cases, the overall survival rate was 68.3% (69/101), with an average survival time of (29.8 ± 1.3) months. Survival analysis showed the cases with MYCN amplification had worse prognosis (P < 0.05). NT were commonly diagnosed in early ages and easily to metastasize. Most of cases with favorable histology. The cases of MYCN amplification showed unfavorable histology, and the majority cases with high MKI; The patients with MYCN gene amplification had poor prognosis.

  9. Clusters of orthologous genes for 41 archaeal genomes and implications for evolutionary genomics of archaea

    Directory of Open Access Journals (Sweden)

    Wolf Yuri I

    2007-11-01

    Full Text Available Abstract Background An evolutionary classification of genes from sequenced genomes that distinguishes between orthologs and paralogs is indispensable for genome annotation and evolutionary reconstruction. Shortly after multiple genome sequences of bacteria, archaea, and unicellular eukaryotes became available, an attempt on such a classification was implemented in Clusters of Orthologous Groups of proteins (COGs. Rapid accumulation of genome sequences creates opportunities for refining COGs but also represents a challenge because of error amplification. One of the practical strategies involves construction of refined COGs for phylogenetically compact subsets of genomes. Results New Archaeal Clusters of Orthologous Genes (arCOGs were constructed for 41 archaeal genomes (13 Crenarchaeota, 27 Euryarchaeota and one Nanoarchaeon using an improved procedure that employs a similarity tree between smaller, group-specific clusters, semi-automatically partitions orthology domains in multidomain proteins, and uses profile searches for identification of remote orthologs. The annotation of arCOGs is a consensus between three assignments based on the COGs, the CDD database, and the annotations of homologs in the NR database. The 7538 arCOGs, on average, cover ~88% of the genes in a genome compared to a ~76% coverage in COGs. The finer granularity of ortholog identification in the arCOGs is apparent from the fact that 4538 arCOGs correspond to 2362 COGs; ~40% of the arCOGs are new. The archaeal gene core (protein-coding genes found in all 41 genome consists of 166 arCOGs. The arCOGs were used to reconstruct gene loss and gene gain events during archaeal evolution and gene sets of ancestral forms. The Last Archaeal Common Ancestor (LACA is conservatively estimated to possess 996 genes compared to 1245 and 1335 genes for the last common ancestors of Crenarchaeota and Euryarchaeota, respectively. It is inferred that LACA was a chemoautotrophic hyperthermophile

  10. Development and preliminary evaluation of a multiplexed amplification and next generation sequencing method for viral hemorrhagic fever diagnostics.

    Directory of Open Access Journals (Sweden)

    Annika Brinkmann

    2017-11-01

    Full Text Available We describe the development and evaluation of a novel method for targeted amplification and Next Generation Sequencing (NGS-based identification of viral hemorrhagic fever (VHF agents and assess the feasibility of this approach in diagnostics.An ultrahigh-multiplex panel was designed with primers to amplify all known variants of VHF-associated viruses and relevant controls. The performance of the panel was evaluated via serially quantified nucleic acids from Yellow fever virus, Rift Valley fever virus, Crimean-Congo hemorrhagic fever (CCHF virus, Ebola virus, Junin virus and Chikungunya virus in a semiconductor-based sequencing platform. A comparison of direct NGS and targeted amplification-NGS was performed. The panel was further tested via a real-time nanopore sequencing-based platform, using clinical specimens from CCHF patients.The multiplex primer panel comprises two pools of 285 and 256 primer pairs for the identification of 46 virus species causing hemorrhagic fevers, encompassing 6,130 genetic variants of the strains involved. In silico validation revealed that the panel detected over 97% of all known genetic variants of the targeted virus species. High levels of specificity and sensitivity were observed for the tested virus strains. Targeted amplification ensured viral read detection in specimens with the lowest virus concentration (1-10 genome equivalents and enabled significant increases in specific reads over background for all viruses investigated. In clinical specimens, the panel enabled detection of the causative agent and its characterization within 10 minutes of sequencing, with sample-to-result time of less than 3.5 hours.Virus enrichment via targeted amplification followed by NGS is an applicable strategy for the diagnosis of VHFs which can be adapted for high-throughput or nanopore sequencing platforms and employed for surveillance or outbreak monitoring.

  11. Development and preliminary evaluation of a multiplexed amplification and next generation sequencing method for viral hemorrhagic fever diagnostics.

    Science.gov (United States)

    Brinkmann, Annika; Ergünay, Koray; Radonić, Aleksandar; Kocak Tufan, Zeliha; Domingo, Cristina; Nitsche, Andreas

    2017-11-01

    We describe the development and evaluation of a novel method for targeted amplification and Next Generation Sequencing (NGS)-based identification of viral hemorrhagic fever (VHF) agents and assess the feasibility of this approach in diagnostics. An ultrahigh-multiplex panel was designed with primers to amplify all known variants of VHF-associated viruses and relevant controls. The performance of the panel was evaluated via serially quantified nucleic acids from Yellow fever virus, Rift Valley fever virus, Crimean-Congo hemorrhagic fever (CCHF) virus, Ebola virus, Junin virus and Chikungunya virus in a semiconductor-based sequencing platform. A comparison of direct NGS and targeted amplification-NGS was performed. The panel was further tested via a real-time nanopore sequencing-based platform, using clinical specimens from CCHF patients. The multiplex primer panel comprises two pools of 285 and 256 primer pairs for the identification of 46 virus species causing hemorrhagic fevers, encompassing 6,130 genetic variants of the strains involved. In silico validation revealed that the panel detected over 97% of all known genetic variants of the targeted virus species. High levels of specificity and sensitivity were observed for the tested virus strains. Targeted amplification ensured viral read detection in specimens with the lowest virus concentration (1-10 genome equivalents) and enabled significant increases in specific reads over background for all viruses investigated. In clinical specimens, the panel enabled detection of the causative agent and its characterization within 10 minutes of sequencing, with sample-to-result time of less than 3.5 hours. Virus enrichment via targeted amplification followed by NGS is an applicable strategy for the diagnosis of VHFs which can be adapted for high-throughput or nanopore sequencing platforms and employed for surveillance or outbreak monitoring.

  12. The peculiar landscape of repetitive sequences in the olive (Olea europaea L.) genome.

    Science.gov (United States)

    Barghini, Elena; Natali, Lucia; Cossu, Rosa Maria; Giordani, Tommaso; Pindo, Massimo; Cattonaro, Federica; Scalabrin, Simone; Velasco, Riccardo; Morgante, Michele; Cavallini, Andrea

    2014-04-01

    Analyzing genome structure in different species allows to gain an insight into the evolution of plant genome size. Olive (Olea europaea L.) has a medium-sized haploid genome of 1.4 Gb, whose structure is largely uncharacterized, despite the growing importance of this tree as oil crop. Next-generation sequencing technologies and different computational procedures have been used to study the composition of the olive genome and its repetitive fraction. A total of 2.03 and 2.3 genome equivalents of Illumina and 454 reads from genomic DNA, respectively, were assembled following different procedures, which produced more than 200,000 differently redundant contigs, with mean length higher than 1,000 nt. Mapping Illumina reads onto the assembled sequences was used to estimate their redundancy. The genome data set was subdivided into highly and medium redundant and nonredundant contigs. By combining identification and mapping of repeated sequences, it was established that tandem repeats represent a very large portion of the olive genome (∼31% of the whole genome), consisting of six main families of different length, two of which were first discovered in these experiments. The other large redundant class in the olive genome is represented by transposable elements (especially long terminal repeat-retrotransposons). On the whole, the results of our analyses show the peculiar landscape of the olive genome, related to the massive amplification of tandem repeats, more than that reported for any other sequenced plant genome.

  13. Diversity among Cynodon accessions and taxa based on DNA amplification fingerprinting.

    Science.gov (United States)

    Assefa, S; Taliaferro, C M; Anderson, M P; de los Reyes, B G; Edwards, R M

    1999-06-01

    The genus Cynodon (Gramineae), comprised of 9 species, is geographically widely distributed and genetically diverse. Information on the amounts of molecular genetic variation among and within Cynodon taxa is needed to enhance understanding of phylogenetic relations and facilitate germplasm management and breeding improvement efforts. Genetic relatedness among 62 Cynodon accessions, representing eight species, was assessed using DNA amplification fingerprinting (DAF). Ten 8-mer oligonucleotides were used to amplify specific Cynodon genomic sequences. The DNA amplification products of individual accessions were scored for presence (1) or absence (0) of bands. Similarity matrices were developed and the accessions were grouped by cluster (UPGMA) and principal coordinate analysis. Analyses were conducted within ploidy level (2x = 18 and 4x = 36) and over ploidy levels. Each primer revealed polymorphic loci among accessions within species. Of 539 loci (bands) scored, 496 (92%) were polymorphic. Cynodon arcuatus was clearly separated from other species by numerous monomorphic bands. The strongest species similarities were between C. aethiopicus and C. arcuatus, C. transvaalensis and C. plectostachyus, and C. incompletus and C. nlemfuensis. Intraspecific variation was least for C. aethiopicus, C. arcuatus, and C. transvaalensis, and greatest for C. dactylon. Accessions of like taxonomic classification were generally clustered, except the cosmopolitan C. dactylon var. dactylon and C. dactylon var. afganicus. Within taxa, accessions differing in chromosome number clustered in all instances indicating the 2x and 4x forms to be closely related. Little, if any, relationship was found between relatedness as indicated by the DAF profiles and previous estimates of hybridization potential between the different taxa.

  14. A CTAB Procedure Of Total Genomic DNA Extraction For Medicinal Mushrooms

    International Nuclear Information System (INIS)

    Azhar Mohamad; Muhammad Hussaini Mohd Mustafa; Muhammad Hanif Azhari Noor; Rosnani Abdul Rashid; Hasan Hamdani Hasan Mutaat; Meswan Meskom; Mat Rasol Awang

    2014-01-01

    Medicinal mushroom is defined as mushrooms used in medicine or medical research. Isolation of intact, high-molecular-mass genomic DNA is essential for many molecular biology applications including Polymerase Chain Reaction (PCR), endonuclease restriction digestion, Southern blot analysis, and genomic library construction. The most important and prerequisite towards reliable molecular biology work is the total genomic DNA of a sample must be in good quality. Five freshly samples of medicinal mushroom were used in this work known as Auriculariapolytricha, Lentinus edode, Pleurotus sayorcaju, Sczhizopyllum commune and Ganodermalucidum. 5 mg of each sample were used to extraction the DNA, prepared in 3 replications and repeated twice. PCR based technique by using ISSR markers were used in checking the amplification ability of the total genomic extraction. A standard Doyle and Doyle protocol for genomic DNA extraction was modified in optimizing the total genomic DNA from the medicinal mushroom.The modification parameters were percentage of CTAB, incubation period and temperature. The results reveal that each sample required a certain combinations of time and period of incubation. Besides, percentage of CTAB in the buffer was found significant in giving a high yielding of extracted total genomic DNA. The extracted total genomic DNA from the medicinal mushroom yielded from 39.7 ng/ μl to 919.1 ng/ μl. The different yield among the samples found to be corresponded to polysaccharide content in the medicinal mushrooms. The objective of this works is to optimize total genomic DNA extraction of medicinal mushrooms towards a high quality intact genomic DNA for molecular activities. (author)

  15. Hybridization chain reaction amplification for highly sensitive fluorescence detection of DNA with dextran coated microarrays.

    Science.gov (United States)

    Chao, Jie; Li, Zhenhua; Li, Jing; Peng, Hongzhen; Su, Shao; Li, Qian; Zhu, Changfeng; Zuo, Xiaolei; Song, Shiping; Wang, Lianhui; Wang, Lihua

    2016-07-15

    Microarrays of biomolecules hold great promise in the fields of genomics, proteomics, and clinical assays on account of their remarkably parallel and high-throughput assay capability. However, the fluorescence detection used in most conventional DNA microarrays is still limited by sensitivity. In this study, we have demonstrated a novel universal and highly sensitive platform for fluorescent detection of sequence specific DNA at the femtomolar level by combining dextran-coated microarrays with hybridization chain reaction (HCR) signal amplification. Three-dimensional dextran matrix was covalently coated on glass surface as the scaffold to immobilize DNA recognition probes to increase the surface binding capacity and accessibility. DNA nanowire tentacles were formed on the matrix surface for efficient signal amplification by capturing multiple fluorescent molecules in a highly ordered way. By quantifying microscopic fluorescent signals, the synergetic effects of dextran and HCR greatly improved sensitivity of DNA microarrays, with a detection limit of 10fM (1×10(5) molecules). This detection assay could recognize one-base mismatch with fluorescence signals dropped down to ~20%. This cost-effective microarray platform also worked well with samples in serum and thus shows great potential for clinical diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Loop-mediated isothermal amplification (LAMP) based detection of Colletotrichum falcatum causing red rot in sugarcane.

    Science.gov (United States)

    Chandra, Amaresh; Keizerweerd, Amber T; Que, Youxiong; Grisham, Michael P

    2015-08-01

    Red rot, caused by Colletotrichum falcatum, is a destructive disease prevalent in most sugarcane-producing countries. Disease-free sugarcane planting materials (setts) are essential as the pathogen spreads primarily through infected setts. The present study was undertaken to develop a loop-mediated isothermal amplification (LAMP) assay for the detection of C. falcatum. C. falcatum genomic DNA was isolated from pure mycelium culture and infected tissues. Four sets of primers corresponding to a unique DNA sequence specific to C. falcatum were designed. Specificity of the LAMP test was checked with DNA of another fungal pathogen of sugarcane, Puccinia melanocephala, as well as two closely-related species, Colletotrichum fructivorum and Colletotrichum acutatum. No reaction was found with the three pathogens. When C. falcatum DNA from pure culture was used in a detection limit analysis, sensitivity of the LAMP method was observed to be ten times higher than that of conventional PCR; however, sensitivity was only 5 times higher when DNA from C. falcatum-infected tissues was used. Using the LAMP assay, C. falcatum DNA is amplified with high specificity, efficiency, and rapidity under isothermal conditions. Moreover, visual judgment of color change in <1 h without further post-amplification processing makes the LAMP method convenient, economical, and useful in diagnostic laboratories and the field.

  17. CDK4 amplification predicts recurrence of well-differentiated liposarcoma of the abdomen.

    Directory of Open Access Journals (Sweden)

    Sanghoon Lee

    Full Text Available The absence of CDK4 amplification in liposarcomas is associated with favorable prognosis. We aimed to identify the factors associated with tumor recurrence in patients with well-differentiated (WD and dedifferentiated (DD liposarcomas.From 2000 to 2010, surgical resections for 101 WD and DD liposarcomas were performed. Cases in which complete surgical resections with curative intent were carried out were selected. MDM2 and CDK4 gene amplification were analyzed by quantitative real-time polymerase chain reaction (Q-PCR.There were 31 WD and 17 DD liposarcomas. Locoregional recurrence was observed in 11 WD and 3 DD liposarcomas. WD liposarcomas showed better patient survival compared to DD liposarcomas (P<0.05. Q-PCR analysis of the liposarcomas revealed the presence of CDK4 amplification in 44 cases (91.7% and MDM2 amplification in 46 cases (95.8%. WD liposarcomas with recurrence after surgical resection had significantly higher levels of CDK4 amplification compared to those without recurrence (P = 0.041. High level of CDK4 amplification (cases with CDK4 amplification higher than the median 7.54 was associated with poor recurrence-free survival compared to low CDK4 amplification in both univariate (P = 0.012 and multivariate analyses (P = 0.020.Level of CDK4 amplification determined by Q-PCR was associated with the recurrence of WD liposarcomas after surgical resection.

  18. Gene copy number variation throughout the Plasmodium falciparum genome

    Directory of Open Access Journals (Sweden)

    Stewart Lindsay B

    2009-08-01

    Full Text Available Abstract Background Gene copy number variation (CNV is responsible for several important phenotypes of the malaria parasite Plasmodium falciparum, including drug resistance, loss of infected erythrocyte cytoadherence and alteration of receptor usage for erythrocyte invasion. Despite the known effects of CNV, little is known about its extent throughout the genome. Results We performed a whole-genome survey of CNV genes in P. falciparum using comparative genome hybridisation of a diverse set of 16 laboratory culture-adapted isolates to a custom designed high density Affymetrix GeneChip array. Overall, 186 genes showed hybridisation signals consistent with deletion or amplification in one or more isolate. There is a strong association of CNV with gene length, genomic location, and low orthology to genes in other Plasmodium species. Sub-telomeric regions of all chromosomes are strongly associated with CNV genes independent from members of previously described multigene families. However, ~40% of CNV genes were located in more central regions of the chromosomes. Among the previously undescribed CNV genes, several that are of potential phenotypic relevance are identified. Conclusion CNV represents a major form of genetic variation within the P. falciparum genome; the distribution of gene features indicates the involvement of highly non-random mutational and selective processes. Additional studies should be directed at examining CNV in natural parasite populations to extend conclusions to clinical settings.

  19. Explanatory Model for Sound Amplification in a Stethoscope

    Science.gov (United States)

    Eshach, H.; Volfson, A.

    2015-01-01

    In the present paper we suggest an original physical explanatory model that explains the mechanism of the sound amplification process in a stethoscope. We discuss the amplification of a single pulse, a continuous wave of certain frequency, and finally we address the resonant frequencies. It is our belief that this model may provide students with…

  20. Exponential isothermal amplification of nucleic acids and amplified assays for proteins, cells, and enzyme activities.

    Science.gov (United States)

    Reid, Michael S; Le, X Chris; Zhang, Hongquan

    2018-04-27

    Isothermal exponential amplification techniques, such as strand-displacement amplification (SDA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HDA), and recombinase polymerase amplification (RPA), have great potential for on-site, point-of-care, and in-situ assay applications. These amplification techniques eliminate the need for temperature cycling required for polymerase chain reaction (PCR) while achieving comparable amplification yield. We highlight here recent advances in exponential amplification reaction (EXPAR) for the detection of nucleic acids, proteins, enzyme activities, cells, and metal ions. We discuss design strategies, enzyme reactions, detection techniques, and key features. Incorporation of fluorescence, colorimetric, chemiluminescence, Raman, and electrochemical approaches enables highly sensitive detection of a variety of targets. Remaining issues, such as undesirable background amplification resulting from non-specific template interactions, must be addressed to further improve isothermal and exponential amplification techniques. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Radioactive wastes and the social amplification of risk

    International Nuclear Information System (INIS)

    Kasperson, R.E.; Emel, J.; Goble, R.; Hohenemser, C.; Kasperson, J.X.; Renn, O.

    1987-01-01

    A significant problem in radioactive waste facility siting is that apparent small risks or minor risks events produce substantial public concern and social impacts. The reasons for this difference in public health and societal impacts is not well understood. This paper explores the issues involved in the social amplification of risk, using the risk associated with site characterization as the example. Noteworthy as sources of amplification are the information flow associated with risks and risk events including the large volume of information, the extent of dispute, and misinformation and rumor. Such information passes through the mass media and interpersonal networks. The major mechanisms involved in risk amplifications are discussed and their likely impacts on society described

  2. A novel method of genomic DNA extraction for Cactaceae1

    Science.gov (United States)

    Fehlberg, Shannon D.; Allen, Jessica M.; Church, Kathleen

    2013-01-01

    • Premise of the study: Genetic studies of Cactaceae can at times be impeded by difficult sampling logistics and/or high mucilage content in tissues. Simplifying sampling and DNA isolation through the use of cactus spines has not previously been investigated. • Methods and Results: Several protocols for extracting DNA from spines were tested and modified to maximize yield, amplification, and sequencing. Sampling of and extraction from spines resulted in a simplified protocol overall and complete avoidance of mucilage as compared to typical tissue extractions. Sequences from one nuclear and three plastid regions were obtained across eight genera and 20 species of cacti using DNA extracted from spines. • Conclusions: Genomic DNA useful for amplification and sequencing can be obtained from cactus spines. The protocols described here are valuable for any cactus species, but are particularly useful for investigators interested in sampling living collections, extensive field sampling, and/or conservation genetic studies. PMID:25202521

  3. Improved multiplex ligation-dependent probe amplification analysis identifies a deleterious PMS2 allele generated by recombination with crossover between PMS2 and PMS2CL

    OpenAIRE

    Wernstedt, Annekatrin; Valtorta, Emanuele; Armelao, Franco; Togni, Roberto; Girlando, Salvatore; Baudis, Michael; Heinimann, Karl; Messiaen, Ludwine; Staehli, Noemie; Zschocke, Johannes; Marra, Giancarlo; Wimmer, Katharina

    2012-01-01

    Heterozygous PMS2 germline mutations are associated with Lynch syndrome. Up to one third of these mutations are genomic deletions. Their detection is complicated by a pseudogene (PMS2CL), which – owing to extensive interparalog sequence exchange – closely resembles PMS2 downstream of exon 12. A recently redesigned multiplex ligation-dependent probe amplification (MLPA) assay identifies PMS2 copy number alterations with improved reliability when used with reference DNAs containing equal number...

  4. Genomic screening for blood-borne viruses in transfusion settings.

    Science.gov (United States)

    Allain, J P

    2000-02-01

    The residual risk of post-transfusion human immunodeficiency virus (HIV) infection is low but slightly higher for hepatitis B virus (HBV) and hepatitis C virus (HCV), the main reason being viraemia during the window period preceding antibody or antigen detection by enzyme immunoassays. Immunosilent-infected individuals and carriers of distant viral variants also play an unquantifiable role. Multiple techniques, e.g. reverse transcription-polymerase chain reaction (RT-PCR), PCR, ligase-chain reaction, nucleic acid sequence-based amplification (NASBA) and transcription-mediated amplification (TMA) have been developed to amplify and detect viral genomes as single or multiplex assays. Equipment providing various degrees of automation has been adapted to these techniques. Applying nucleic acid amplification techniques (NAT) to blood screening, two main approaches have been advocated: plasma pool and single-donation testing. Pool testing presents the advantage of lower cost and readily available equipment although it is prone to false negative and positive reactions. The time required to identify infected donations is incompatible with blood component release, and may lead to product waste. Single-unit testing, although appealing, is not yet fully automated and potentially very costly unless a systematic multiplex approach is taken. Although technically feasible, NAT applied to the blood supply needs to be clinically evaluated and its cost efficiency assessed in the general public health context. However, pool NAT is currently implemented in continental Europe and the USA.

  5. Basin amplification of seismic waves in the city of Pahrump, Nevada.

    Energy Technology Data Exchange (ETDEWEB)

    Abbott, Robert E.

    2005-07-01

    Sedimentary basins can increase the magnitude and extend the duration of seismic shaking. This potential for seismic amplification is investigated for Pahrump Valley, Nevada-California. The Pahrump Valley is located approximately 50 km northwest of Las Vegas and 75 km south of the Nevada Test Site. Gravity data suggest that the city of Pahrump sits atop a narrow, approximately 5 km deep sub-basin within the valley. The seismic amplification, or ''site effect'', was investigated using a combination of in situ velocity modeling and comparison of the waveforms and spectra of weak ground motion recorded in the city of Pahrump, Nevada, and those recorded in the nearby mountains. Resulting spectral ratios indicate seismic amplification factors of 3-6 over the deepest portion of Pahrump Valley. This amplification predominantly occurs at 2-2.5 Hz. Amplification over the deep sub-basin is lower than amplification at the sub-basin edge, location of the John Blume and Associates PAHA seismic station, which recorded many underground nuclear tests at the Nevada Test Site. A comprehensive analysis of basin amplification for the city of Pahrump should include 3-D basin modeling, due to the extreme basement topography of the Pahrump Valley.

  6. Whole-genome sequencing of cultivated and wild peppers provides insights into Capsicum domestication and specialization

    Science.gov (United States)

    Qin, Cheng; Yu, Changshui; Shen, Yaou; Fang, Xiaodong; Chen, Lang; Min, Jiumeng; Cheng, Jiaowen; Zhao, Shancen; Xu, Meng; Luo, Yong; Yang, Yulan; Wu, Zhiming; Mao, Likai; Wu, Haiyang; Ling-Hu, Changying; Zhou, Huangkai; Lin, Haijian; González-Morales, Sandra; Trejo-Saavedra, Diana L.; Tian, Hao; Tang, Xin; Zhao, Maojun; Huang, Zhiyong; Zhou, Anwei; Yao, Xiaoming; Cui, Junjie; Li, Wenqi; Chen, Zhe; Feng, Yongqiang; Niu, Yongchao; Bi, Shimin; Yang, Xiuwei; Li, Weipeng; Cai, Huimin; Luo, Xirong; Montes-Hernández, Salvador; Leyva-González, Marco A.; Xiong, Zhiqiang; He, Xiujing; Bai, Lijun; Tan, Shu; Tang, Xiangqun; Liu, Dan; Liu, Jinwen; Zhang, Shangxing; Chen, Maoshan; Zhang, Lu; Zhang, Li; Zhang, Yinchao; Liao, Weiqin; Zhang, Yan; Wang, Min; Lv, Xiaodan; Wen, Bo; Liu, Hongjun; Luan, Hemi; Zhang, Yonggang; Yang, Shuang; Wang, Xiaodian; Xu, Jiaohui; Li, Xueqin; Li, Shuaicheng; Wang, Junyi; Palloix, Alain; Bosland, Paul W.; Li, Yingrui; Krogh, Anders; Rivera-Bustamante, Rafael F.; Herrera-Estrella, Luis; Yin, Ye; Yu, Jiping; Hu, Kailin; Zhang, Zhiming

    2014-01-01

    As an economic crop, pepper satisfies people’s spicy taste and has medicinal uses worldwide. To gain a better understanding of Capsicum evolution, domestication, and specialization, we present here the genome sequence of the cultivated pepper Zunla-1 (C. annuum L.) and its wild progenitor Chiltepin (C. annuum var. glabriusculum). We estimate that the pepper genome expanded ∼0.3 Mya (with respect to the genome of other Solanaceae) by a rapid amplification of retrotransposons elements, resulting in a genome comprised of ∼81% repetitive sequences. Approximately 79% of 3.48-Gb scaffolds containing 34,476 protein-coding genes were anchored to chromosomes by a high-density genetic map. Comparison of cultivated and wild pepper genomes with 20 resequencing accessions revealed molecular footprints of artificial selection, providing us with a list of candidate domestication genes. We also found that dosage compensation effect of tandem duplication genes probably contributed to the pungent diversification in pepper. The Capsicum reference genome provides crucial information for the study of not only the evolution of the pepper genome but also, the Solanaceae family, and it will facilitate the establishment of more effective pepper breeding programs. PMID:24591624

  7. Genome-wide conserved non-coding microsatellite (CNMS) marker-based integrative genetical genomics for quantitative dissection of seed weight in chickpea.

    Science.gov (United States)

    Bajaj, Deepak; Saxena, Maneesha S; Kujur, Alice; Das, Shouvik; Badoni, Saurabh; Tripathi, Shailesh; Upadhyaya, Hari D; Gowda, C L L; Sharma, Shivali; Singh, Sube; Tyagi, Akhilesh K; Parida, Swarup K

    2015-03-01

    Phylogenetic footprinting identified 666 genome-wide paralogous and orthologous CNMS (conserved non-coding microsatellite) markers from 5'-untranslated and regulatory regions (URRs) of 603 protein-coding chickpea genes. The (CT)n and (GA)n CNMS carrying CTRMCAMV35S and GAGA8BKN3 regulatory elements, respectively, are abundant in the chickpea genome. The mapped genic CNMS markers with robust amplification efficiencies (94.7%) detected higher intraspecific polymorphic potential (37.6%) among genotypes, implying their immense utility in chickpea breeding and genetic analyses. Seventeen differentially expressed CNMS marker-associated genes showing strong preferential and seed tissue/developmental stage-specific expression in contrasting genotypes were selected to narrow down the gene targets underlying seed weight quantitative trait loci (QTLs)/eQTLs (expression QTLs) through integrative genetical genomics. The integration of transcript profiling with seed weight QTL/eQTL mapping, molecular haplotyping, and association analyses identified potential molecular tags (GAGA8BKN3 and RAV1AAT regulatory elements and alleles/haplotypes) in the LOB-domain-containing protein- and KANADI protein-encoding transcription factor genes controlling the cis-regulated expression for seed weight in the chickpea. This emphasizes the potential of CNMS marker-based integrative genetical genomics for the quantitative genetic dissection of complex seed weight in chickpea. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  8. ESTIMATION OF AMPLIFICATION FACTOR IN EARTHQUAKE ENGINEERING

    Directory of Open Access Journals (Sweden)

    Nazarov Yuriy Pavlovich

    2015-03-01

    Full Text Available The authors are the developers of Odyssey Software (Eurosoft Co. for the analysis of seismological data and computing of seismic loads and their parameters. While communicating with the users of the software, the authors have revealed some uncertainty about both understanding of the term "amplification factor (AF" and calculation of the amplification factor using various methods. In this article, a simple example shows that the determination of the amplification factor as the ratio of the acceleration’s spectrum to the maximal acceleration is derived from the classical definition of AF in the form of the ratio of maximal dynamic displacement to the displacement by the action of static load. Deterministic and probabilistic ap-proaches for the calculating of the AF were discussed. There was an example of AFs calculation and their envelopes for translational and rotational components of seismic impact by using Odyssey Software.

  9. Breast tumor copy number aberration phenotypes and genomic instability

    International Nuclear Information System (INIS)

    Fridlyand, Jane; Jain, Ajay N; McLennan, Jane; Ziegler, John; Chin, Koei; Devries, Sandy; Feiler, Heidi; Gray, Joe W; Waldman, Frederic; Pinkel, Daniel; Albertson, Donna G; Snijders, Antoine M; Ylstra, Bauke; Li, Hua; Olshen, Adam; Segraves, Richard; Dairkee, Shanaz; Tokuyasu, Taku; Ljung, Britt Marie

    2006-01-01

    Genomic DNA copy number aberrations are frequent in solid tumors, although the underlying causes of chromosomal instability in tumors remain obscure. Genes likely to have genomic instability phenotypes when mutated (e.g. those involved in mitosis, replication, repair, and telomeres) are rarely mutated in chromosomally unstable sporadic tumors, even though such mutations are associated with some heritable cancer prone syndromes. We applied array comparative genomic hybridization (CGH) to the analysis of breast tumors. The variation in the levels of genomic instability amongst tumors prompted us to investigate whether alterations in processes/genes involved in maintenance and/or manipulation of the genome were associated with particular types of genomic instability. We discriminated three breast tumor subtypes based on genomic DNA copy number alterations. The subtypes varied with respect to level of genomic instability. We find that shorter telomeres and altered telomere related gene expression are associated with amplification, implicating telomere attrition as a promoter of this type of aberration in breast cancer. On the other hand, the numbers of chromosomal alterations, particularly low level changes, are associated with altered expression of genes in other functional classes (mitosis, cell cycle, DNA replication and repair). Further, although loss of function instability phenotypes have been demonstrated for many of the genes in model systems, we observed enhanced expression of most genes in tumors, indicating that over expression, rather than deficiency underlies instability. Many of the genes associated with higher frequency of copy number aberrations are direct targets of E2F, supporting the hypothesis that deregulation of the Rb pathway is a major contributor to chromosomal instability in breast tumors. These observations are consistent with failure to find mutations in sporadic tumors in genes that have roles in maintenance or manipulation of the genome

  10. Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area.

    Science.gov (United States)

    Nakano, Kazuma; Shiroma, Akino; Shimoji, Makiko; Tamotsu, Hinako; Ashimine, Noriko; Ohki, Shun; Shinzato, Misuzu; Minami, Maiko; Nakanishi, Tetsuhiro; Teruya, Kuniko; Satou, Kazuhito; Hirano, Takashi

    2017-07-01

    PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II's sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.

  11. Fast and robust methods for full genome sequencing of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Type 1 and Type 2

    DEFF Research Database (Denmark)

    Kvisgaard, Lise Kirstine; Hjulsager, Charlotte Kristiane; Fahnøe, Ulrik

    . In the present study, fast and robust methods for long range RT-PCR amplification and subsequent next generation sequencing (NGS) of PRRSV Type 1 and Type 2 viruses were developed and validated on nine Type 1 and nine Type 2 PRRSV viruses. The methods were shown to generate robust and reliable sequences both...... on primary material and cell culture adapted viruses and the protocols were shown to perform well on all three NGS platforms tested (Roche 454 FLX, Illumina HiSeq 2000, and Ion Torrent PGM™ Sequencer). To complete the sequences at the 5’ end, 5’ Rapid Amplification of cDNA Ends (5’ RACE) was conducted...... followed by cycle sequencing of clones. The genome lengths were determined to be 14,876-15,098 and 15,342-15,408 nucleotides long for the Type 1 and Type 2 strains, respectively. These methods will greatly facilitate the generation of more complete genome PRRSV sequences globally which in turn may lead...

  12. Identifying genomic changes associated with insecticide resistance in the dengue mosquito Aedes aegypti by deep targeted sequencing

    Science.gov (United States)

    Faucon, Frederic; Dusfour, Isabelle; Gaude, Thierry; Navratil, Vincent; Boyer, Frederic; Chandre, Fabrice; Sirisopa, Patcharawan; Thanispong, Kanutcharee; Juntarajumnong, Waraporn; Poupardin, Rodolphe; Chareonviriyaphap, Theeraphap; Girod, Romain; Corbel, Vincent; Reynaud, Stephane; David, Jean-Philippe

    2015-01-01

    The capacity of mosquitoes to resist insecticides threatens the control of diseases such as dengue and malaria. Until alternative control tools are implemented, characterizing resistance mechanisms is crucial for managing resistance in natural populations. Insecticide biodegradation by detoxification enzymes is a common resistance mechanism; however, the genomic changes underlying this mechanism have rarely been identified, precluding individual resistance genotyping. In particular, the role of copy number variations (CNVs) and polymorphisms of detoxification enzymes have never been investigated at the genome level, although they can represent robust markers of metabolic resistance. In this context, we combined target enrichment with high-throughput sequencing for conducting the first comprehensive screening of gene amplifications and polymorphisms associated with insecticide resistance in mosquitoes. More than 760 candidate genes were captured and deep sequenced in several populations of the dengue mosquito Ae. aegypti displaying distinct genetic backgrounds and contrasted resistance levels to the insecticide deltamethrin. CNV analysis identified 41 gene amplifications associated with resistance, most affecting cytochrome P450s overtranscribed in resistant populations. Polymorphism analysis detected more than 30,000 variants and strong selection footprints in specific genomic regions. Combining Bayesian and allele frequency filtering approaches identified 55 nonsynonymous variants strongly associated with resistance. Both CNVs and polymorphisms were conserved within regions but differed across continents, confirming that genomic changes underlying metabolic resistance to insecticides are not universal. By identifying novel DNA markers of insecticide resistance, this study opens the way for tracking down metabolic changes developed by mosquitoes to resist insecticides within and among populations. PMID:26206155

  13. Random Tagging Genotyping by Sequencing (rtGBS, an Unbiased Approach to Locate Restriction Enzyme Sites across the Target Genome.

    Directory of Open Access Journals (Sweden)

    Elena Hilario

    Full Text Available Genotyping by sequencing (GBS is a restriction enzyme based targeted approach developed to reduce the genome complexity and discover genetic markers when a priori sequence information is unavailable. Sufficient coverage at each locus is essential to distinguish heterozygous from homozygous sites accurately. The number of GBS samples able to be pooled in one sequencing lane is limited by the number of restriction sites present in the genome and the read depth required at each site per sample for accurate calling of single-nucleotide polymorphisms. Loci bias was observed using a slight modification of the Elshire et al.some restriction enzyme sites were represented in higher proportions while others were poorly represented or absent. This bias could be due to the quality of genomic DNA, the endonuclease and ligase reaction efficiency, the distance between restriction sites, the preferential amplification of small library restriction fragments, or bias towards cluster formation of small amplicons during the sequencing process. To overcome these issues, we have developed a GBS method based on randomly tagging genomic DNA (rtGBS. By randomly landing on the genome, we can, with less bias, find restriction sites that are far apart, and undetected by the standard GBS (stdGBS method. The study comprises two types of biological replicates: six different kiwifruit plants and two independent DNA extractions per plant; and three types of technical replicates: four samples of each DNA extraction, stdGBS vs. rtGBS methods, and two independent library amplifications, each sequenced in separate lanes. A statistically significant unbiased distribution of restriction fragment size by rtGBS showed that this method targeted 49% (39,145 of BamH I sites shared with the reference genome, compared to only 14% (11,513 by stdGBS.

  14. Isothermal amplification detection of nucleic acids by a double-nicked beacon.

    Science.gov (United States)

    Shi, Chao; Zhou, Meiling; Pan, Mei; Zhong, Guilin; Ma, Cuiping

    2016-03-01

    Isothermal and rapid amplification detection of nucleic acids is an important technology in environmental monitoring, foodborne pathogen detection, and point-of-care clinical diagnostics. Here we have developed a novel method of isothermal signal amplification for single-stranded DNA (ssDNA) detection. The ssDNA target could be used as an initiator, coupled with a double-nicked molecular beacon, to originate amplification cycles, achieving cascade signal amplification. In addition, the method showed good specificity and strong anti-jamming capability. Overall, it is a one-pot and isothermal strand displacement amplification method without the requirement of a stepwise procedure, which greatly simplifies the experimental procedure and decreases the probability of contamination of samples. With its advantages, the method would be very useful to detect nucleic acids in point-of-care or field use. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Amplification of surface acoustic waves by transverse electric current in piezoelectric semiconductors

    DEFF Research Database (Denmark)

    Gulyaev, Yuri V.

    1974-01-01

    acoustoelectric effect but also lead to amplification of surface acoustic waves by electron drift perpendicular to the surface. For Love waves in a piezoelectric semiconductor film on a highly conducting substrate, the amplification coefficient is found and the conditions necessary for amplification...

  16. A method for release and multiple strand amplification of small quantities of DNA from endospores of the fastidious bacterium Pasteuria penetrans.

    Science.gov (United States)

    Mauchline, T H; Mohan, S; Davies, K G; Schaff, J E; Opperman, C H; Kerry, B R; Hirsch, P R

    2010-05-01

    To establish a reliable protocol to extract DNA from Pasteuria penetrans endospores for use as template in multiple strand amplification, thus providing sufficient material for genetic analyses. To develop a highly sensitive PCR-based diagnostic tool for P. penetrans. An optimized method to decontaminate endospores, release and purify DNA enabled multiple strand amplification. DNA purity was assessed by cloning and sequencing gyrB and 16S rRNA gene fragments obtained from PCR using generic primers. Samples indicated to be 100%P. penetrans by the gyrB assay were estimated at 46% using the 16S rRNA gene. No bias was detected on cloning and sequencing 12 housekeeping and sporulation gene fragments from amplified DNA. The detection limit by PCR with Pasteuria-specific 16S rRNA gene primers following multiple strand amplification of DNA extracted using the method was a single endospore. Generation of large quantities DNA will facilitate genomic sequencing of P. penetrans. Apparent differences in sample purity are explained by variations in 16S rRNA gene copy number in Eubacteria leading to exaggerated estimations of sample contamination. Detection of single endospores will facilitate investigations of P. penetrans molecular ecology. These methods will advance studies on P. penetrans and facilitate research on other obligate and fastidious micro-organisms where it is currently impractical to obtain DNA in sufficient quantity and quality.

  17. Telomerase Repeated Amplification Protocol (TRAP).

    Science.gov (United States)

    Mender, Ilgen; Shay, Jerry W

    2015-11-20

    Telomeres are found at the end of eukaryotic linear chromosomes, and proteins that bind to telomeres protect DNA from being recognized as double-strand breaks thus preventing end-to-end fusions (Griffith et al. , 1999). However, due to the end replication problem and other factors such as oxidative damage, the limited life span of cultured cells (Hayflick limit) results in progressive shortening of these protective structures (Hayflick and Moorhead, 1961; Olovnikov, 1973). The ribonucleoprotein enzyme complex telomerase-consisting of a protein catalytic component hTERT and a functional RNA component hTR or hTERC - counteracts telomere shortening by adding telomeric repeats to the end of chromosomes in ~90% of primary human tumors and in some transiently proliferating stem-like cells (Shay and Wright, 1996; Shay and Wright, 2001). This results in continuous proliferation of cells which is a hallmark of cancer. Therefore, telomere biology has a central role in aging, cancer progression/metastasis as well as targeted cancer therapies. There are commonly used methods in telomere biology such as Telomere Restriction Fragment (TRF) (Mender and Shay, 2015b), Telomere Repeat Amplification Protocol (TRAP) and Telomere dysfunction Induced Foci (TIF) analysis (Mender and Shay, 2015a). In this detailed protocol we describe Telomere Repeat Amplification Protocol (TRAP). The TRAP assay is a popular method to determine telomerase activity in mammalian cells and tissue samples (Kim et al. , 1994). The TRAP assay includes three steps: extension, amplification, and detection of telomerase products. In the extension step, telomeric repeats are added to the telomerase substrate (which is actually a non telomeric oligonucleotide, TS) by telomerase. In the amplification step, the extension products are amplified by the polymerase chain reaction (PCR) using specific primers (TS upstream primer and ACX downstream primer) and in the detection step, the presence or absence of telomerase is

  18. Probabilistic Sensitivity Amplification Control for Lower Extremity Exoskeleton

    Directory of Open Access Journals (Sweden)

    Likun Wang

    2018-03-01

    Full Text Available To achieve ideal force control of a functional autonomous exoskeleton, sensitivity amplification control is widely used in human strength augmentation applications. The original sensitivity amplification control aims to increase the closed-loop control system sensitivity based on positive feedback without any sensors between the pilot and the exoskeleton. Thus, the measurement system can be greatly simplified. Nevertheless, the controller lacks the ability to reject disturbance and has little robustness to the variation of the parameters. Consequently, a relatively precise dynamic model of the exoskeleton system is desired. Moreover, the human-robot interaction (HRI cannot be interpreted merely as a particular part of the driven torque quantitatively. Therefore, a novel control methodology, so-called probabilistic sensitivity amplification control, is presented in this paper. The innovation of the proposed control algorithm is two-fold: distributed hidden-state identification based on sensor observations and evolving learning of sensitivity factors for the purpose of dealing with the variational HRI. Compared to the other state-of-the-art algorithms, we verify the feasibility of the probabilistic sensitivity amplification control with several experiments, i.e., distributed identification model learning and walking with a human subject. The experimental result shows potential application feasibility.

  19. Multiple displacement amplification as an adjunct to PCR-based detection of Staphylococcus aureus in synovial fluid

    Directory of Open Access Journals (Sweden)

    Johnson Sandra

    2010-10-01

    Full Text Available Abstract Background Detection of bacterial nucleic acids in synovial fluid following total joint arthroplasty with suspected infection can be difficult; among other technical challenges, inhibitors in the specimens require extensive sample preparation and can diminish assay sensitivity even using polymerase chain reaction (PCR-based methods. To address this problem a simple protocol for prior use of multiple displacement amplification (MDA as an adjunct to PCR was established and tested on both purified S. aureus DNA as well as on clinical samples known to contain S. aureus nucleic acids. Findings A single round of MDA on purified nucleic acids resulted in a > 300 thousand-fold increase in template DNA on subsequent quantitative PCR (qPCR analysis. MDA use on clinical samples resulted in at least a 100-fold increase in sensitivity on subsequent qPCR and required no sample preparation other than a simple alkali/heat lysis step. Mixed samples of S. aureus DNA with a 103 - 104-fold excess of human genomic DNA still allowed for MDA amplification of the minor bacterial component to the threshold of detectability. Conclusion MDA is a promising technique that may serve to significantly enhance the sensitivity of molecular assays in cases of suspected joint infection while simultaneously reducing the specimen handling required.

  20. Detection of Puccinia kuehnii Causing Sugarcane Orange Rust with a Loop-Mediated Isothermal Amplification-Based Assay.

    Science.gov (United States)

    Chandra, Amaresh; Keizerweerd, Amber T; Grisham, Michael P

    2016-03-01

    Puccinia kuehnii is a fungal pathogen that causes orange rust in sugarcane, which is now prevalent in many countries. At the early stage of disease, it is almost indistinguishable from brown rust, which is caused by Puccinia melanocephala. Although several PCR assays are available to detect these diseases, the loop-mediated isothermal amplification (LAMP)-based assay has been reported to be more economical and easier to perform. Under isothermal conditions, DNA is amplified with high specificity and rapidity. Moreover, visual judgment of color change without further post-amplification processing makes the method convenient. The present study was undertaken to detect P. kuehnii genomic DNA using four primers corresponding to a unique DNA sequence of P. kuehnii. The LAMP assay was found to be optimal when 8 mM MgSO4 was used and the reaction was incubated at 63 °C for 90 min. Positive samples showed a color change from orange to green upon SYBR Green I dye addition. Specificity of the LAMP test was checked with DNA of P. melanocephala, which showed no reaction. Sensitivity of the LAMP method was observed to be the same as real-time PCR at 0.1 ng, thus providing a rapid and more affordable option for early disease detection.

  1. Insights into the binding specificity of wild type and mutated wheat germ agglutinin towards Neu5Acα(2-3)Gal: a study by in silico mutations and molecular dynamics simulations.

    Science.gov (United States)

    Parasuraman, Ponnusamy; Murugan, Veeramani; Selvin, Jeyasigamani F A; Gromiha, M Michael; Fukui, Kazuhiko; Veluraja, Kasinadar

    2014-08-01

    Wheat germ agglutinin (WGA) is a plant lectin, which specifically recognizes the sugars NeuNAc and GlcNAc. Mutated WGA with enhanced binding specificity can be used as biomarkers for cancer. In silico mutations are performed at the active site of WGA to enhance the binding specificity towards sialylglycans, and molecular dynamics simulations of 20 ns are carried out for wild type and mutated WGAs (WGA1, WGA2, and WGA3) in complex with sialylgalactose to examine the change in binding specificity. MD simulations reveal the change in binding specificity of wild type and mutated WGAs towards sialylgalactose and bound conformational flexibility of sialylgalactose. The mutated polar amino acid residues Asn114 (S114N), Lys118 (G118K), and Arg118 (G118R) make direct and water mediated hydrogen bonds and hydrophobic interactions with sialylgalactose. An analysis of possible hydrogen bonds, hydrophobic interactions, total pair wise interaction energy between active site residues and sialylgalactose and MM-PBSA free energy calculation reveals the plausible binding modes and the role of water in stabilizing different binding modes. An interesting observation is that the binding specificity of mutated WGAs (cyborg lectin) towards sialylgalactose is found to be higher in double point mutation (WGA3). One of the substituted residues Arg118 plays a crucial role in sugar binding. Based on the interactions and energy calculations, it is concluded that the order of binding specificity of WGAs towards sialylgalactose is WGA3 > WGA1 > WGA2 > WGA. On comparing with the wild type, double point mutated WGA (WGA3) exhibits increased specificity towards sialylgalactose, and thus, it can be effectively used in targeted drug delivery and as biological cell marker in cancer therapeutics. Copyright © 2014 John Wiley & Sons, Ltd.

  2. Next generation Chirped Pulse Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Nees, J; Biswal, S; Mourou, G [Univ. Michigan, Center for Ultrafast Optical Science, Ann Arbor, MI (United States); Nishimura, Akihiko; Takuma, Hiroshi

    1998-03-01

    The limiting factors of Chirped Pulse Amplification (CPA) are discussed and experimental results of CPA in Yb:glass regenerative amplifier are given. Scaling of Yb:glass to the petawatt level is briefly discussed. (author)

  3. Laser amplification in excited dielectrics

    Science.gov (United States)

    Winkler, Thomas; Haahr-Lillevang, Lasse; Sarpe, Cristian; Zielinski, Bastian; Götte, Nadine; Senftleben, Arne; Balling, Peter; Baumert, Thomas

    2018-01-01

    Wide-bandgap dielectrics such as glasses or water are transparent at visible and infrared wavelengths. This changes when they are exposed to ultrashort and highly intense laser pulses. Different interaction mechanisms lead to the appearance of various transient nonlinear optical phenomena. Using these, the optical properties of dielectrics can be controlled from the transparent to the metal-like state. Here we expand this range by a yet unexplored mechanism in excited dielectrics: amplification. In a two-colour pump-probe experiment, we show that a 400 nm femtosecond laser pulse is coherently amplified inside an excited sapphire sample on a scale of a few micrometres. Simulations strongly support the proposed two-photon stimulated emission process, which is temporally and spatially controllable. Consequently, we expect applications in all fields that demand strongly localized amplification.

  4. Development of a highly sensitive loop-mediated isothermal amplification (LAMP) method for the detection of Loa loa.

    Science.gov (United States)

    Fernández-Soto, Pedro; Mvoulouga, Prosper Obolo; Akue, Jean Paul; Abán, Julio López; Santiago, Belén Vicente; Sánchez, Miguel Cordero; Muro, Antonio

    2014-01-01

    The filarial parasite Loa loa, the causative agent of loiasis, is endemic in Central and Western Africa infecting 3-13 million people. L. loa has been associated with fatal encephalopathic reactions in high Loa-infected individuals receiving ivermectin during mass drug administration programs for the control of onchocerciasis and lymphatic filariasis. In endemic areas, the only diagnostic method routinely used is the microscopic examination of mid-day blood samples by thick blood film. Improved methods for detection of L. loa are needed in endemic regions with limited resources, where delayed diagnosis results in high mortality. We have investigated the use of a loop-mediated isothermal amplification (LAMP) assay to facilitate rapid, inexpensive, molecular diagnosis of loiasis. Primers for LAMP were designed from a species-specific repetitive DNA sequence from L. loa retrieved from GenBank. Genomic DNA of a L. loa adult worm was used to optimize the LAMP conditions using a thermocycler or a conventional heating block. Amplification of DNA in the LAMP mixture was visually inspected for turbidity as well as addition of fluorescent dye. LAMP specificity was evaluated using DNA from other parasites; sensitivity was evaluated using DNA from L. loa 10-fold serially diluted. Simulated human blood samples spiked with DNA from L. loa were also tested for sensitivity. Upon addition of fluorescent dye, all positive reactions turned green while the negative controls remained orange under ambient light. After electrophoresis on agarose gels, a ladder of multiple bands of different sizes could be observed in positive samples. The detection limit of the assay was found to be as little as 0.5 ag of L. loa genomic DNA when using a heating block. We have designed, for the first time, a highly sensitive LAMP assay for the detection of L. loa which is potentially adaptable for field diagnosis and disease surveillance in loiasis-endemic areas.

  5. Fast implementation of length-adaptive privacy amplification in quantum key distribution

    International Nuclear Information System (INIS)

    Zhang Chun-Mei; Li Mo; Huang Jing-Zheng; Li Hong-Wei; Li Fang-Yi; Wang Chuan; Yin Zhen-Qiang; Chen Wei; Han Zhen-Fu; Treeviriyanupab Patcharapong; Sripimanwat Keattisak

    2014-01-01

    Post-processing is indispensable in quantum key distribution (QKD), which is aimed at sharing secret keys between two distant parties. It mainly consists of key reconciliation and privacy amplification, which is used for sharing the same keys and for distilling unconditional secret keys. In this paper, we focus on speeding up the privacy amplification process by choosing a simple multiplicative universal class of hash functions. By constructing an optimal multiplication algorithm based on four basic multiplication algorithms, we give a fast software implementation of length-adaptive privacy amplification. “Length-adaptive” indicates that the implementation of privacy amplification automatically adapts to different lengths of input blocks. When the lengths of the input blocks are 1 Mbit and 10 Mbit, the speed of privacy amplification can be as fast as 14.86 Mbps and 10.88 Mbps, respectively. Thus, it is practical for GHz or even higher repetition frequency QKD systems. (general)

  6. Analysis of the N-glycosylation of the serum glycoproteins defined by Con A, PHA-E, RCA1 and WGA in Chinese patients with gastrointestinal diseases.

    Science.gov (United States)

    Zhang, J; Chen, Y; Dai, X; Wang, S

    1996-03-01

    Glycoproteins from tumor cells isolated by Con A, PHA-E and RCA1, were coupled to horse radish peroxidase and then used as reporters to evaluate the oligosaccharides change on serum glycoproteins in digestive tract diseases and individual healthy persons. A value 2 standard deviation above the mean of the healthy person group was considered positive. The results indicated that the positive rates of the oligosaccharides bound with Con A, PHA-E and RCA1 were 51% (61/119), 70.6% (84/119), and 76.4% (91/119) in patients with gastric cancer; 53.3% (16/30), 46.7% (14/30) and 66.7% (20/30) in esophageal cancers; 28.5% (15/49), 49.0% (24/49) and 46.9% (23/49) in colorectal cancer; 78.1% (25/32), 81.3% (26/32) and 31.3% (10/32) in gallbladder tumors; 60.4% (29/48), 54.1% (26/48) and 39.5% (19/48) in hepatocellular cancer. The positive rates among the patients with benign diseases were also found to be 12.7% (16/126), 15.5% (20/126) and 26.2% (33/126). However, the expression of the oligosaccharide moieties recognized by WGA on the glycoprotein bound with Con A, was significantly lower in the serum of patients with tumors than in those with benign disease and in healthy individuals (P < 0.01, P < 0.01).

  7. Direct detection of chicken genomic DNA for gender determination by thymine-DNA glycosylase.

    Science.gov (United States)

    Porat, N; Bogdanov, K; Danielli, A; Arie, A; Samina, I; Hadani, A

    2011-02-01

    1. Birds, especially nestlings, are generally difficult to sex by morphology and early detection of chick gender in ovo in the hatchery would facilitate removal of unwanted chicks and diminish welfare objections regarding culling after hatch. 2. We describe a method to determine chicken gender without the need for PCR via use of Thymine-DNA Glycosylase (TDG). TDG restores thymine (T)/guanine (G) mismatches to cytosine (C)/G. We show here, that like DNA Polymerase, TDG can recognise, bind and function on a primer hybridised to chicken genomic DNA. 3. The primer contained a T to mismatch a G in a chicken genomic template and the T/G was cleaved with high fidelity by TDG. Thus, the chicken genomic DNA can be identified without PCR amplification via direct and linear detection. Sensitivity was increased using gender specific sequences from the chicken genome. 4. Currently, these are laboratory results, but we anticipate that further development will allow this method to be used in non-laboratory settings, where PCR cannot be employed.

  8. The clinical application of single-sperm-based SNP haplotyping for PGD of osteogenesis imperfecta.

    Science.gov (United States)

    Chen, Linjun; Diao, Zhenyu; Xu, Zhipeng; Zhou, Jianjun; Yan, Guijun; Sun, Haixiang

    2018-05-15

    : short tandem repeat; TE: trophectoderm; WGA: whole-genome amplification.

  9. GtfA and GtfB Are Both Required for Protein O-Glycosylation in Lactobacillus plantarum

    Science.gov (United States)

    Lee, I-Chiao; van Swam, Iris I.; Tomita, Satoru; Morsomme, Pierre; Rolain, Thomas; Hols, Pascal; Bron, Peter A.

    2014-01-01

    Acm2, the major autolysin of Lactobacillus plantarum WCFS1, was recently found to be O-glycosylated with N-acetylhexosamine, likely N-acetylglucosamine (GlcNAc). In this study, we set out to identify the glycosylation machinery by employing a comparative genomics approach to identify Gtf1 homologues, which are involved in fimbria-associated protein 1 (Fap1) glycosylation in Streptococcus parasanguinis. This in silico approach resulted in the identification of 6 candidate L. plantarum WCFS1 genes with significant homology to Gtf1, namely, tagE1 to tagE6. These candidate genes were targeted by systematic gene deletion, followed by assessment of the consequences on glycosylation of Acm2. We observed a changed mobility of Acm2 on SDS-PAGE in the tagE5E6 deletion strain, while deletion of other tagE genes resulted in Acm2 mobility comparable to that of the wild type. Subsequent mass spectrometry analysis of excised and in-gel-digested Acm2 confirmed the loss of glycosylation on Acm2 in the tagE5E6 deletion mutant, whereas a lectin blot using GlcNAc-specific succinylated wheat germ agglutinin (sWGA) revealed that besides Acm2, tagE5E6 deletion also abolished all but one other sWGA-reactive, protease-sensitive signal. Only complementation of both tagE5 and tagE6 restored those sWGA lectin signals, establishing that TagE5 and TagE6 are both required for the glycosylation of Acm2 as well as the vast majority of other sWGA-reactive proteins. Finally, sWGA lectin blotting experiments using a panel of 8 other L. plantarum strains revealed that protein glycosylation is a common feature in L. plantarum strains. With the establishment of these enzymes as protein glycosyltransferases, we propose to rename TagE5 and TagE6 as GtfA and GtfB, respectively. PMID:24532775

  10. Sparse representation and Bayesian detection of genome copy number alterations from microarray data.

    Science.gov (United States)

    Pique-Regi, Roger; Monso-Varona, Jordi; Ortega, Antonio; Seeger, Robert C; Triche, Timothy J; Asgharzadeh, Shahab

    2008-02-01

    Genomic instability in cancer leads to abnormal genome copy number alterations (CNA) that are associated with the development and behavior of tumors. Advances in microarray technology have allowed for greater resolution in detection of DNA copy number changes (amplifications or deletions) across the genome. However, the increase in number of measured signals and accompanying noise from the array probes present a challenge in accurate and fast identification of breakpoints that define CNA. This article proposes a novel detection technique that exploits the use of piece wise constant (PWC) vectors to represent genome copy number and sparse Bayesian learning (SBL) to detect CNA breakpoints. First, a compact linear algebra representation for the genome copy number is developed from normalized probe intensities. Second, SBL is applied and optimized to infer locations where copy number changes occur. Third, a backward elimination (BE) procedure is used to rank the inferred breakpoints; and a cut-off point can be efficiently adjusted in this procedure to control for the false discovery rate (FDR). The performance of our algorithm is evaluated using simulated and real genome datasets and compared to other existing techniques. Our approach achieves the highest accuracy and lowest FDR while improving computational speed by several orders of magnitude. The proposed algorithm has been developed into a free standing software application (GADA, Genome Alteration Detection Algorithm). http://biron.usc.edu/~piquereg/GADA

  11. Amplification-free liquid biopsy by fluorescence approach

    DEFF Research Database (Denmark)

    Uhd, Jesper; Okholm, Anders; Kjems, Jargen

    2017-01-01

    Liquid biopsy is an attractive new paradigm of modern cancer research and clinical oncology. Synergy of fluorescence microscopy with mutation specific molecular probes is a method that we developed for the detection of tumor related circulating DNA, ctDNA. The present detection methods of ctDNA...... samples include amplification-based techniques that have multiple challenges, are often time consuming and rather expensive. In this work, we successfully applied the hybridization assay and advanced microscopy for the reliable amplification-free detection and quantification of cancer associated mutations...... in ctDNA....

  12. Amplification of LAPTM4B and YWHAZ contributes to chemotherapy resistance and recurrence of breast cancer

    DEFF Research Database (Denmark)

    Szallasi, Zoltan Imre; Li, Yang; Zou, Lihua

    2010-01-01

    Adjuvant chemotherapy for breast cancer after surgery has effectively lowered metastatic recurrence rates. However, a considerable proportion of women suffer recurrent cancer at distant metastatic sites despite adjuvant treatment. Identification of the genes crucial for tumor response to specific...... chemotherapy drugs is a challenge but is necessary to improve outcomes. By using integrated genomics, we identified a small number of overexpressed and amplified genes from chromosome 8q22 that were associated with early disease recurrence despite anthracycline-based adjuvant chemotherapy. We confirmed...... that 8q22 amplification and overexpression of LAPTM4B and YWHAZ contribute to de novo chemoresistance to anthracyclines and are permissive for metastatic recurrence. Overexpression of these two genes may predict anthracycline resistance and influence selection of chemotherapy....

  13. Identification of SEC62 as a potential marker for 3q amplification and cellular migration in dysplastic cervical lesions

    International Nuclear Information System (INIS)

    Linxweiler, Maximilian; Bochen, Florian; Schick, Bernhard; Wemmert, Silke; Al Kadah, Basel; Greiner, Markus; Hasenfus, Andrea; Bohle, Rainer-Maria; Juhasz-Böss, Ingolf; Solomayer, Erich-Franz; Takacs, Zoltan Ferenc

    2016-01-01

    Chromosome 3 amplification affecting the 3q26 region is a common genomic alteration in cervical cancer, typically marking the transition of precancerous intraepithelial lesions to an invasive phenotype. Though potential 3q encoded target genes of this amplification have been identified, a functional correlation of potential oncogenic function is still missing. In this study, we investigated copy number changes and the expression level of SEC62 encoded at 3q26.2 as a new potential 3q oncogene in dysplastic cervical lesions and analyzed its role in cervical cancer cell biology. Expression levels of Sec62 and vimentin were analyzed in liquid based cytology specimens from 107 women with varying grades of cervical dysplasia ranging from normal cases to cancer by immunofluorescence cytology. Additionally, a subset of 20 representative cases was used for FISH analyses targeting SEC62. To further explore the functional role of Sec62 in cervical cancer, HeLa cells were transfected with a SEC62 plasmid or SEC62 siRNA and analyzed for their proliferation and migration potential using real-time monitoring and trans-well systems as well as changes in the expression of EMT markers. FISH analyses of the swabbed cells showed a rising number of SEC62 gains and amplifications correlating to the grade of dysplasia with the highest incidence in high grade squamous intraepithelial lesions and squamous cell carcinomas. When analyzing the expression level of Sec62 and vimentin, we found a gradually increasing expression level of both proteins according to the severity of the dysplasia. In functional analyses, SEC62 silencing inhibited and SEC62 overexpression stimulated the migration of HeLa cells with only marginal effects on cell proliferation, the expression level of EMT markers and the cytoskeleton structure. Our study suggests SEC62 as a target gene of 3q26 amplification and a stimulator of cellular migration in dysplastic cervical lesions. Hence, SEC62 could serve as a potential

  14. Human minisatellite alleles detectable only after PCR amplification.

    Science.gov (United States)

    Armour, J A; Crosier, M; Jeffreys, A J

    1992-01-01

    We present evidence that a proportion of alleles at two human minisatellite loci is undetected by standard Southern blot hybridization. In each case the missing allele(s) can be identified after PCR amplification and correspond to tandem arrays too short to detect by hybridization. At one locus, there is only one undetected allele (population frequency 0.3), which contains just three repeat units. At the second locus, there are at least five undetected alleles (total population frequency 0.9) containing 60-120 repeats; they are not detected because these tandem repeats give very poor signals when used as a probe in standard Southern blot hybridization, and also cross-hybridize with other sequences in the genome. Under these circumstances only signals from the longest tandemly repeated alleles are detectable above the nonspecific background. The structures of these loci have been compared in human and primate DNA, and at one locus the short human allele containing three repeat units is shown to be an intermediate state in the expansion of a monomeric precursor allele in primates to high copy number in the longer human arrays. We discuss the implications of such loci for studies of human populations, minisatellite isolation by cloning, and the evolution of highly variable tandem arrays.

  15. Breakdown of hot-spot model in determining convective amplification in large homogeneous systems

    International Nuclear Information System (INIS)

    Mounaix, Philippe; Divol, Laurent

    2004-01-01

    Convective amplification in large homogeneous systems is studied, both analytically and numerically, in the case of a linear diffraction-free stochastic amplifier. Overall amplification does not result from successive amplifications in small scale high intensity hot spots, but from a single amplification in a delocalized mode of the driver field spreading over the whole interaction length. For this model, the hot-spot approach is found to systematically underestimate the gain factor by more than 50%

  16. Increased centrosome amplification in aged stem cells of the Drosophila midgut

    International Nuclear Information System (INIS)

    Park, Joung-Sun; Pyo, Jung-Hoon; Na, Hyun-Jin; Jeon, Ho-Jun; Kim, Young-Shin; Arking, Robert; Yoo, Mi-Ae

    2014-01-01

    Highlights: • Increased centrosome amplification in ISCs of aged Drosophila midguts. • Increased centrosome amplification in ISCs of oxidative stressed Drosophila midguts. • Increased centrosome amplification in ISCs by overexpression of PVR, EGFR, and AKT. • Supernumerary centrosomes can be responsible for abnormal ISC polyploid cells. • Supernumerary centrosomes can be a useful marker for aging stem cells. - Abstract: Age-related changes in long-lived tissue-resident stem cells may be tightly linked to aging and age-related diseases such as cancer. Centrosomes play key roles in cell proliferation, differentiation and migration. Supernumerary centrosomes are known to be an early event in tumorigenesis and senescence. However, the age-related changes of centrosome duplication in tissue-resident stem cells in vivo remain unknown. Here, using anti-γ-tubulin and anti-PH3, we analyzed mitotic intestinal stem cells with supernumerary centrosomes in the adult Drosophila midgut, which may be a versatile model system for stem cell biology. The results showed increased centrosome amplification in intestinal stem cells of aged and oxidatively stressed Drosophila midguts. Increased centrosome amplification was detected by overexpression of PVR, EGFR, and AKT in intestinal stem cells/enteroblasts, known to mimic age-related changes including hyperproliferation of intestinal stem cells and hyperplasia in the midgut. Our data show the first direct evidence for the age-related increase of centrosome amplification in intestinal stem cells and suggest that the Drosophila midgut is an excellent model for studying molecular mechanisms underlying centrosome amplification in aging adult stem cells in vivo

  17. Increased centrosome amplification in aged stem cells of the Drosophila midgut

    Energy Technology Data Exchange (ETDEWEB)

    Park, Joung-Sun; Pyo, Jung-Hoon; Na, Hyun-Jin; Jeon, Ho-Jun; Kim, Young-Shin [Department of Molecular Biology, Pusan National University, Busan 609-735 (Korea, Republic of); Arking, Robert, E-mail: aa2210@wayne.edu [Department of Biological Sciences, Wayne State University, Detroit, MI 48202 (United States); Yoo, Mi-Ae, E-mail: mayoo@pusan.ac.kr [Department of Molecular Biology, Pusan National University, Busan 609-735 (Korea, Republic of)

    2014-07-25

    Highlights: • Increased centrosome amplification in ISCs of aged Drosophila midguts. • Increased centrosome amplification in ISCs of oxidative stressed Drosophila midguts. • Increased centrosome amplification in ISCs by overexpression of PVR, EGFR, and AKT. • Supernumerary centrosomes can be responsible for abnormal ISC polyploid cells. • Supernumerary centrosomes can be a useful marker for aging stem cells. - Abstract: Age-related changes in long-lived tissue-resident stem cells may be tightly linked to aging and age-related diseases such as cancer. Centrosomes play key roles in cell proliferation, differentiation and migration. Supernumerary centrosomes are known to be an early event in tumorigenesis and senescence. However, the age-related changes of centrosome duplication in tissue-resident stem cells in vivo remain unknown. Here, using anti-γ-tubulin and anti-PH3, we analyzed mitotic intestinal stem cells with supernumerary centrosomes in the adult Drosophila midgut, which may be a versatile model system for stem cell biology. The results showed increased centrosome amplification in intestinal stem cells of aged and oxidatively stressed Drosophila midguts. Increased centrosome amplification was detected by overexpression of PVR, EGFR, and AKT in intestinal stem cells/enteroblasts, known to mimic age-related changes including hyperproliferation of intestinal stem cells and hyperplasia in the midgut. Our data show the first direct evidence for the age-related increase of centrosome amplification in intestinal stem cells and suggest that the Drosophila midgut is an excellent model for studying molecular mechanisms underlying centrosome amplification in aging adult stem cells in vivo.

  18. The first complete chloroplast genome sequence of a lycophyte,Huperzia lucidula (Lycopodiaceae)

    Energy Technology Data Exchange (ETDEWEB)

    Wolf, Paul G.; Karol, Kenneth G.; Mandoli, Dina F.; Kuehl,Jennifer V.; Arumuganathan, K.; Ellis, Mark W.; Mishler, Brent D.; Kelch,Dean G.; Olmstead, Richard G.; Boore, Jeffrey L.

    2005-02-01

    We used a unique combination of techniques to sequence the first complete chloroplast genome of a lycophyte, Huperzia lucidula. This plant belongs to a significant clade hypothesized to represent the sister group to all other vascular plants. We used fluorescence-activated cell sorting (FACS) to isolate the organelles, rolling circle amplification (RCA) to amplify the genome, and shotgun sequencing to 8x depth coverage to obtain the complete chloroplast genome sequence. The genome is 154,373bp, containing inverted repeats of 15,314 bp each, a large single-copy region of 104,088 bp, and a small single-copy region of 19,671 bp. Gene order is more similar to those of mosses, liverworts, and hornworts than to gene order for other vascular plants. For example, the Huperziachloroplast genome possesses the bryophyte gene order for a previously characterized 30 kb inversion, thus supporting the hypothesis that lycophytes are sister to all other extant vascular plants. The lycophytechloroplast genome data also enable a better reconstruction of the basaltracheophyte genome, which is useful for inferring relationships among bryophyte lineages. Several unique characters are observed in Huperzia, such as movement of the gene ndhF from the small single copy region into the inverted repeat. We present several analyses of evolutionary relationships among land plants by using nucleotide data, amino acid sequences, and by comparing gene arrangements from chloroplast genomes. The results, while still tentative pending the large number of chloroplast genomes from other key lineages that are soon to be sequenced, are intriguing in themselves, and contribute to a growing comparative database of genomic and morphological data across the green plants.

  19. Optimized thermal amplification in a radiative transistor

    Energy Technology Data Exchange (ETDEWEB)

    Prod' homme, Hugo; Ordonez-Miranda, Jose; Ezzahri, Younes, E-mail: younes.ezzahri@univ-poitiers.fr; Drevillon, Jeremie; Joulain, Karl [Institut Pprime, CNRS, Université de Poitiers, ISAE-ENSMA, F-86962 Futuroscope Chasseneuil (France)

    2016-05-21

    The thermal performance of a far-field radiative transistor made up of a VO{sub 2} base in between a blackbody collector and a blackbody emitter is theoretically studied and optimized. This is done by using the grey approximation on the emissivity of VO{sub 2} and deriving analytical expressions for the involved heat fluxes and transistor amplification factor. It is shown that this amplification factor can be maximized by tuning the base temperature close to its critical one, which is determined by the temperature derivative of the VO{sub 2} emissivity and the equilibrium temperatures of the collector and emitter. This maximization is the result of the presence of two bi-stable temperatures appearing during the heating and cooling processes of the VO{sub 2} base and enables a thermal switching (temperature jump) characterized by a sizeable variation of the collector-to-base and base-to-emitter heat fluxes associated with a slight change of the applied power to the base. This switching effect leads to the optimization of the amplification factor and therefore it could be used for thermal modulation purposes.

  20. Empirical Site Amplification Factors Incorporating Soil Nonlinearity in Taiwan

    Science.gov (United States)

    Kuo, C. H.; Chung, C. H.; Che-Min, L.; Huang, J. Y.; Wen, K. L.

    2017-12-01

    Characteristics of site amplifications caused by both crustal and subduction earthquakes are important in Taiwan. For example, seismic waves were amplified and led to significant building damages in the Taipei Basin by the 1986 Hualien offshore (subduction interface) and the 1999 Chi-Chi earthquakes (crustal), for which the epicentral distances were about 100 km. To understand local site amplifications in Taiwan, empirical site amplification factors for horizontal ground motions are studied using recently constructed strong motion and site databases for the free-field TSMIP stations in Taiwan. Records of large magnitude earthquakes of ML larger than six from 1994 to 2014 were selected for this study. Site amplification factors at site conditions with Vs30 of 120 m/s to 1500 m/s and base accelerations up to 0.7g were inferred from intensity ratios of station pairs within specific distances. The reference site condition is assumed as Vs30 of 760 m/s (B/C boundary). Preliminary results indicate: 1. Soil nonlinearity is more obviously at short periods (PGA, Sa0.3) than long periods (PGV, Sa1.0). 2. Soil nonlinearity is significant for stations belong to site classes of B, C, D, and E in Taiwan. 3. Effect of station-pair distance is seen at short periods (PGA and Sa0.3). 4. No significant different is found in site amplifications of crustal and subduction earthquakes. The result could be a reference for the Fa and Fv in Taiwan's building code.

  1. Virology: The Next Generation from Digital PCR to Single Virion Genomics

    Energy Technology Data Exchange (ETDEWEB)

    White, Richard A.; Brazelton De Cardenas, Jessica N.; Hayden, Randall T.

    2015-10-01

    In the past 25 years, virology has had major technology breakthroughs stemming first from the introduction of nucleic acid amplification testing, but more recently from the use of next-generation sequencing, digital PCR, and the possibility of single virion genomics. These technologies have and will improve diagnosis and disease state monitoring in clinical settings, aid in environmental monitoring, and reveal the vast genetic potential of viruses. Using the principle of limiting dilution, digital PCR amplifies single molecules of DNA in highly partitioned endpoint reactions and reads each of those reactions as either positive or negative based on the presence or absence of target fluorophore. In this review, digital PCR will be highlighted along with current studies, advantages/disadvantages, and future perspectives with regard to digital PCR, viral load testing, and the possibility of single virion genomics.

  2. Solution-based targeted genomic enrichment for precious DNA samples

    Directory of Open Access Journals (Sweden)

    Shearer Aiden

    2012-05-01

    Full Text Available Abstract Background Solution-based targeted genomic enrichment (TGE protocols permit selective sequencing of genomic regions of interest on a massively parallel scale. These protocols could be improved by: 1 modifying or eliminating time consuming steps; 2 increasing yield to reduce input DNA and excessive PCR cycling; and 3 enhancing reproducible. Results We developed a solution-based TGE method for downstream Illumina sequencing in a non-automated workflow, adding standard Illumina barcode indexes during the post-hybridization amplification to allow for sample pooling prior to sequencing. The method utilizes Agilent SureSelect baits, primers and hybridization reagents for the capture, off-the-shelf reagents for the library preparation steps, and adaptor oligonucleotides for Illumina paired-end sequencing purchased directly from an oligonucleotide manufacturing company. Conclusions This solution-based TGE method for Illumina sequencing is optimized for small- or medium-sized laboratories and addresses the weaknesses of standard protocols by reducing the amount of input DNA required, increasing capture yield, optimizing efficiency, and improving reproducibility.

  3. Deciphering the Code of the Cancer Genome: Mechanisms of Chromosome Rearrangement

    Science.gov (United States)

    Willis, Nicholas A.; Rass, Emilie; Scully, Ralph

    2015-01-01

    Chromosome rearrangement plays a causal role in tumorigenesis by contributing to the inactivation of tumor suppressor genes, the dysregulated expression or amplification of oncogenes and the generation of novel gene fusions. Chromosome breaks are important intermediates in this process. How, when and where these breaks arise and the specific mechanisms engaged in their repair strongly influence the resulting patterns of chromosome rearrangement. Here, we review recent progress in understanding how certain distinctive features of the cancer genome, including clustered mutagenesis, tandem segmental duplications, complex breakpoints, chromothripsis, chromoplexy and chromoanasynthesis may arise. PMID:26726318

  4. Laser light triggers increased Raman amplification in the regime of nonlinear Landau damping

    International Nuclear Information System (INIS)

    Depierreux, S.; Goyon, C.; Masson-Laborde, P.E.; Yahia, V.; Loisel, G.; Labaune, C.

    2014-01-01

    Stimulated Raman backscattering (SRS) has many unwanted effects in megajoule-scale inertially confined fusion (ICF) plasmas. Moreover, attempts to harness SRS to amplify short laser pulses through backward Raman amplification have achieved limited success. In high temperature fusion plasmas, SRS usually occurs in a kinetic regime where the nonlinear response of the Langmuir wave to the laser drive and its host of complicating factors make it difficult to predict the degree of amplification that can be achieved under given experimental conditions. Here we present experimental evidence of reduced Landau damping with increasing Langmuir wave amplitude and determine its effects on Raman amplification. The threshold for trapping effects to influence the amplification is shown to be very low. Above threshold, the complex SRS dynamics results in increased amplification factors, which partly explains previous ICF experiments. These insights could aid the development of more efficient backward Raman amplification schemes in this regime. (authors)

  5. Optical parametric amplification and oscillation assisted by low-frequency stimulated emission

    OpenAIRE

    Longhi, Stefano

    2016-01-01

    Optical parametric amplification/oscillation provide a powerful tool for coherent light generation in spectral regions inaccessible to lasers. Parametric gain is based on a frequency {\\it down-conversion} process, and thus it can not be realized for signal waves at a frequency $\\omega_3$ {\\it higher} than the frequency of the pump wave $\\omega_1$. In this work we suggest a route toward the realization of {\\it up-conversion} optical parametric amplification and oscillation, i.e. amplification ...

  6. Mutation of mapped TIA-1/TIAR binding sites in the 3' terminal stem-loop of West Nile virus minus-strand RNA in an infectious clone negatively affects genomic RNA amplification.

    Science.gov (United States)

    Emara, Mohamed M; Liu, Hsuan; Davis, William G; Brinton, Margo A

    2008-11-01

    Previous data showed that the cellular proteins TIA-1 and TIAR bound specifically to the West Nile virus 3' minus-strand stem-loop [WNV3'(-)SL] RNA (37) and colocalized with flavivirus replication complexes in WNV- and dengue virus-infected cells (21). In the present study, the sites on the WNV3'(-)SL RNA required for efficient in vitro T-cell intracellular antigen-related (TIAR) and T-cell intracellular antigen-1 (TIA-1) protein binding were mapped to short AU sequences (UAAUU) located in two internal loops of the WNV3'(-)SL RNA structure. Infectious clone RNAs with all or most of the binding site nucleotides in one of the 3' (-)SL loops deleted or substituted did not produce detectable virus after transfection or subsequent passage. With one exception, deletion/mutation of a single terminal nucleotide in one of the binding sequences had little effect on the efficiency of protein binding or virus production, but mutation of a nucleotide in the middle of a binding sequence reduced both the in vitro protein binding efficiency and virus production. Plaque size, intracellular genomic RNA levels, and virus production progressively decreased with decreasing in vitro TIAR/TIA-1 binding activity, but the translation efficiency of the various mutant RNAs was similar to that of the parental RNA. Several of the mutant RNAs that inefficiently interacted with TIAR/TIA-1 in vitro rapidly reverted in vivo, indicating that they could replicate at a low level and suggesting that an interaction between TIAR/TIA-1 and the viral 3'(-)SL RNA is not required for initial low-level symmetric RNA replication but instead facilitates the subsequent asymmetric amplification of genome RNA from the minus-strand template.

  7. Multi-chamber nucleic acid amplification and detection device

    Science.gov (United States)

    Dugan, Lawrence

    2017-10-25

    A nucleic acid amplification and detection device includes an amplification cartridge with a plurality of reaction chambers for containing an amplification reagent and a visual detection reagent, and a plurality of optically transparent view ports for viewing inside the reaction chambers. The cartridge also includes a sample receiving port which is adapted to receive a fluid sample and fluidically connected to distribute the fluid sample to the reaction chamber, and in one embodiment, a plunger is carried by the cartridge for occluding fluidic communication to the reaction chambers. The device also includes a heating apparatus having a heating element which is activated by controller to generate heat when a trigger event is detected. The heating apparatus includes a cartridge-mounting section which positioned a cartridge in thermal communication with the heating element so that visual changes to the contents of the reaction chambers are viewable through the view ports.

  8. Plasmon enhanced light amplification in metal–insulator–metal waveguides with gain

    International Nuclear Information System (INIS)

    Zhong, Xiao-Lan; Li, Zhi-Yuan

    2012-01-01

    In this paper we study the loss compensation and light amplification properties of metal–insulator–metal (MIM) waveguides that are doped with gain material in the dielectric core. An analytical approach based on Maxwell’s equations is developed to evaluate quantitatively the influence of the gain coefficient on the loss compensation and light amplification efficiencies of the waveguide under different values of the waveguide width and working wavelengths. The analytical results agree excellently with all-numerical calculations that directly solve Maxwell’s equations. The results show that the light amplification efficiency obeys a strict linear relationship with the gain coefficient, and MIM waveguides with narrower widths and under shorter wavelengths have better efficiencies. In addition, the MIM waveguides have higher light amplification efficiencies than usual dielectric waveguides, which suggests a very positive role of the plasmonic structure in enhancing the light amplification when gain is introduced. These loss and gain behaviors can be well explained by looking at the modal profile of each transport mode and the corresponding light energy confinement effect and slow light effect. (paper)

  9. Signal amplification of microRNAs with modified strand displacement-based cycling probe technology.

    Science.gov (United States)

    Jia, Huning; Bu, Ying; Zou, Bingjie; Wang, Jianping; Kumar, Shalen; Pitman, Janet L; Zhou, Guohua; Song, Qinxin

    2016-10-24

    Micro ribose nucleic acids (miRNAs) play an important role in biological processes such as cell differentiation, proliferation and apoptosis. Therefore, miRNAs are potentially a powerful marker for monitoring cancer and diagnosis. Here, we present sensitive signal amplification for miRNAs based on modified cycling probe technology with strand displacement amplification. miRNA was captured by the template coupled with beads, and then the first cycle based on SDA was repeatedly extended to the nicking end, which was produced by the extension reaction of miRNA. The products generated by SDA are captured by a molecular beacon (MB), which is designed to initiate the second amplification cycle, with a similar principle to the cycling probe technology (CPT), which is based on repeated digestion of the DNA-RNA hybrid by the RNase H. After one sample enrichment and two steps of signal amplification, 0.1 pM of let-7a can be detected. The miRNA assay exhibits a great dynamic range of over 100 orders of magnitude and high specificity to clearly discriminate a single base difference in miRNA sequences. This isothermal amplification does not require any special temperature control instrument. The assay is also about signal amplification rather than template amplification, therefore minimising contamination issues. In addition, there is no need for the reverse transcription (RT) process. Thus the amplification is suitable for miRNA detection.

  10. Identification of genomic insertion and flanking sequence of G2-EPSPS and GAT transgenes in soybean using whole genome sequencing method

    Directory of Open Access Journals (Sweden)

    Bingfu Guo

    2016-07-01

    Full Text Available Molecular characterization of sequences flanking exogenous fragment insertions is essential for safety assessment and labeling of genetically modified organisms (GMO. In this study, the T-DNA insertion sites and flanking sequences were identified in two newly developed transgenic glyphosate-tolerant soybeans GE-J16 and ZH10-6 based on whole genome sequencing (WGS method. About 21 Gb sequence data (~21× coverage for each line was generated on Illumina HiSeq 2500 platform. The junction reads mapped to boundary of T-DNA and flanking sequences in these two events were identified by comparing all sequencing reads with soybean reference genome and sequence of transgenic vector. The putative insertion loci and flanking sequences were further confirmed by PCR amplification, Sanger sequencing, and co-segregation analysis. All these analyses supported that exogenous T-DNA fragments were integrated in positions of Chr19: 50543767-50543792 and Chr17: 7980527-7980541 in these two transgenic lines. Identification of the genomic insertion site of the G2-EPSPS and GAT transgenes will facilitate the use of their glyphosate-tolerant traits in soybean breeding program. These results also demonstrated that WGS is a cost-effective and rapid method of identifying sites of T-DNA insertions and flanking sequences in soybean.

  11. Implementing sponge physiological and genomic information to enhance the diversity of its culturable associated bacteria.

    Science.gov (United States)

    Lavy, Adi; Keren, Ray; Haber, Markus; Schwartz, Inbar; Ilan, Micha

    2014-02-01

    In recent years new approaches have emerged for culturing marine environmental bacteria. They include the use of novel culture media, sometimes with very low-nutrient content, and a variety of growth conditions such as temperature, oxygen levels, and different atmospheric pressures. These approaches have largely been neglected when it came to the cultivation of sponge-associated bacteria. Here, we used physiological and environmental conditions to reflect the environment of sponge-associated bacteria along with genomic data of the prominent sponge symbiont Candidatus Poribacteria sp. WGA-4E, to cultivate bacteria from the Red Sea sponge Theonella swinhoei. Designing culturing conditions to fit the metabolic needs of major bacterial taxa present in the sponge, through a combined use of diverse culture media compositions with aerobic and microaerophilic states, and addition of antibiotics, yielded higher diversity of the cultured bacteria and led to the isolation of novel sponge-associated and sponge-specific bacteria. In this work, 59 OTUs of six phyla were isolated. Of these, 22 have no close type strains at the species level (< 97% similarity of 16S rRNA gene sequence), representing novel bacteria species, and some are probably new genera and even families. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  12. Particle-in-cell Simulations of Raman Laser Amplification in Preformed Plasmas

    International Nuclear Information System (INIS)

    Clark, Daniel S.; Fisch, Nathaniel J.

    2003-01-01

    Two critical issues in the amplification of laser pulses by backward Raman scattering in plasma slabs are the saturation mechanism of the amplification effect (which determines the maximum attainable output intensity of a Raman amplifier) and the optimal plasma density for amplification. Previous investigations [V.M. Malkin, et al., Phys. Rev. Lett., 82 (22):4448-4451, 1999] identified forward Raman scattering and modulational instabilities of the amplifying seed as the likely saturation mechanisms and lead to an estimated unfocused output intensities of 10 17 W/cm 2 . The optimal density for amplification is determined by the competing constraints of minimizing the plasma density so as to minimize the growth rate of the instabilities leading to saturation but also maintaining the plasma sufficiently dense that the driven Langmuir wave responsible for backscattering does not break prematurely. Here, particle-in-cell code are simulations presented which verify that saturation of backward Raman amplification does occur at intensities of ∼10 17 W/cm 2 by forward Raman scattering and modulational instabilities. The optimal density for amplification in a plasma with the representative temperature of T(sub)e = 200 eV is also shown in these simulations to be intermediate between the cold plasma wave-breaking density and the density limit found by assuming a water bag electron distribution function

  13. Error amplification to promote motor learning and motivation in therapy robotics.

    Science.gov (United States)

    Shirzad, Navid; Van der Loos, H F Machiel

    2012-01-01

    To study the effects of different feedback error amplification methods on a subject's upper-limb motor learning and affect during a point-to-point reaching exercise, we developed a real-time controller for a robotic manipulandum. The reaching environment was visually distorted by implementing a thirty degrees rotation between the coordinate systems of the robot's end-effector and the visual display. Feedback error amplification was provided to subjects as they trained to learn reaching within the visually rotated environment. Error amplification was provided either visually or through both haptic and visual means, each method with two different amplification gains. Subjects' performance (i.e., trajectory error) and self-reports to a questionnaire were used to study the speed and amount of adaptation promoted by each error amplification method and subjects' emotional changes. We found that providing haptic and visual feedback promotes faster adaptation to the distortion and increases subjects' satisfaction with the task, leading to a higher level of attentiveness during the exercise. This finding can be used to design a novel exercise regimen, where alternating between error amplification methods is used to both increase a subject's motor learning and maintain a minimum level of motivational engagement in the exercise. In future experiments, we will test whether such exercise methods will lead to a faster learning time and greater motivation to pursue a therapy exercise regimen.

  14. Centrosome Amplification Is Sufficient to Promote Spontaneous Tumorigenesis in Mammals

    NARCIS (Netherlands)

    Levine, Michelle S.; Bakker, Bjorn; Boeckx, Bram; Moyett, Julia; Lu, James; Vitre, Benjamin; Spierings, Diana C.; Lansdorp, Peter M.; Cleveland, Don W.; Lambrechts, Diether; Foijer, Floris; Holland, Andrew J.

    2017-01-01

    Centrosome amplification is a common feature of human tumors, but whether this is a cause or a consequence of cancer remains unclear. Here, we test the consequence of centrosome amplification by creating mice in which centrosome number can be chronically increased in the absence of additional

  15. An enzymatic signal amplification system for calorimetric studies of cellobiohydrolases

    DEFF Research Database (Denmark)

    Murphy, Leigh; Baumann, Martin Johannes; Borch, Kim

    2010-01-01

    amplification method has been developed to measure even slow hydrolytically active enzymes such as cellobiohydrolases. This method is explained in detail for the amplification of the heat signal by more than 130 times by using glucose oxidase and catalase. The kinetics of this complex coupled reaction system...

  16. Rapid detection of Piper yellow mottle virus and Cucumber mosaic virus infecting black pepper (Piper nigrum) by loop-mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Bhat, A I; Siljo, A; Deeshma, K P

    2013-10-01

    The loop-mediated isothermal amplification (LAMP) assay for Piper yellow mottle virus and the reverse transcription (RT) LAMP assay for Cucumber mosaic virus each consisted of a set of five primers designed against the conserved sequences in the viral genome. Both RNA and DNA isolated from black pepper were used as a template for the assay. The results were assessed visually by checking turbidity, green fluorescence and pellet formation in the reaction tube and also by gel electrophoresis. The assay successfully detected both viruses in infected plants whereas no cross-reactions were recorded with healthy plants. Optimum conditions for successful amplification were determined in terms of the concentrations of magnesium sulphate and betaine, temperature, and duration. The detection limit for both LAMP and RT-LAMP was up to 100 times that for conventional PCR and up to one-hundredth of that for real-time PCR. The optimal conditions arrived at were validated by testing field samples of infected vines of three species from different regions. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Development of multiple cross displacement amplification label-based gold nanoparticles lateral flow biosensor for detection of Shigella spp.

    Directory of Open Access Journals (Sweden)

    Yi Wang

    2016-11-01

    Full Text Available Shigella spp., the etiological agent of shigellosis or bacillary dysentery, are responsible for considerable morbidity and mortality in excess of a million deaths globally per year. Although PCR-based techniques (such as PCR-based dipstick biosensors have been used for the molecular diagnosis of infectious disease, these assays were restricted due to the need for a sophisticated thermal cycling apparatus to denature target templates. To facilitate simple and rapid detection of target pathogens, we successfully devised an inexpensive, reliable and nearly instrument-free molecular technique, which incorporates multiple cross displacement amplification (MCDA combined with a newly designed lateral flow biosensor (LFB for visual, sensitive and specific detection of Shigella. The MCDA-LFB assay was conducted at 65 ˚C for only 20 min during the amplification stage, and then products were directly analyzed on the biosensor, alleviating the use of special reagents, electrophoresis equipment and amplicon detection instruments. The entire process, including specimen processing (35 min, amplification (20 and detection (2-5 min, can be finished within 1 h. The MCDA-LFB assay demonstrated high specificity for Shigella detection. The analytical sensitivity of the assay was 10 fg of genomic templates per reaction in pure culture and 5.86 CFU per tube in human fecal samples, which was consistent with MCDA by colorimetric indicator, gel electrophoresis, real time turbidity and fluorescence detection. Hence, the simplicity, rapidity and nearly instrument-free platform of the MCDA-LFB assay make it practical for ‘on-site’ diagnosis, point-of-care testing and more. Moreover, the proof-of-concept approach can be reconfigured to detect a wide variety of target sequences by re-designing the specific MCDA primers.

  18. DNA amplification fingerprinting using 10 x polymerase chain reaction buffer with ammonium sulfate for human identification

    International Nuclear Information System (INIS)

    Baransel, Asyun; Dugler, Hikmat E.; Tokdemir, Mehmet

    2004-01-01

    The polymerase chain reaction (PCR) - based DNA amplification fingerprinting (DAF) or randomly amplified polymorphic DNA (RAPD) is based on a strategy using a single arbitrary oligonucleotide primer to generate anonymous amplification of genomic DNA. On this basic strategy, in this study, we aimed to test individual differences and usefulness of 2 basic primers (5-CGCGCCGG-3 and 5-TGCCGAGCTG-3) and examined whether there is a positive effect on results of 10 x PCR buffer with ammonium sulfate. A new approach in DNA fingerprinting, 10 x PCR buffer with ammonium sulfate, is presented in the study. Primers with single 8 and 10 nucleotides in length and 2 different PCR buffers with or without ammonium sulfate were used to identify 135 volunteers with no blood relationship. This study was carried out at the Pharmacology Laboratory, University of Gaziantep, School of Medicine, Turkey between 1999 and 2000. An average of 10 major bands representing 500-1500 base pair (bp) in length was determined as amplified DNA products on standard agarose gels for these volunteers. The use of ammonium sulfate in 10 x PCR buffers has increased to 92% success ratio of individual difference obtained from the 8 nucleotides primer. With this study, more reliable results can be obtained by using ammonium sulfate in 10 x PCR buffers. (author)

  19. Preimplantation genetic diagnosis for Duchenne muscular dystrophy by multiple displacement amplification.

    Science.gov (United States)

    Ren, Zi; Zeng, Hai-tao; Xu, Yan-wen; Zhuang, Guang-lun; Deng, Jie; Zhang, Cheng; Zhou, Can-quan

    2009-02-01

    To evaluate the use of multiple displacement amplification (MDA) in preimplantation genetic diagnosis (PGD) for female carriers with Duchenne muscular dystrophy (DMD). MDA was used to amplify a whole genome of single cells. Following the setup on single cells, the test was applied in two clinical cases of PGD. One mutant exon, six short tandem repeats (STR) markers within the dystrophin gene, and amelogenin were incorporated into singleplex polymerase chain reaction (PCR) assays on MDA products of single blastomeres. Center for reproductive medicine in First Affiliated Hospital, Sun Yat-sen University, China. Two female carriers with a duplication of exons 3-11 and a deletion of exons 47-50, respectively. The MDA of single cells and fluorescent PCR assays for PGD. The ability to analyze single blastomeres for DMD using MDA. The protocol setup previously allowed for the accurate diagnosis of each embryo. Two clinical cases resulted in a healthy girl, which was the first successful clinical application of MDA in PGD for DMD. We suggest that this protocol is reliable to increase the accuracy of the PGD for DMD.

  20. Tropical temperature altitude amplification in the hiatus period (1998-2012

    Directory of Open Access Journals (Sweden)

    Ducić Vladan D.

    2015-01-01

    Full Text Available In the period 1998-2012 there was a stagnation in temperature rise, despite the GHGs radiation forcing is increased (hiatus period. According to Global Circulation Models simulations, expected response on the rise of GHGs forcing is tropical temperature altitude amplification - temperature increases faster in higher troposphere than in lower troposphere. In this paper, two satellite data sets, UAH MSU and RSS, were used to test altitude temperature amplification in tropic (20°N-20°S in the hiatus period. We compared data from satellite data sets from lower troposphere (TLT and middle troposphere (TMT in general and particularly for land and ocean (for UAH MSU. The results from both satellite measurements showed the presence of hiatus, i.e. slowdown of the temperature rise in the period 1998-2012 compared to period 1979-2012 (UAH MSU and temperature fall for RSS data. Smaller increase, i.e. temperature fall over ocean showed that hiatus is an ocean phenomenon above all. Data for UAH MSU showed that temperature altitude amplification in tropic was not present either for period 1979-2012, or 1998-2012. RSS data set also do not show temperature altitude amplification either for longer (1979-2012, or for shorter period (1998-2012. RSS data for successive 15-year periods from 1979-1993 till 1998-2012 does not show tropical temperature altitude amplification and in one case negative trend is registered in TLT and in two cases in TMT. In general, our results do not show presence of temperature altitude amplification in tropic in the hiatus period. [Projekat Ministarstva nauke Republike Srbije, br. III47007

  1. Voltage-dependent amplification of synaptic inputs in respiratory motoneurones

    Science.gov (United States)

    Enríquez Denton, M; Wienecke, J; Zhang, M; Hultborn, H; Kirkwood, P A

    2012-01-01

    The role of persistent inward currents (PICs) in cat respiratory motoneurones (phrenic inspiratory and thoracic expiratory) was investigated by studying the voltage-dependent amplification of central respiratory drive potentials (CRDPs), recorded intracellularly, with action potentials blocked with the local anaesthetic derivative, QX-314. Decerebrate unanaesthetized or barbiturate-anaesthetized preparations were used. In expiratory motoneurones, plateau potentials were observed in the decerebrates, but not under anaesthesia. For phrenic motoneurones, no plateau potentials were observed in either state (except in one motoneurone after the abolition of the respiratory drive by means of a medullary lesion), but all motoneurones showed voltage-dependent amplification of the CRDPs, over a wide range of membrane potentials, too wide to result mainly from PIC activation. The measurements of the amplification were restricted to the phase of excitation, thus excluding the inhibitory phase. Amplification was found to be greatest for the smallest CRDPs in the lowest resistance motoneurones and was reduced or abolished following intracellular injection of the NMDA channel blocker, MK-801. Plateau potentials were readily evoked in non-phrenic cervical motoneurones in the same (decerebrate) preparations. We conclude that the voltage-dependent amplification of synaptic excitation in phrenic motoneurones is mainly the result of NMDA channel modulation rather than the activation of Ca2+ channel mediated PICs, despite phrenic motoneurones being strongly immunohistochemically labelled for CaV1.3 channels. The differential PIC activation in different motoneurones, all of which are CaV1.3 positive, leads us to postulate that the descending modulation of PICs is more selective than has hitherto been believed. PMID:22495582

  2. Applicability of SCAR markers to food genomics: olive oil traceability.

    Science.gov (United States)

    Pafundo, Simona; Agrimonti, Caterina; Maestri, Elena; Marmiroli, Nelson

    2007-07-25

    DNA analysis with molecular markers has opened a shortcut toward a genomic comprehension of complex organisms. The availability of micro-DNA extraction methods, coupled with selective amplification of the smallest extracted fragments with molecular markers, could equally bring a breakthrough in food genomics: the identification of original components in food. Amplified fragment length polymorphisms (AFLPs) have been instrumental in plant genomics because they may allow rapid and reliable analysis of multiple and potentially polymorphic sites. Nevertheless, their direct application to the analysis of DNA extracted from food matrixes is complicated by the low quality of DNA extracted: its high degradation and the presence of inhibitors of enzymatic reactions. The conversion of an AFLP fragment to a robust and specific single-locus PCR-based marker, therefore, could extend the use of molecular markers to large-scale analysis of complex agro-food matrixes. In the present study is reported the development of sequence characterized amplified regions (SCARs) starting from AFLP profiles of monovarietal olive oils analyzed on agarose gel; one of these was used to identify differences among 56 olive cultivars. All the developed markers were purposefully amplified in olive oils to apply them to olive oil traceability.

  3. Device-independent randomness amplification with a single device

    International Nuclear Information System (INIS)

    Plesch, Martin; Pivoluska, Matej

    2014-01-01

    Expansion and amplification of weak randomness with untrusted quantum devices has recently become a very fruitful topic of research. Here we contribute with a procedure for amplifying a single weak random source using tri-partite GHZ-type entangled states. If the quality of the source reaches a fixed threshold R=log 2 ⁡(10), perfect random bits can be produced. This technique can be used to extract randomness from sources that can't be extracted neither classically, nor by existing procedures developed for Santha–Vazirani sources. Our protocol works with a single fault-free device decomposable into three non-communicating parts, that is repeatedly reused throughout the amplification process. - Highlights: • We propose a protocol for device independent randomness amplification. • Our protocol repeatedly re-uses a single device decomposable into three parts. • Weak random sources with min-entropy rate greater than 1/4 log 2 ⁡(10) can be amplified. • Security against all-quantum adversaries is achieved

  4. DNA methylation alteration is a major consequence of genome doubling in autotetraploid Brassica rapa

    Directory of Open Access Journals (Sweden)

    Xu Yanhao

    2017-01-01

    Full Text Available Polyploids are typically classified as autopolyploids or allopolyploids based on the origin of their chromosome sets. Autopolyploidy is much more common than traditionally believed. Allopolyploidization, accompanied by genomic and transcriptomic changes, has been well investigated. In this study, genetic, DNA methylation and gene expression changes in autotetraploid Brassica rapa were investigated. No genetic alteration was detected using an amplified fragment length polymorphism (AFLP approach. Using a cDNA-AFLP approach, approximately 0.58% of fragments showed changes in gene expression in autotetraploid B. rapa. The methylation-sensitive amplification polymorphism (MSAP analysis showed that approximately 1.7% of the fragments underwent DNA methylation changes upon genome doubling, with hypermethylation and demethylation changes equally affected. Fragments displaying changes in gene expression and methylation status were isolated and then sequenced and characterized, respectively. This study showed that variation in cytosine methylation is a major consequence of genome doubling in autotetraploid Brassica rapa.

  5. Four-Wave Optical Parametric Amplification in a Raman-Active Gas

    Directory of Open Access Journals (Sweden)

    Yuichiro Kida

    2015-08-01

    Full Text Available Four-wave optical parametric amplification (FWOPA in a Raman-active medium is experimentally investigated by use of an air-filled hollow fiber. A femtosecond pump pulse shorter than the period of molecular motion excites the coherent molecular motion of the Raman-active molecules during the parametric amplification of a signal pulse. The excited coherent motion modulates the frequency of the signal pulse during the parametric amplification, and shifts it to lower frequencies. The magnitude of the frequency redshift depends on the pump intensity, resulting in intensity-dependent spectral characteristics that are different from those in the FWOPA induced in a noble-gas-filled hollow fiber.

  6. A large ungated TPC with GEM amplification

    Science.gov (United States)

    Berger, M.; Ball, M.; Fabbietti, L.; Ketzer, B.; Arora, R.; Beck, R.; Böhmer, F. V.; Chen, J.-C.; Cusanno, F.; Dørheim, S.; García, F.; Hehner, J.; Herrmann, N.; Höppner, C.; Kaiser, D.; Kis̆, M.; Kleipa, V.; Konorov, I.; Kunkel, J.; Kurz, N.; Leifels, Y.; Müllner, P.; Münzer, R.; Neubert, S.; Rauch, J.; Schmidt, C. J.; Schmitz, R.; Soyk, D.; Vandenbroucke, M.; Voss, B.; Walther, D.; Zmeskal, J.

    2017-10-01

    A Time Projection Chamber (TPC) is an ideal device for the detection of charged particle tracks in a large volume covering a solid angle of almost 4 π. The high density of hits on a given particle track facilitates the task of pattern recognition in a high-occupancy environment and in addition provides particle identification by measuring the specific energy loss for each track. For these reasons, TPCs with Multiwire Proportional Chamber (MWPC) amplification have been and are widely used in experiments recording heavy-ion collisions. A significant drawback, however, is the large dead time of the order of 1 ms per event generated by the use of a gating grid, which is mandatory to prevent ions created in the amplification region from drifting back into the drift volume, where they would severely distort the drift path of subsequent tracks. For experiments with higher event rates this concept of a conventional TPC operating with a triggered gating grid can therefore not be applied without a significant loss of data. A continuous readout of the signals is the more appropriate way of operation. This, however, constitutes a change of paradigm with considerable challenges to be met concerning the amplification region, the design and bandwidth of the readout electronics, and the data handling. A mandatory prerequisite for such an operation is a sufficiently good suppression of the ion backflow from the avalanche region, which otherwise limits the tracking and particle identification capabilities of such a detector. Gas Electron Multipliers (GEM) are a promising candidate to combine excellent spatial resolution with an intrinsic suppression of ions. In this paper we describe the design, construction and the commissioning of a large TPC with GEM amplification and without gating grid (GEM-TPC). The design requirements have driven innovations in the construction of a light-weight field-cage, a supporting media flange, the GEM amplification and the readout system, which are

  7. EuMicroSatdb: A database for microsatellites in the sequenced genomes of eukaryotes

    Directory of Open Access Journals (Sweden)

    Grover Atul

    2007-07-01

    Full Text Available Abstract Background Microsatellites have immense utility as molecular markers in different fields like genome characterization and mapping, phylogeny and evolutionary biology. Existing microsatellite databases are of limited utility for experimental and computational biologists with regard to their content and information output. EuMicroSatdb (Eukaryotic MicroSatellite database http://ipu.ac.in/usbt/EuMicroSatdb.htm is a web based relational database for easy and efficient positional mining of microsatellites from sequenced eukaryotic genomes. Description A user friendly web interface has been developed for microsatellite data retrieval using Active Server Pages (ASP. The backend database codes for data extraction and assembly have been written using Perl based scripts and C++. Precise need based microsatellites data retrieval is possible using different input parameters like microsatellite type (simple perfect or compound perfect, repeat unit length (mono- to hexa-nucleotide, repeat number, microsatellite length and chromosomal location in the genome. Furthermore, information about clustering of different microsatellites in the genome can also be retrieved. Finally, to facilitate primer designing for PCR amplification of any desired microsatellite locus, 200 bp upstream and downstream sequences are provided. Conclusion The database allows easy systematic retrieval of comprehensive information about simple and compound microsatellites, microsatellite clusters and their locus coordinates in 31 sequenced eukaryotic genomes. The information content of the database is useful in different areas of research like gene tagging, genome mapping, population genetics, germplasm characterization and in understanding microsatellite dynamics in eukaryotic genomes.

  8. Robust and efficient direct multiplex amplification method for large-scale DNA detection of blood samples on FTA cards

    International Nuclear Information System (INIS)

    Jiang Bowei; Xiang Fawei; Zhao Xingchun; Wang Lihua; Fan Chunhai

    2013-01-01

    Deoxyribonucleic acid (DNA) damage arising from radiations widely occurred along with the development of nuclear weapons and clinically wide application of computed tomography (CT) scan and nuclear medicine. All ionizing radiations (X-rays, γ-rays, alpha particles, etc.) and ultraviolet (UV) radiation lead to the DNA damage. Polymerase chain reaction (PCR) is one of the most wildly used techniques for detecting DNA damage as the amplification stops at the site of the damage. Improvements to enhance the efficiency of PCR are always required and remain a great challenge. Here we establish a multiplex PCR assay system (MPAS) that is served as a robust and efficient method for direct detection of target DNA sequences in genomic DNA. The establishment of the system is performed by adding a combination of PCR enhancers to standard PCR buffer, The performance of MPAS was demonstrated by carrying out the direct PCR amplification on l.2 mm human blood punch using commercially available primer sets which include multiple primer pairs. The optimized PCR system resulted in high quality genotyping results without any inhibitory effect indicated and led to a full-profile success rate of 98.13%. Our studies demonstrate that the MPAS provides an efficient and robust method for obtaining sensitive, reliable and reproducible PCR results from human blood samples. (authors)

  9. Distilled single-cell genome sequencing and de novo assembly for sparse microbial communities.

    Science.gov (United States)

    Taghavi, Zeinab; Movahedi, Narjes S; Draghici, Sorin; Chitsaz, Hamidreza

    2013-10-01

    Identification of every single genome present in a microbial sample is an important and challenging task with crucial applications. It is challenging because there are typically millions of cells in a microbial sample, the vast majority of which elude cultivation. The most accurate method to date is exhaustive single-cell sequencing using multiple displacement amplification, which is simply intractable for a large number of cells. However, there is hope for breaking this barrier, as the number of different cell types with distinct genome sequences is usually much smaller than the number of cells. Here, we present a novel divide and conquer method to sequence and de novo assemble all distinct genomes present in a microbial sample with a sequencing cost and computational complexity proportional to the number of genome types, rather than the number of cells. The method is implemented in a tool called Squeezambler. We evaluated Squeezambler on simulated data. The proposed divide and conquer method successfully reduces the cost of sequencing in comparison with the naïve exhaustive approach. Squeezambler and datasets are available at http://compbio.cs.wayne.edu/software/squeezambler/.

  10. Mitogenomes of polar bodies and corresponding oocytes.

    Directory of Open Access Journals (Sweden)

    Luca Gianaroli

    Full Text Available The objective of the present study was to develop an approach that could assess the chromosomal status and the mitochondrial DNA (mtDNA content of oocytes and their corresponding polar bodies (PBs with the goal of obtaining a comparative picture of the segregation process both for nuclear and mtDNA. After Whole Genome Amplification (WGA, sequencing of the whole mitochondrial genome was attempted to analyze the segregation of mutant and wild-type mtDNA during human meiosis. Three triads, composed of oocyte and corresponding PBs, were analyzed and their chromosome status was successfully assessed. The complete mitochondrial genome (mitogenome was almost entirely sequenced in the oocytes (95.99% compared to 98.43% in blood, while the percentage of sequences obtained in the corresponding PB1 and PB2 was lower (69.70% and 69.04% respectively. The comparison with the mtDNA sequence in blood revealed no changes in the D-loop region for any of the cells of each triad. In the coding region of blood mtDNA and oocyte mtDNA sequences showed full correspondence, whereas all PBs had at least one change with respect to the blood-oocyte pairs. In all, 9 changes were found, either in PB1 or PB2: 4 in MT-ND5, 2 in MT-RNR2, and 1 each in MT-ATP8, MT-ND4, MT-CYTB. The full concordance between oocyte and blood in the 3 triads, and the relegation of changes to PBs, revealed the unexpected coexistence of different variants, giving a refined estimation of mitochondrial heteroplasmy. Should these findings be confirmed by additional data, an active mechanism could be postulated in the oocyte to preserve a condition of 'normality'.

  11. Effects of wheat germ agglutinin on human gastrointestinal epithelium: Insights from an experimental model of immune/epithelial cell interaction

    International Nuclear Information System (INIS)

    Pellegrina, Chiara Dalla; Perbellini, Omar; Scupoli, Maria Teresa; Tomelleri, Carlo; Zanetti, Chiara; Zoccatelli, Gianni; Fusi, Marina; Peruffo, Angelo; Rizzi, Corrado; Chignola, Roberto

    2009-01-01

    Wheat germ agglutinin (WGA) is a plant protein that binds specifically to sugars expressed, among many others, by human gastrointestinal epithelial and immune cells. WGA is a toxic compound and an anti-nutritional factor, but recent works have shown that it may have potential as an anti-tumor drug and as a carrier for oral drugs. To quantitate the toxicity threshold for WGA on normal epithelial cells we previously investigated the effects of the lectin on differentiated Caco2 cells, and showed that in the micromolar range of concentrations WGA could alter the integrity of the epithelium layer and increase its permeability to both mannitol and dextran. WGA was shown to be uptaken by Caco2 cells and only ∼ 0.1% molecules were observed to cross the epithelium layer by transcytosis. Here we show that at nanomolar concentrations WGA is unexpectedly bioactive on immune cells. The supernatants of WGA-stimulated peripheral blood mononuclear cells (PBMC) can alter the integrity of the epithelium layer when administered to the basolateral side of differentiated Caco2 cells and the effects can be partially inhibited by monoclonal antibodies against IL1, IL6 and IL8. At nanomolar concentrations WGA stimulates the synthesis of pro-inflammatory cytokines and thus the biological activity of WGA should be reconsidered by taking into account the effects of WGA on the immune system at the gastrointestinal interface. These results shed new light onto the molecular mechanisms underlying the onset of gastrointestinal disorders observed in vivo upon dietary intake of wheat-based foods.

  12. NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs.

    Science.gov (United States)

    Morisset, Dany; Dobnik, David; Hamels, Sandrine; Zel, Jana; Gruden, Kristina

    2008-10-01

    We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the mult