Sample records for gene amplification

  1. Gene amplification in carcinogenesis

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    Lucimari Bizari


    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.

  2. Heat induces gene amplification in cancer cells

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    Yan, Bin, E-mail: [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Mercy Cancer Center, Mercy Medical Center-North Iowa, Mason City, IA 50401 (United States); Ouyang, Ruoyun [Department of Respiratory Medicine, The Second Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410011 (China); Huang, Chenghui [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Department of Oncology, The Third Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410013 (China); Liu, Franklin [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States); Neill, Daniel [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Li, Chuanyuan [Dermatology, Duke University Medical Center, Durham, NC 27710 (United States); Dewhirst, Mark [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States)


    Highlights: Black-Right-Pointing-Pointer This study discovered that heat exposure (hyperthermia) results in gene amplification in cancer cells. Black-Right-Pointing-Pointer Hyperthermia induces DNA double strand breaks. Black-Right-Pointing-Pointer DNA double strand breaks are considered to be required for the initiation of gene amplification. Black-Right-Pointing-Pointer The underlying mechanism of heat-induced gene amplification is generation of DNA double strand breaks. -- Abstract: Background: Hyperthermia plays an important role in cancer therapy. However, as with radiation, it can cause DNA damage and therefore genetic instability. We studied whether hyperthermia can induce gene amplification in cancer cells and explored potential underlying molecular mechanisms. Materials and methods: (1) Hyperthermia: HCT116 colon cancer cells received water-submerged heating treatment at 42 or 44 Degree-Sign C for 30 min; (2) gene amplification assay using N-(phosphoacetyl)-L-aspartate (PALA) selection of cabamyl-P-synthetase, aspartate transcarbarmylase, dihydro-orotase (cad) gene amplified cells; (3) southern blotting for confirmation of increased cad gene copies in PALA-resistant cells; (4) {gamma}H2AX immunostaining to detect {gamma}H2AX foci as an indication for DNA double strand breaks. Results: (1) Heat exposure at 42 or 44 Degree-Sign C for 30 min induces gene amplification. The frequency of cad gene amplification increased by 2.8 and 6.5 folds respectively; (2) heat exposure at both 42 and 44 Degree-Sign C for 30 min induces DNA double strand breaks in HCT116 cells as shown by {gamma}H2AX immunostaining. Conclusion: This study shows that heat exposure can induce gene amplification in cancer cells, likely through the generation of DNA double strand breaks, which are believed to be required for the initiation of gene amplification. This process may be promoted by heat when cellular proteins that are responsible for checkpoints, DNA replication, DNA repair and

  3. RNA amplification for successful gene profiling analysis

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    Wang Ena


    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

  4. Amplification and characterization of eukaryotic structural genes. (United States)

    Maniatis, T; Efstratiadis, A; Sim, G K; Kafatos, F


    An approach to the study of eukaryotic structural genes which are differentially expressed during development is described. This approach involves the isolation and amplification of mRNA sequences by in vitro conversion of mRNA to double-stranded cDNA followed by molecular cloning in bacterial plasmids. This procedure provides highly specific hybridization probes that can be used to identify genes and their contiguous DNA sequences in genomic DNA, and to detect specific RNA transcripts during development. The nature of the method allows the isolation of individual mRNA sequences from a complex population of molecules at different stages of development.

  5. Mutualism breakdown by amplification of Wolbachia genes. (United States)

    Chrostek, Ewa; Teixeira, Luis


    Most insect species are associated with vertically transmitted endosymbionts. Because of the mode of transmission, the fitness of these symbionts is dependent on the fitness of the hosts. Therefore, these endosymbionts need to control their proliferation in order to minimize their cost for the host. The genetic bases and mechanisms of this regulation remain largely undetermined. The maternally inherited bacteria of the genus Wolbachia are the most common endosymbionts of insects, providing some of them with fitness benefits. In Drosophila melanogaster, Wolbachia wMelPop is a unique virulent variant that proliferates massively in the hosts and shortens their lifespan. The genetic bases of wMelPop virulence are unknown, and their identification would allow a better understanding of how Wolbachia levels are regulated. Here we show that amplification of a region containing eight Wolbachia genes, called Octomom, is responsible for wMelPop virulence. Using Drosophila lines selected for carrying Wolbachia with different Octomom copy numbers, we demonstrate that the number of Octomom copies determines Wolbachia titers and the strength of the lethal phenotype. Octomom amplification is unstable, and reversion of copy number to one reverts all the phenotypes. Our results provide a link between genotype and phenotype in Wolbachia and identify a genomic region regulating Wolbachia proliferation. We also prove that these bacteria can evolve rapidly. Rapid evolution by changes in gene copy number may be common in endosymbionts with a high number of mobile elements and other repeated regions. Understanding wMelPop pathogenicity and variability also allows researchers to better control and predict the outcome of releasing mosquitoes transinfected with this variant to block human vector-borne diseases. Our results show that transition from a mutualist to a pathogen may occur because of a single genomic change in the endosymbiont. This implies that there must be constant selection on

  6. Mechanism of Gene Amplification via Yeast Autonomously Replicating Sequences

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    Shelly Sehgal


    Full Text Available The present investigation was aimed at understanding the molecular mechanism of gene amplification. Interplay of fragile sites in promoting gene amplification was also elucidated. The amplification promoting sequences were chosen from the Saccharomyces cerevisiae ARS, 5S rRNA regions of Plantago ovata and P. lagopus, proposed sites of replication pausing at Ste20 gene locus of S. cerevisiae, and the bend DNA sequences within fragile site FRA11A in humans. The gene amplification assays showed that plasmid bearing APS from yeast and human beings led to enhanced protein concentration as compared to the wild type. Both the in silico and in vitro analyses were pointed out at the strong bending potential of these APS. In addition, high mitotic stability and presence of TTTT repeats and SAR amongst these sequences encourage gene amplification. Phylogenetic analysis of S. cerevisiae ARS was also conducted. The combinatorial power of different aspects of APS analyzed in the present investigation was harnessed to reach a consensus about the factors which stimulate gene expression, in presence of these sequences. It was concluded that the mechanism of gene amplification was that AT rich tracts present in fragile sites of yeast serve as binding sites for MAR/SAR and DNA unwinding elements. The DNA protein interactions necessary for ORC activation are facilitated by DNA bending. These specific bindings at ORC promote repeated rounds of DNA replication leading to gene amplification.

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

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    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

  8. Identification of genetic elements associated with EPSPs gene amplification.

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    Todd A Gaines

    Full Text Available Weed populations can have high genetic plasticity and rapid responses to environmental selection pressures. For example, 100-fold amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS gene evolved in the weed species Amaranthus palmeri to confer resistance to glyphosate, the world's most important herbicide. However, the gene amplification mechanism is unknown. We sequenced the EPSPS gene and genomic regions flanking EPSPS loci in A. palmeri, and searched for mobile genetic elements or repetitive sequences. The EPSPS gene was 10,229 bp, containing 8 exons and 7 introns. The gene amplification likely proceeded through a DNA-mediated mechanism, as introns exist in the amplified gene copies and the entire amplified sequence is at least 30 kb in length. Our data support the presence of two EPSPS loci in susceptible (S A. palmeri, and that only one of these was amplified in glyphosate-resistant (R A. palmeri. The EPSPS gene amplification event likely occurred recently, as no sequence polymorphisms were found within introns of amplified EPSPS copies from R individuals. Sequences with homology to miniature inverted-repeat transposable elements (MITEs were identified next to EPSPS gene copies only in R individuals. Additionally, a putative Activator (Ac transposase and a repetitive sequence region were associated with amplified EPSPS genes. The mechanism controlling this DNA-mediated amplification remains unknown. Further investigation is necessary to determine if the gene amplification may have proceeded via DNA transposon-mediated replication, and/or unequal recombination between different genomic regions resulting in replication of the EPSPS gene.

  9. Somatic recombination, gene amplification and cancer. (United States)

    Ramel, C; Cederberg, H; Magnusson, J; Vogel, E; Natarajan, A T; Mullender, L H; Nivard, J M; Parry, J M; Leyson, A; Comendador, M A; Sierra, L M; Ferreiro, J A; Consuegra, S


    The principle objective of this research programme, to analyse chemical induction of somatic recombination and related endpoints, i.e., mobilization of transposing elements and gene amplification, has been approached by means of several assay systems. These have included Drosophila, Saccharomyces and mammalian cell cultures. 6.1. Screening assays for mitotic recombination. A large number of chemicals have been investigated in the three Drosophila assay systems employed--the multiple wing hair/flare wing spot system developed by Graf et al., 1984, the white-ivory system developed by Green et al., 1986 and the white/white+ eye spot assay developed by Vogel (Vogel and Nivard, 1993). Particularly the screening of 181 chemicals, covering a wide array of chemical classes, by the last mentioned assay has shown that measurement of somatic recombination in Drosophila constitutes a sensitive and efficient short-term test which shows a remarkably good correlation with the agent score of 83 short-term tests analysed by ICPEMC (Mendelsohn et al., 1992; Table 2) as well as the assay performance in international collaborative programmes measuring carcinogen/non-carcinogens (de Serres and Ashby, 1981; Ashby et al., 1985, 1988). Also the wing spot assay has gained wide international recognition as a similarly sensitive test. These two assay systems in Drosophila measure both intrachromosomal events and interchromosomal recombination. The white-ivory system on the other hand is based on the loss of a tandem duplication in the white locus, the mechanism of which is less known, but probably involves intrachromosomal recombination. The difference in the mechanism between this assay and the former two was indicated by the lack of response to methotrexate in the white-ivory assay, while this compound was strongly recombinogenic in both the wing spot and white/white+ assays. The use of different strains of Drosophila with the white/white+ assay demonstrated the importance of the

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

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    Pravas Ranjan Sahoo


    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.

  11. Loop mediated isothermal amplification: An innovative gene amplification technique for animal diseases. (United States)

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


    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. Amplification of kinetic oscillations in gene expression (United States)

    Zhdanov, V. P.


    Because of the feedbacks between the DNA transcription and mRNA translation, the gene expression in cells may exhibit bistability and oscillations. The deterministic and stochastic calculations presented illustrate how the bistable kinetics of expression of one gene in a cell can be influenced by the kinetic oscillations in the expression of another gene. Due to stability of the states of the bistable kinetics of gene 1 and the relatively small difference between the maximum and minimum protein amounts during the oscillations of gene 2, the induced oscillations of gene 1 are found to typically be related either to the low-or high-reactive state of this gene. The quality of the induced oscillations may be appreciably better than that of the inducing oscillations. This means that gene 1 can serve as an amplifier of the kinetic oscillations of gene 2.

  13. Amplification biases: possible differences among deviating gene expressions

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    Piumi Francois


    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

  14. Identification of genetic elements associated with EPSPS gene amplification (United States)

    Weed populations can have high genetic plasticity and rapid responses to environmental selection pressures. For example, 100-fold amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene evolved to confer resistance to glyphosate, the world's most important herbicide, in the wee...

  15. Hormonal Involvement in Breast Cancer Gene Amplification (United States)


    and s ubsequently amp lified at the Yale University sequenc ing facility for Illumina sequencing. However, it required a lot of effort to obtain this...and Polyak K. (2008). Genome-wide functi onal synergy between amp lified and mutated genes in human breast cancer. Cancer Res. 68: 9532-9540...east cancer patient samples. Other co-amp lified genes, within the HER2 amplicon and/or at other regions, could serve as additional novel target s for

  16. Whole genome amplification of DNA for genotyping pharmacogenetics candidate genes.

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    Santosh ePhilips


    Full Text Available Whole genome amplification (WGA technologies can be used to amplify genomic DNA when only small amounts of DNA are available. The Multiple Displacement Amplification Phi polymerase based amplification has been shown to accurately amplify DNA for a variety of genotyping assays; however, it has not been tested for genotyping many of the clinically relevant genes important for pharmacogenetic studies, such as the cytochrome P450 genes, that are typically difficult to genotype due to multiple pseudogenes, copy number variations, and high similarity to other related genes. We evaluated whole genome amplified samples for Taqman™ genotyping of SNPs in a variety of pharmacogenetic genes. In 24 DNA samples from the Coriell human diversity panel, the call rates and concordance between amplified (~200-fold amplification and unamplified samples was 100% for two SNPs in CYP2D6 and one in ESR1. In samples from a breast cancer clinical trial (Trial 1, we compared the genotyping results in samples before and after WGA for four SNPs in CYP2D6, one SNP in CYP2C19, one SNP in CYP19A1, two SNPs in ESR1, and two SNPs in ESR2. The concordance rates were all >97%. Finally, we compared the allele frequencies of 143 SNPs determined in Trial 1 (whole genome amplified DNA to the allele frequencies determined in unamplified DNA samples from a separate trial (Trial 2 that enrolled a similar population. The call rates and allele frequencies between the two trials were 98% and 99.7%, respectively. We conclude that the whole genome amplified DNA is suitable for Taqman™ genotyping for a wide variety of pharmacogenetically relevant SNPs.

  17. Genome-wide gene amplification during differentiation of neural progenitor cells in vitro. (United States)

    Fischer, Ulrike; Keller, Andreas; Voss, Meike; Backes, Christina; Welter, Cornelius; Meese, Eckart


    DNA sequence amplification is a phenomenon that occurs predictably at defined stages during normal development in some organisms. Developmental gene amplification was first described in amphibians during gametogenesis and has not yet been described in humans. To date gene amplification in humans is a hallmark of many tumors. We used array-CGH (comparative genomic hybridization) and FISH (fluorescence in situ hybridization) to discover gene amplifications during in vitro differentiation of human neural progenitor cells. Here we report a complex gene amplification pattern two and five days after induction of differentiation of human neural progenitor cells. We identified several amplified genes in neural progenitor cells that are known to be amplified in malignant tumors. There is also a striking overlap of amplified chromosomal regions between differentiating neural progenitor cells and malignant tumor cells derived from astrocytes. Gene amplifications in normal human cells as physiological process has not been reported yet and may bear resemblance to developmental gene amplifications in amphibians and insects.

  18. Copy number change: evolving views on gene amplification. (United States)

    Elliott, Kathryn T; Cuff, Laura E; Neidle, Ellen L


    The rapid pace of genomic sequence analysis is increasing the awareness of intrinsically dynamic genetic landscapes. Gene duplication and amplification (GDA) contribute to adaptation and evolution by allowing DNA regions to expand and contract in an accordion-like fashion. This process affects diverse aspects of bacterial infection, including antibiotic resistance and host-pathogen interactions. In this review, microbial GDA is discussed, primarily using recent bacterial examples that demonstrate medical and evolutionary consequences. Interplay between GDA and horizontal gene transfer further impact evolutionary trajectories. Complementing the discovery of gene duplication in clinical and environmental settings, experimental evolution provides a powerful method to document genetic change over time. New methods for GDA detection highlight both its importance and its potential application for genetic engineering, synthetic biology and biotechnology.

  19. Gene amplification during differentiation of mammalian neural stem cells in vitro and in vivo. (United States)

    Fischer, Ulrike; Backes, Christina; Raslan, Abdulrahman; Keller, Andreas; Meier, Carola; Meese, Eckart


    In development of amphibians and flies, gene amplification is one of mechanisms to increase gene expression. In mammalian cells, gene amplification seems to be restricted to tumorigenesis and acquiring of drug-resistance in cancer cells. Here, we report a complex gene amplification pattern in mouse neural progenitor cells during differentiation with approximately 10% of the genome involved. Half of the amplified mouse chromosome regions overlap with amplified regions previously reported in human neural progenitor cells, indicating conserved mechanisms during differentiation. Using fluorescence in situ hybridization, we verified the amplification in single cells of primary mouse mesencephalon E14 (embryonic stage) neurosphere cells during differentiation. In vivo we confirmed gene amplifications of the TRP53 gene in cryosections from mouse embryos at stage E11.5. Gene amplification is not only a cancer-related mechanism but is also conserved in evolution, occurring during differentiation of mammalian neural stem cells.

  20. Gene amplification of the Hps locus in Glycine max

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    Kuflu Kuflom


    Full Text Available Abstract Background Hydrophobic protein from soybean (HPS is an 8 kD cysteine-rich polypeptide that causes asthma in persons allergic to soybean dust. HPS is synthesized in the pod endocarp and deposited on the seed surface during development. Past evidence suggests that the protein may mediate the adherence or dehiscence of endocarp tissues during maturation and affect the lustre, or glossiness of the seed surface. Results A comparison of soybean germplasm by genomic DNA blot hybridization shows that the copy number and structure of the Hps locus is polymorphic among soybean cultivars and related species. Changes in Hps gene copy number were also detected by comparative genomic DNA hybridization using cDNA microarrays. The Hps copy number polymorphisms co-segregated with seed lustre phenotype and HPS surface protein in a cross between dull- and shiny-seeded soybeans. In soybean cultivar Harosoy 63, a minimum of 27 ± 5 copies of the Hps gene were estimated to be present in each haploid genome. The isolation and analysis of genomic clones indicates that the core Hps locus is comprised of a tandem array of reiterated units, with each 8.6 kb unit containing a single HPS open reading frame. Conclusion This study shows that polymorphisms at the Hps locus arise from changes in the gene copy number via gene amplification. We present a model whereby Hps copy number modulates protein expression levels and seed lustre, and we suggest that gene amplification may result from selection pressures imposed on crop plants.

  1. Amplification of LDH gene from Indian strains of Plasmodium vivax

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    Ritu Berwal, N. Gopalan, Kshitij Chandel, Shri Prakash ,K. Sekhar


    Full Text Available Background & objectives: Plasmodium vivax is geographically widespread and responsible for >50% of malaria cases in India. Increased drug resistance of the parasite highlights the immediaterequirement of early and accurate diagnosis as well as new therapeutics. In view of this, the presentstudy was undertaken to amplify P. vivax (Indian strains lactate dehydrogenase gene (PvLDHwhich has been identified as a good target for antimalarials as well as diagnostics.Methods: P. vivax infected clinical blood samples were collected from southern part of India andwere tested with established diagnostic parameters (ICT, Giemsa staining. Total DNA was extractedfrom blood samples and subjected to PCR using two sets of primers, one for the amplification of fullPvLDH gene (951bp and the other for a partial PvLDH gene fragment (422bp, covering a variableantigenic region (140aa as compared to other plasmodial species.Results & conclusion: PCRs for both the full and partial gene targets were optimised and found to beconsistent when tested on several P. vivax positive clinical samples. In addition, full gene PCR wasfound to specifically detect only P. vivax DNA and could be used as a specific molecular diagnostictool. These amplified products can be cloned and expressed as a recombinant protein that might beuseful for the development and screening of antimalarials as well as for diagnostic purposes.

  2. No fitness cost of glyphosate resistance endowed by massive EPSPS gene amplification in Amaranthus palmeri. (United States)

    Vila-Aiub, Martin M; Goh, Sou S; Gaines, Todd A; Han, Heping; Busi, Roberto; Yu, Qin; Powles, Stephen B


    Amplification of the EPSPS gene has been previously identified as the glyphosate resistance mechanism in many populations of Amaranthus palmeri, a major weed pest in US agriculture. Here, we evaluate the effects of EPSPS gene amplification on both the level of glyphosate resistance and fitness cost of resistance. A. palmeri individuals resistant to glyphosate by expressing a wide range of EPSPS gene copy numbers were evaluated under competitive conditions in the presence or absence of glyphosate. Survival rates to glyphosate and fitness traits of plants under intra-specific competition were assessed. Plants with higher amplification of the EPSPS gene (53-fold) showed high levels of glyphosate resistance, whereas less amplification of the EPSPS gene (21-fold) endowed a lower level of glyphosate resistance. Without glyphosate but under competitive conditions, plants exhibiting up to 76-fold EPSPS gene amplification exhibited similar height, and biomass allocation to vegetative and reproductive organs, compared to glyphosate susceptible A. palmeri plants with no amplification of the EPSPS gene. Both the additive effects of EPSPS gene amplification on the level of glyphosate resistance and the lack of associated fitness costs are key factors contributing to EPSPS gene amplification as a widespread and important glyphosate resistance mechanism likely to become much more evident in weed plant species.

  3. Gene Amplification of the Multidrug Resistance 1 Gene of Plasmodium vivax Isolates from Thailand, Laos, and Myanmar▿ (United States)

    Imwong, Mallika; Pukrittayakamee, Sasithon; Pongtavornpinyo, Wirichada; Nakeesathit, Supatchara; Nair, Shalini; Newton, Paul; Nosten, Francois; Anderson, Timothy J. C.; Dondorp, Arjen; Day, Nicholas P. J.; White, Nicholas J.


    Plasmodium vivax mdr1 gene amplification, quantified by real-time PCR, was significantly more common on the western Thailand border (6 of 66 samples), where mefloquine pressure has been intense, than elsewhere in southeast Asia (3 of 149; P = 0.02). Five coding mutations in pvmdr1, independent of gene amplification, were also found. PMID:18443118

  4. The Study on Gene Amplification of EGFR in Bronchioloalveolar Carcinoma and Conventional Adenocarcinoma of the Lung

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    Xin SONG


    Full Text Available Background and objective Patients with adenocarcinoma of the lung have disproportionately response to the epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI. The aim of this study is to analyze the difference of EGFR gene amplification in bronchioloalveolar carcinoma (BAC, adenocarcinma mixed subtype and conventional adenocarcinoma of the lung and provide some information to clinical therapies. Methods Lung cancer cases were collected and reviewed from the archives of the Department of Pathology, Chinese PLA General Hospital during the time period from 2004 to 2006. The definite diagnosis of BAC based on 2004 WHO classification of lung tumors was made by two pathologists. Fluorescence in situ hybridization (FISH was performed to detect EGFR gene amplification in pure BAC, adenocarcinma mixed subtype and conventional adenocarcinoma. Results Conventional adenocarcinoma had higher EGFR amplification compared with pure BAC and adenocarcinma mixed subtype (χ2=11.632, P<0.05. EGFR gene amplification was found in 45.45% of conventional adenocarcinoma, 14.81% in pure BACs, and 22.58% in adenocarcinma mixed subtype. EGFR gene amplification was observed as scattered signals in most cases. Conclusion EGFR gene amplification was seen more frequently in the invasive components than in BAC. EGFR gene amplification might be associated with the development of adenocarcinoma of the lung.

  5. KRAS and MAPK1 Gene Amplification in Type II Ovarian Carcinomas

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    Noriyuki Ishikawa


    Full Text Available In this study, we examined the clinical significance of KRAS and MAPK1 amplification and assessed whether these amplified genes were potential therapeutic targets in type II ovarian carcinoma. Using fluorescence in situ hybridization, immunohistochemistry, and retrospectively collected clinical data, KRAS and MAPK1 amplifications were identified in 9 (13.2% and 5 (7.4% of 68 type II ovarian carcinoma tissue samples, respectively. Interestingly, co-amplification of KRAS and MAPK1 seemed to be absent in the type II ovarian carcinomas tested, except one case. Active phospho-ERK1/2 was identified in 26 (38.2% out of 68 type II ovarian carcinomas and did not correlate with KRAS or MAPK1 amplification. There was no significant relationship between KRAS amplification and overall or progression-free survival in patients with type II ovarian carcinoma. However, patients with MAPK1 amplification had significantly poorer progression-free survival than patients without MAPK1 amplification. Moreover, type II ovarian carcinoma cells with concomitant KRAS amplification and mutation exhibited dramatic growth reduction following treatment with the MEK inhibitor PD0325901. These findings indicate that KRAS/MAPK1 amplification is critical for the growth of a subset of type II ovarian carcinomas. Additionally, RAS/RAF/MEK/ERK pathway-targeted therapy may benefit selected patients with type II ovarian carcinoma harboring KRAS/MAPK1 amplifications.


    Sperm RNA Amplification for Gene Expression Profiling by DNA Microarray TechnologyHongzu Ren, Kary E. Thompson, Judith E. Schmid and David J. Dix, Reproductive Toxicology Division, NHEERL, Office of Research and Development, US Environmental Protection Agency, Research Triang...

  7. Study on the protein expression and amplification of HER2 gene in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Sunan Wang; Yingying Li; Zhengshun Xu; Wenzhao Zhao; Tian Yun; Wuling Zhu; Yangkun Wang


    Objective:The aim of the study was to investigate the human epidermal growth factor receptor 2 (HER2) gene amplification and protein expression and interpretation points in the stomach mixed carcinomas. Methods:Immunohisto-chemistry (IHC) and fluorescence in situ hybridization (FISH) technique were used to detect HER2 gene amplification and ex-pression of HER2 protein in 442 cases of gastric mixed carcinoma. Results:The expression rate of HER2 protein was 41.2%(182/442):the HER2 protein expression IHC 3+extensive type in 18 cases, partial type in 21 cases, focal type in 8 cases, accounting for 10.6%(47/442);the HER2 protein expression IHC 2+extensive type in 23 cases, partial type in 28 cases, focal type in 11 cases, accounting for 14.0%(62/442);the HER2 protein expression IHC 1+extensive type in 27 cases, partial type in 31 cases, focal type in 15 cases, accounting for 16.5%(73/442). HER2 gene amplification rate of 442 cases was 16.1%(71/442). In 182 cases of HER2 protein positive expression, the HER2 gene cluster amplification rate was 14.8%(27/182), large granular amplification rate 11.0%(20/182), punctate amplification rate 6.0%(11/182) and high polysomy 7.1%(13/182). In 71 cases of HER2 gene amplification, there was 42 cases of HER2 protein expression IHC 3+, 22 cases of HER2 protein expression IHC 2+, and 7 cases of IHC 1+. Conclusion:HER2 detection of gastric mixed carcinoma has great heterogeneity, HER2 protein positive expression is divided into extensive type, partial type and focal type, and HER2 gene positive amplifica-tion is divided into cluster amplification, large granular amplification, punctate amplification and high polysomy. These typing of HER2 protein expression and HER2 gene amplification provide reference index to quantify for targeted therapeutic ef ect of anticancer drugs.

  8. Her2 expression and gene amplification is rarely detectable in patients with oral squamous cell carcinomas. (United States)

    Hanken, Henning; Gaudin, Robert; Gröbe, Alexander; Fraederich, Meike; Eichhorn, Wolfgang; Smeets, Ralf; Simon, Ronald; Sauter, Guido; Grupp, Katharina; Izbicki, Jacob R; Sehner, Susanne; Heiland, Max; Blessmann, Marco


    Her2 (ErbB2) transforms cells when overexpressed and is an important therapeutic target in breast cancer. Contrary to breast cancer, studies on Her2 overexpression and gene amplification in squamous cell carcinomas of the head and neck region described largely different results. This study was undertaken to learn more on the prevalence and clinical significance of HER2 amplification and overexpression in squamous cell carcinomas of the head and neck. Her2 expression and gene amplification was analyzed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) on two tissue microarrays composed of 427 squamous cell carcinomas of the head and neck region and 222 oral squamous cell carcinomas. Results were compared with clinicopathological features. Her2 expression and gene amplification was rarely detectable in squamous cell carcinomas of the head and neck region and unrelated to tumor phenotype or survival of the patients with oral squamous carcinoma. Our results demonstrate that Her2 protein and gene amplification was only detectable in a small subset of squamous cell carcinomas of the head and neck region as well as oral squamous cell carcinomas. However, it can be speculated that those few patients with Her2 overexpressing and gene amplificated tumors may possibly benefit from an anti-Her2 therapy. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. Analysis of HER2 gene amplification using Differential PCR in breast cancer patients of Isfahan Province

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    Zohreh Hojati


    Full Text Available Background: Amplification of HER2 is seen in 20-30% of breast cancer cases. Measurement of HER2 gene amplification appears to be of vital importance in planning the treatment schedule for patients with breast carcinoma. The aim of our study was to evaluate HER2 amplification status in malignant and benign breast tumors by differential PCR (dPCR. Materials and Methods: The genomic DNA was extracted using the phenol/chloroform extraction procedure from 76 different breast tissues. Differential PCR was performed using the DNA samples isolated from fresh and paraffin- embedded breast cancer tissues. The relative copy number ratio of target gene (HER2 to control gene ( INF-γ was measured. dPCR products were then separated by electrophoresis using 2% agarose gel. The intensity of HER2 and INFγ bands were determined for each sample by ImageJ software. Results: According to the ratio between the band intensity of HER2 to INFγ in tumour and also normal samples, 7% and 26% rates of HER2 amplification were observed in benign and malignant samples respectively. The ratio showed a 2-5 fold increase in HER2 gene copy number for tissues with HER2 amplification whereas, a one-fold increase was found in other samples. Conclusion: Differential PCR provides a relatively rapid and inexpensive technique to assess the HER2 gene amplification, especially alongside immunohistochemistry as a routine assessing method .

  10. Flux variability scanning based on enforced objective flux for identifying gene amplification targets

    Directory of Open Access Journals (Sweden)

    Park Jong


    Full Text Available Abstract Background In order to reduce time and efforts to develop microbial strains with better capability of producing desired bioproducts, genome-scale metabolic simulations have proven useful in identifying gene knockout and amplification targets. Constraints-based flux analysis has successfully been employed for such simulation, but is limited in its ability to properly describe the complex nature of biological systems. Gene knockout simulations are relatively straightforward to implement, simply by constraining the flux values of the target reaction to zero, but the identification of reliable gene amplification targets is rather difficult. Here, we report a new algorithm which incorporates physiological data into a model to improve the model’s prediction capabilities and to capitalize on the relationships between genes and metabolic fluxes. Results We developed an algorithm, flux variability scanning based on enforced objective flux (FVSEOF with grouping reaction (GR constraints, in an effort to identify gene amplification targets by considering reactions that co-carry flux values based on physiological omics data via “GR constraints”. This method scans changes in the variabilities of metabolic fluxes in response to an artificially enforced objective flux of product formation. The gene amplification targets predicted using this method were validated by comparing the predicted effects with the previous experimental results obtained for the production of shikimic acid and putrescine in Escherichia coli. Moreover, new gene amplification targets for further enhancing putrescine production were validated through experiments involving the overexpression of each identified targeted gene under condition-controlled batch cultivation. Conclusions FVSEOF with GR constraints allows identification of gene amplification targets for metabolic engineering of microbial strains in order to enhance the production of desired bioproducts. The algorithm

  11. On the mechanism of gene amplification induced under stress in Escherichia coli.

    Directory of Open Access Journals (Sweden)


    Full Text Available Gene amplification is a collection of processes whereby a DNA segment is reiterated to multiple copies per genome. It is important in carcinogenesis and resistance to chemotherapeutic agents, and can underlie adaptive evolution via increased expression of an amplified gene, evolution of new gene functions, and genome evolution. Though first described in the model organism Escherichia coli in the early 1960s, only scant information on the mechanism(s of amplification in this system has been obtained, and many models for mechanism(s were possible. More recently, some gene amplifications in E. coli were shown to be stress-inducible and to confer a selective advantage to cells under stress (adaptive amplifications, potentially accelerating evolution specifically when cells are poorly adapted to their environment. We focus on stress-induced amplification in E. coli and report several findings that indicate a novel molecular mechanism, and we suggest that most amplifications might be stress-induced, not spontaneous. First, as often hypothesized, but not shown previously, certain proteins used for DNA double-strand-break repair and homologous recombination are required for amplification. Second, in contrast with previous models in which homologous recombination between repeated sequences caused duplications that lead to amplification, the amplified DNAs are present in situ as tandem, direct repeats of 7-32 kilobases bordered by only 4 to 15 base pairs of G-rich homology, indicating an initial non-homologous recombination event. Sequences at the rearrangement junctions suggest nonhomologous recombination mechanisms that occur via template switching during DNA replication, but unlike previously described template switching events, these must occur over long distances. Third, we provide evidence that 3'-single-strand DNA ends are intermediates in the process, supporting a template-switching mechanism. Fourth, we provide evidence that lagging

  12. Carboxylesterase gene amplifications associated with insecticide resistance in Aedes albopictus: Geographical distribution and evolutionary origin.

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    Linda Grigoraki


    Full Text Available Aedes albopictus is one of the most invasive human disease vectors. Its control has been largely based on insecticides, such as the larvicide temephos. Temephos resistance has been associated with the up-regulation, through gene amplification, of two carboxylesterase (CCE genes closely linked on the genome, capable of sequestering and metabolizing temephos oxon, the activated form of temephos.Here, we investigated the occurrence, geographical distribution and origin of the CCE amplicon in Ae. albopictus populations from several geographical regions worldwide. The haplotypic diversity at the CCEae3a locus revealed high polymorphism, while phylogenetic analysis showed an absence of correlation between haplotype similarity and geographic origin. Two types of esterase amplifications were found, in two locations only (Athens and Florida: one, previously described, results in the amplification of both CCEae3a and CCEae6a; the second is being described for the first time and results in the amplification of CCEae3a only. The two amplification events are independent, as confirmed by sequence analysis. All individuals from Athens and Florida carrying the CCEae3a-CCEae6a co-amplicon share a common haplotype, indicating a single amplification event, which spread between the two countries.The importance of passive transportation of disease vectors, including individuals carrying resistance mechanisms, is discussed in the light of efficient and sustainable vector control strategies.

  13. Inducible amplification of gene copy number and heterologous protein production in the yeast Kluyveromyces lactis. (United States)

    Morlino, G B; Tizzani, L; Fleer, R; Frontali, L; Bianchi, M M


    Heterologous protein production can be doubled by increasing the copy number of the corresponding heterologous gene. We constructed a host-vector system in the yeast Kluyveromyces lactis that was able to induce copy number amplification of pKD1 plasmid-based vectors upon expression of an integrated copy of the plasmid recombinase gene. We increased the production and secretion of two heterologous proteins, glucoamylase from the yeast Arxula adeninivorans and mammalian interleukin-1beta, following gene dosage amplification when the heterologous genes were carried by pKD1-based vectors. The choice of the promoters for expression of the integrated recombinase gene and of the episomal heterologous genes are critical for the mitotic stability of the host-vector system.

  14. Amplification of epidermal growth factor receptor gene in renal cell carcinoma

    DEFF Research Database (Denmark)

    Harper, Peter; El-Hariry, Iman; Powles, Thomas;


    Expression of epidermal growth factor receptor (EGFR) may be of prognostic value in renal cell cancer (RCC). Gene amplification of EGFR was investigated in a cohort of 315 patients with advanced RCC from a previously reported randomised study. Using fluorescent in situ hybridisation, only 2...

  15. Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification

    NARCIS (Netherlands)

    Ziesemer, K.A.; Mann, A.E.; Sankaranarayanan, K.; Schroeder, H.; Ozga, A.T.; Brandt, B.W.; Zaura, E.; Waters-Rist, A.; Hoogland, M.; Salazar-García, D.C.; Aldenderfer, M.; Speller, C.; Hendy, J.; Weston, D.A.; MacDonald, S.J.; Thomas, G.H.; Collins, M.J.; Lewis, C.M.; Hofman, C.; Warinner, C.


    To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341-534) of this gen

  16. Complex Oncogenic Translocations with Gene Amplification are Initiated by Specific DNA Breaks in Lymphocytes



    Chromosomal instability is a hallmark of many tumor types. Complex chromosomal rearrangements with associated gene amplification, known as complicons, characterize many hematologic and solid cancers. While chromosomal aberrations, including complicons, are useful diagnostic and prognostic cancer markers, their molecular origins are not known. Although accumulating evidence has implicated DNA double strand break repair in suppression of oncogenic genome instability, the genomic elements requir...

  17. Expression of FGFR3 Protein and Gene Amplification in Urinary Bladder Lesions in Relation to Schistosomiasis

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    Olfat Hammam


    CONCLUSIONS: FGFR3 overexpression in malignant cases was significantly higher than in chronic cystitis. FGFR3 gene amplification was reported mainly in low grade and NNMBIC tumours. FGFR3 may be further studied as a subject for target therapy of bladder cancer.

  18. Amplification and overexpression of the EGF receptor gene in primary human glioblastomas. (United States)

    Libermann, T A; Nusbaum, H R; Razon, N; Kris, R; Lax, I; Soreq, H; Whittle, N; Waterfield, M D; Ullrich, A; Schlessinger, J


    The expression of epidermal growth factor (EGF) receptor in brain tumours of glial origin was studied at the protein, mRNA and genomic levels. Four out of 10 glioblastomas that overexpress EGF receptor also have gene amplification. The amplified genes appear to be rearranged, generating an aberrant mRNA in at least one of these tumours. Such receptor defects may be relevant to tumorigenesis of human glioblastomas.

  19. Super resolution imaging of HER2 gene amplification (United States)

    Okada, Masaya; Kubo, Takuya; Masumoto, Kanako; Iwanaga, Shigeki


    HER2 positive breast cancer is currently examined by counting HER2 genes using fluorescence in situ hybridization (FISH)-stained breast carcinoma samples. In this research, two-dimensional super resolution fluorescence microscopy based on stochastic optical reconstruction microscopy (STORM), with a spatial resolution of approximately 20 nm in the lateral direction, was used to more precisely distinguish and count HER2 genes in a FISH-stained tissue section. Furthermore, by introducing double-helix point spread function (DH-PSF), an optical phase modulation technique, to super resolution microscopy, three-dimensional images were obtained of HER2 in a breast carcinoma sample approximately 4 μm thick.

  20. Concurrent AURKA and MYCN Gene Amplifications Are Harbingers of Lethal TreatmentRelated Neuroendocrine Prostate Cancer

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    Juan Miguel Mosquera


    Full Text Available Neuroendocrine prostate cancer (NEPC, also referred to as anaplastic prostate cancer, is a lethal tumor that most commonly arises in late stages of prostate adenocarcinoma (PCA with predilection to metastasize to visceral organs. In the current study, we explore for evidence that Aurora kinase A (AURKA and N-myc (MYCN gene abnormalities are harbingers of treatment-related NEPC (t-NEPC. We studied primary prostate tissue from 15 hormone naïve PCAs, 51 castration-resistant prostate cancers, and 15 metastatic tumors from 72 patients at different stages of disease progression to t-NEPC, some with multiple specimens. Histologic evaluation, immunohistochemistry, and fluorescence in situ hybridization were performed and correlated with clinical variables. AURKA amplification was identified in overall 65% of PCAs (hormone naïve and treated from patients that developed t-NEPC and in 86% of metastases. Concurrent amplification of MYCN was present in 70% of primary PCAs, 69% of treated PCAs, and 83% of metastases. In contrast, in an unselected PCA cohort, AURKA and MYCN amplifications were identified in only 5% of 169 cases. When metastatic t-NEPC was compared to primary PCA from the same patients, there was 100% concordance of ERG rearrangement, 100% concordance of AURKA amplification, and 60% concordance of MYCN amplification. In tumors with mixed features, there was also 100% concordance of ERG rearrangement and 94% concordance of AURKA and MYCN co-amplification between areas of NEPC and adenocarcinoma. AURKA and MYCN amplifications may be prognostic and predictive biomarkers, as they are harbingers of tumors at risk of progressing to t-NEPC after hormonal therapy.

  1. Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification. (United States)

    Ziesemer, Kirsten A; Mann, Allison E; Sankaranarayanan, Krithivasan; Schroeder, Hannes; Ozga, Andrew T; Brandt, Bernd W; Zaura, Egija; Waters-Rist, Andrea; Hoogland, Menno; Salazar-García, Domingo C; Aldenderfer, Mark; Speller, Camilla; Hendy, Jessica; Weston, Darlene A; MacDonald, Sandy J; Thomas, Gavin H; Collins, Matthew J; Lewis, Cecil M; Hofman, Corinne; Warinner, Christina


    To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341-534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions.

  2. 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


    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.

  3. Cyclin E gene (CCNE) amplification and hCDC4 mutations in endometrial carcinoma. (United States)

    Cassia, Raúl; Moreno-Bueno, Gema; Rodríguez-Perales, Sandra; Hardisson, David; Cigudosa, Juan C; Palacios, José


    Cyclin E overexpression occurs in a subset of endometrial carcinomas (ECs), but the molecular mechanisms underlying this alteration remain to be established. The present study has analysed amplification of the cyclin E gene (CCNE) and mutation in hCDC4, the gene coding for the F-box protein, which tags phosphorylated cyclin E for proteosomal degradation, to ascertain whether these alterations might be responsible for cyclin E overexpression in ECs. Cyclin E and p53 expression was studied by immunohistochemistry in eight atypical endometrial hyperplasias (AEHs), 51 endometrioid endometrial carcinomas (EECs), and 22 non-endometrioid endometrial carcinomas (NEECs). CCNE amplification was analysed by fluorescence in situ hybridization (FISH). Mutations in exons 2-11 of the hCDC4 gene were screened by PCR-SSCP-sequencing. Finally, the polymorphic marker D4S1610 was used to assess loss of heterozygosity (LOH) in the hCDC4 gene. Cyclin E overexpression was found in 26/81 (32%) cases and was associated with the histological type of the lesion, since it was not found in any AEHs but was present in 27% of EECs and 54.5% of NEECs (p=0.035). Cyclin E overexpression was associated with histological grade (p=0.011) and p53 immunostaining in EECs (p=0.033). CCNE amplification was found in 6 of 37 (16%) ECs examined. There was a significant association between CCNE amplification and the histological type of the lesion, since five (83%) of the six cases with amplification were NEECs (p=0.008). One EEC harboured an hCDC4 mutation: a CGA to CAA (Arg/Gln) change at codon 479. In addition, D4S1610 LOH was found in 7 of 23 (30%) informative cases analysed, but no correlation with cyclin E overexpression was found. However, the tumour with hCDC4 mutation also showed LOH. This is the first study demonstrating that cyclin E overexpression is associated with gene amplification in ECs, these alterations being more frequent in NEECs. Although hCDC4 exhibits a low mutation frequency in ECs

  4. The Association between EGFR Gene Amplification and the Prognosis in Non-small Cell Lung Cancer: A meta-analysis

    Directory of Open Access Journals (Sweden)

    Jie WANG


    Full Text Available Background and objective Many studies concluded that epidermal growth factor receptor (EGFR gene amplification might be related with the prognosis in non-small cell lung cancer. However, the results were not consistent. To identify whether EGFR gene amplification could affect the prognosis, a meta-analysis was conducted based on the published works. Methods According to inclusion and exclusion criteria, articles were selected from medical electronic databases searching, including PubMed, Cochrane library and CNKI. The qualities of included articles were scored based on the European Lung Cancer Working Party scale, and the hazard ratio (HR and the 95% confidence interval (CI were also collected. Meta-analysis was completed using software Review Manager 4.2. Results Forest plot from 12 studies in which 1 221 included patients were all treated with EGFR-TKI showed that the prognosis of patients harboring EGFR gene amplification were superior to negative ones (HR=0.82, 95%CI: 0.68-0.99, P=0.04, and for Caucasian patients who received EGFR-TKI, the survivals were longer in EGFR gene amplification positive cases than those without amplification (HR=0.42, 95%CI: 0.31-0.57, P < 0.001. Meta results from 8 studies in which 658 included patients did not receive EGFR-TKI indicated that the casualty hazard of amplification positive patients was comparable with negative ones. However, for Asia patients who failed to receive EGFR-TKI, the prognosis of patients with EGFR gene amplification were inferior to negative ones (HR=1.88, 95%CI: 1.21-2.93, P=0.005. Conclusion EGFR gene amplification may be a positive predictive factor in terms of survival for Caucasian patients receiving EGFR-TKI rather than Asia patients. However, the prognosis was inferior in Asia patients with EGFR amplification to those without the amplification, if they failed to receive EGFR-TKI.


    Institute of Scientific and Technical Information of China (English)

    刘世喜; 林代诚; 洪邦泰; 黄光琦


    In order to study the ahered molecular events during laryngeal carcinogenesis and elucidate the role of Ha-ras oncogene amplification and mutation, we have examined their profile by polymerase chain reaction (PCR) and selective oligonucleoride hybridization. We analyzed the mutational status of codon 12 of Ha-ras in 22 laryngeal carcinomas and 10 normal tissues, and found that 7 of 22 laryngeal carcinomas con-tained a Ha-ras mutation at codon 12. The frequency of mutation was 32%. None of the normal tissues re-vealed mutation. Moreover, no amplification was found in cancers when compared to the normal. Our findings indicated that the aefivmed Ha-ras gene existed in laryngeal carcinoma, and activation of the Ha-ras gene by mutation at codon 12 might play a key role in laryngeal carcinogenesis.

  6. Biologically relevant effects of mRNA amplification on gene expression profiles

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    Smits Jos FM


    Full Text Available Abstract Background Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results. In the present study we compared microarray data obtained from amplified mRNA derived from biopsies of rat cardiac left ventricle and non-amplified mRNA derived from the same organ. Biopsies were linearly amplified to acquire enough material for a microarray experiment. Both amplified and unamplified samples were hybridized to the Rat Expression Set 230 Array of Affymetrix. Results Analysis of the microarray data showed that unamplified material of two different left ventricles had 99.6% identical gene expression. Gene expression patterns of two biopsies obtained from the same parental organ were 96.3% identical. Similarly, gene expression pattern of two biopsies from dissimilar organs were 92.8% identical to each other. Twenty-one percent of reporters called present in parental left ventricular tissue disappeared after amplification in the biopsies. Those reporters were predominantly seen in the low intensity range. Sequence analysis showed that reporters that disappeared after amplification had a GC-content of 53.7+/-4.0%, while reporters called present in biopsy- and whole LV-samples had an average GC content of 47.8+/-5.5% (P Conclusion This study establishes that the gene expression profile obtained after amplification of mRNA of left ventricular biopsies is representative for the whole left ventricle of the rat heart. However, specific gene transcripts present in parental tissues were undetectable in the minute left

  7. Microarray analysis after RNA amplification can detect pronounced differences in gene expression using limma

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    Orengo Christine


    Full Text Available Abstract Background RNA amplification is necessary for profiling gene expression from small tissue samples. Previous studies have shown that the T7 based amplification techniques are reproducible but may distort the true abundance of targets. However, the consequences of such distortions on the ability to detect biological variation in expression have not been explored sufficiently to define the true extent of usability and limitations of such amplification techniques. Results We show that expression ratios are occasionally distorted by amplification using the Affymetrix small sample protocol version 2 due to a disproportional shift in intensity across biological samples. This occurs when a shift in one sample cannot be reflected in the other sample because the intensity would lie outside the dynamic range of the scanner. Interestingly, such distortions most commonly result in smaller ratios with the consequence of reducing the statistical significance of the ratios. This becomes more critical for less pronounced ratios where the evidence for differential expression is not strong. Indeed, statistical analysis by limma suggests that up to 87% of the genes with the largest and therefore most significant ratios (p -20 in the unamplified group have a p-value below 10e-20 in the amplified group. On the other hand, only 69% of the more moderate ratios (10e-20 -10 in the unamplified group have a p-value below 10e-10 in the amplified group. Our analysis also suggests that, overall, limma shows better overlap of genes found to be significant in the amplified and unamplified groups than the Z-scores statistics. Conclusion We conclude that microarray analysis of amplified samples performs best at detecting differences in gene expression, when these are large and when limma statistics are used.

  8. Patterns of HER2 Gene Amplification and Response to Anti-HER2 Therapies.

    Directory of Open Access Journals (Sweden)

    Rocio Vicario

    Full Text Available A chromosomal region that includes the gene encoding HER2, a receptor tyrosine kinase (RTK, is amplified in 20% of breast cancers. Although these tumors tend to respond to drugs directed against HER2, they frequently become resistant and resume their malignant progression. Gene amplification in double minutes (DMs, which are extrachromosomal entities whose number can be dynamically regulated, has been suggested to facilitate the acquisition of resistance to therapies targeting RTKs. Here we show that ~30% of HER2-positive tumors show amplification in DMs. However, these tumors respond to trastuzumab in a similar fashion than those with amplification of the HER2 gene within chromosomes. Furthermore, in different models of resistance to anti-HER2 therapies, the number of DMs containing HER2 is maintained, even when the acquisition of resistance is concomitant with loss of HER2 protein expression. Thus, both clinical and preclinical data show that, despite expectations, loss of HER2 protein expression due to loss of DMs containing HER2 is not a likely mechanism of resistance to anti-HER2 therapies.

  9. Epidermal growth factor receptor gene amplification and protein expression in glioblastoma multiforme: prognostic significance and relationship to other prognostic factors. (United States)

    Layfield, Lester J; Willmore, Carlynn; Tripp, Sheryl; Jones, Claudia; Jensen, Randy L


    Epidermal growth factor receptor (EGFR) overexpression occurs in a significant percentage of cases of glioblastoma multiforme (GBM), and amplification has been found in approximately 40% of these neoplasms. Controversy exists as to the prognostic significance of EGFR gene amplification: some reports have indicated that amplification is associated with a poor prognosis, while other authors have reported no relationship between gene amplification and prognosis. Some reports have found a poor prognosis to be associated with amplification of the EGFR gene in patients of all ages with GBM, while other authors have found EGFR amplification to be an independent predictor of prolonged survival in patients with GBM who are older than 60 years of age. The authors studied a series of 34 specimens (32 patients) with histologically proven GBM by immunohistochemistry for the presence of EGFR overexpression and by fluorescence in situ hybridization (FISH) for gene amplification of the EGFR gene. Results of these studies and data on patient age, sex, functional status, therapy, and survival were correlated to determine which variables were predictive of survival. p53 expression was also determined by immunohistochemistry and correlated with the other variables and survival.

  10. Amplification of thermostable lipase genes fragment from thermogenic phase of domestic waste composting process (United States)

    Nurhasanah, Nurbaiti, Santi; Madayanti, Fida; Akhmaloka


    Lipases are lipolytic enzymes, catalyze the hydrolysis of fatty acid ester bonds of triglycerides to produce free fatty acids and glycerol. The enzyme is widely used in various fields of biotechnological industry. Hence, lipases with unique properties (e.g.thermostable lipase) are still being explored by variation methods. One of the strategy is by using metagenomic approach to amplify the gene directly from environmental sample. This research was focused on amplification of lipase gene fragment directly from the thermogenic phase of domestic waste composting in aerated trenches. We used domestic waste compost from waste treatment at SABUGA, ITB for the sample. Total chromosomal DNA were directly extracted from several stages at thermogenic phase of compost. The DNA was then directly used as a template for amplification of thermostable lipase gene fragments using a set of internal primers namely Flip-1a and Rlip-1a that has been affixed with a GC clamp in reverse primer. The results showed that the primers amplified the gene from four stages of thermogenic phase with the size of lipase gene fragment of approximately 570 base pairs (bp). These results were further used for Denaturing Gradient Gel Electrophoresis (DGGE) analysis to determine diversity of thermostable lipase gene fragments.

  11. Detection of Clostridium perfringens alpha toxin gene in lambs by loop mediated isothermal amplification

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    B. Radhika


    Full Text Available Aim: The loop mediated isothermal amplification (LAMP was standardized for rapid detection of Clostridium perfringens. Materials and Methods: A total of 120 fecal samples were collected from enterotoxemia suspected lambs were used for screening of C. perfringens cpa gene by LAMP. The specificity of the LAMP amplified products was tested by digesting with restriction enzyme XmnI for alpha toxin gene. Results: Out of 120 samples screened 112 (93.3% samples were positive by both LAMP and polymerase chain reaction (PCR for detection of cpa gene which indicated the equal sensitivity of both the tests. The enzyme produced single cut in 162 base pair amplified product of alpha toxin gene at 81 base pair resulting in a single band in gel electrophoresis. Conclusion: Both LAMP and PCR for detection of cpa gene indicated the equal sensitivity of both the tests. Standardization of LAMP reaction for amplification of epsilon and beta toxin genes will help to identify the C. perfringens toxin types from the clinical samples. The test could be a suitable alternative to the PCR in detection of toxin types without the help of sophisticated machinery like thermal cycler. Considering its simplicity in operation and high sensitivity, there is the potential use of this technique in clinical diagnosis and surveillance of infectious diseases.

  12. CDK4 amplification is an alternative mechanism to p16 gene homozygous deletion in glioma cell lines

    National Research Council Canada - National Science Library

    He, J; Allen, J R; Collins, V P; Allalunis-Turner, M J; Godbout, R; Day, 3rd, R S; James, C D


    ... those established from malignant gliomas. Here we have examined 32 glioma cell lines for amplification-associated overexpression of the CDK4 gene as an alternative mechanism for abrogating the growth-regulatory effects of p16...

  13. Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips. (United States)

    Kosuri, Sriram; Eroshenko, Nikolai; Leproust, Emily M; Super, Michael; Way, Jeffrey; Li, Jin Billy; Church, George M


    Development of cheap, high-throughput and reliable gene synthesis methods will broadly stimulate progress in biology and biotechnology. Currently, the reliance on column-synthesized oligonucleotides as a source of DNA limits further cost reductions in gene synthesis. Oligonucleotides from DNA microchips can reduce costs by at least an order of magnitude, yet efforts to scale their use have been largely unsuccessful owing to the high error rates and complexity of the oligonucleotide mixtures. Here we use high-fidelity DNA microchips, selective oligonucleotide pool amplification, optimized gene assembly protocols and enzymatic error correction to develop a method for highly parallel gene synthesis. We tested our approach by assembling 47 genes, including 42 challenging therapeutic antibody sequences, encoding a total of ∼35 kilobase pairs of DNA. These assemblies were performed from a complex background containing 13,000 oligonucleotides encoding ∼2.5 megabases of DNA, which is at least 50 times larger than in previously published attempts.

  14. Dissection of the beta-globin replication-initiation region reveals specific requirements for replicator elements during gene amplification.

    Directory of Open Access Journals (Sweden)

    Naoya Okada

    Full Text Available Gene amplification plays a pivotal role in malignant transformation of human cells. A plasmid with both a mammalian replication-initiation region (IR/origin/replicator and a nuclear matrix-attachment region (MAR is spontaneously amplified in transfected cells by a mechanism that involves amplification at the extrachromosomal site, followed by amplification at the chromosomal arm, ultimately generating a long homogeneously staining region (HSR. Several observations suggest that replication initiation from IR sequences might mediate amplification. To test this idea, we previously dissected c-myc and DHFR IRs to identify the minimum sequence required to support amplification. In this study, we applied an improved analysis that discriminates between two amplification steps to the ß-globin RepP IR, which contains separate elements already known to be essential for initiation on the chromosome arm. The IR sequence was required at least for the extrachromosomal amplification step. In addition to the vector-encoded MAR, amplification also required an AT-rich region and a MAR-like element, consistent with the results regarding replicator activity on the chromosome. However, amplification did not require the AG-rich tract necessary for replicator activity, but instead required a novel sequence containing another AG-rich tract. The differential sequence requirement might be a consequence of extrachromosomal replication.

  15. Genome-wide copy number profiling to detect gene amplifications in neural progenitor cells

    Directory of Open Access Journals (Sweden)

    U. Fischer


    Full Text Available DNA sequence amplification occurs at defined stages during normal development in amphibians and flies and seems to be restricted in humans to drug-resistant and tumor cells only. We used array-CGH to discover copy number changes including gene amplifications and deletions during differentiation of human neural progenitor cells. Here, we describe cell culture features, DNA extraction, and comparative genomic hybridization (CGH analysis tailored towards the identification of genomic copy number changes. Further detailed analysis of amplified chromosome regions associated with this experiment, was published by Fischer and colleagues in PLOS One in 2012 (Fischer et al., 2012. We provide detailed information on deleted chromosome regions during differentiation and give an overview on copy number changes during differentiation induction for two representative chromosome regions.

  16. Duplication and amplification of antibiotic resistance genes enable increased resistance in isolates of multidrug-resistant Salmonella Typhimurium (United States)

    During normal bacterial DNA replication, gene duplication and amplification (GDA) events occur randomly at a low frequency in the genome throughout a population. In the absence of selection, GDA events that increase the number of copies of a bacterial gene (or a set of genes) are lost. Antibiotic ...

  17. Amplification of complete gag gene sequences from geographically distinct equine infectious anemia virus isolates. (United States)

    Boldbaatar, Bazartseren; Bazartseren, Tsevel; Koba, Ryota; Murakami, Hironobu; Oguma, Keisuke; Murakami, Kenji; Sentsui, Hiroshi


    In the current study, primers described previously and modified versions of these primers were evaluated for amplification of full-length gag genes from different equine infectious anemia virus (EIAV) strains from several countries, including the USA, Germany and Japan. Each strain was inoculated into a primary horse leukocyte culture, and the full-length gag gene was amplified by reverse transcription polymerase chain reaction. Each amplified gag gene was cloned into a plasmid vector for sequencing, and the detectable copy numbers of target DNA were determined. Use of a mixture of two forward primers and one reverse primer in the polymerase chain reaction enabled the amplification of all EIAV strains used in this study. However, further study is required to confirm these primers as universal for all EIAV strains. The nucleotide sequence of gag is considered highly conserved, as evidenced by the use of gag-encoded capsid proteins as a common antigen for the detection of EIAV in serological tests. However, significant sequence variation in the gag genes of different EIAV strains was found in the current study.

  18. Isolation, amplification and characterization of foodborne pathogen disease bacteria gene for rapid kit test development (United States)

    Nurjayadi, M.; Santoso, I.; Kartika, I. R.; Kurniadewi, F.; Saamia, V.; Sofihan, W.; Nurkhasanah, D.


    There is a lot of public concern over food safety. Food-safety cases recently, including many food poisoning cases in both the developed and developing countries, considered to be the national security threats which involved police investigation. Quick and accurate detection methods are needed to handle the food poisoning cases with a big number of sufferers at the same time. Therefore, the research is aimed to develop a specific, sensitive, and rapid result molecular detection tool for foodborne pathogen bacteria. We, thus, propose genomic level approach with Polymerase Chain Reaction. The research has successfully produced a specific primer to perform amplification to fim-C S. typhi, E. coli, and pef Salmonella typhimurium genes. The electrophoresis result shows that amplification products are 95 base pairs, 121 base pairs, and 139 base pairs; and all three genes are in accordance with the size of the in silico to third genes bacteria. In conclusion, the research has been successfully designed a specific detection tool to three foodborne pathogen bacteria genes. Further stages test and the uses of Real-time PCR in the detection are still in the trial process for better detection method.

  19. DNMT3B gene amplification predicts resistance to DNA demethylating drugs. (United States)

    Simó-Riudalbas, Laia; Melo, Sónia A; Esteller, Manel


    Disruption of the DNA methylation landscape is one of the most common features of human tumors. However, genetic alterations of DNA methyltransferases (DNMTs) have not been described in carcinogenesis. Herein, we show that pancreatic and breast cancer cells undergo gene amplification of the DNA methyltransferase 3B (DNMT3B). The presence of extra copies of the DNMT3B gene is linked to higher levels of the corresponding mRNA and protein. Most importantly, the elevated gene dosage of DNMT3B is associated with increased resistance to the growth-inhibitory effect mediated by DNA demethylating agents. In particular, cancer cells harboring DNMT3B gene amplification are less sensitive to the decrease in cell viability caused by 5-azacytidine (Vidaza), 5-aza-2-deoxycytidine (Decitabine), and SGI-1027. Overall, the data confirm DNMT3B as a bona fide oncogene in human cancer and support the incorporation of the DNMT3B copy number assay into current clinical trials assessing the efficacy of DNA demethylating drugs in solid tumors.

  20. Prognostic value of FGFR gene amplification in patients with different types of cancer: a systematic review and meta-analysis.

    Directory of Open Access Journals (Sweden)

    Jinjia Chang

    Full Text Available BACKGROUND: Fibroblast growth factor receptor (FGFR gene amplification has been reported in different types of cancer. We performed an up-to-date meta-analysis to further characterize the prognostic value of FGFR gene amplification in patients with cancer. METHODS: A search of several databases, including MEDLINE (PubMed, EMBASE, Web of Science, and China National Knowledge Infrastructure, was conducted to identify studies examining the association between FGFR gene amplification and cancer. A total of 24 studies met the inclusion criteria, and overall incidence rates, hazard risk (HR, overall survival, disease-free survival, and 95% confidence intervals (CIs were calculated employing fixed- or random-effects models depending on the heterogeneity of the included studies. RESULTS: In the meta-analysis of 24 studies, the prevalence of FGFR gene amplification was FGFR1: 0.11 (95% CI: 0.08-0.13 and FGFR2: 0.04 (95% CI: 0.02-0.06. Overall survival was significantly worse among patients with FGFR gene amplification: FGFR1 [HR 1.57 (95% CI: 1.23-1.99; p = 0.0002] and FGFR2 [HR 2.27 (95% CI: 1.73-3.00; p<0.00001]. CONCLUSIONS: Current evidence supports the conclusion that the outcomes of patients with FGFR gene amplified cancers is worse than for those with non-FGFR gene amplified cancers.

  1. MDM2 gene amplification: a new independent factor of adverse prognosis in non-small cell lung cancer (NSCLC). (United States)

    Dworakowska, Dorota; Jassem, Ewa; Jassem, Jacek; Peters, Brigitte; Dziadziuszko, Rafał; Zylicz, Maciej; Jakóbkiewicz-Banecka, Joanna; Kobierska-Gulida, Grazyna; Szymanowska, Amelia; Skokowski, Jan; Roessner, Albert; Schneider-Stock, Regine


    The prognostic impact of MDM2 amplification in non-small cell lung cancer (NSCLC) remains unknown. In this study, we investigated the occurrence of MDM2 amplification in surgically treated NSCLC patients. Molecular data were correlated with clinicopathological factors and evaluated for their prognostic value. The study group included 116 NSCLC patients who underwent pulmonary resection between 1996 and 1999. MDM2 amplification was assessed by real-time PCR using hybridization probe format on a LightCycler (Roche). The calculated ratio was a MDM2 value normalized to the amplification of the housekeeping gene phenylalaninhydroxylase (PAH). Survival curves were drawn according to the Kaplan-Meier method and compared with the use of the log-rank test. Multivariate analysis was based on Cox regression analysis. MDM2 amplification was found in 24 patients (21%). There was no relationship between MDM2 amplification and clinicopathological factors, such as sex, age and stage of disease, pT, pN, histology and tumor differentiation. Median disease-free survival (DFS) in patients with and without MDM2 amplification was 3 and 31 months, and 5-year DFS 24 and 33%, respectively (log-rank, P = 0.02). Likewise, median overall survival (OS) in patients with and without MDM2 amplification was 9 and 33 months, respectively, and 5-year OS 24 and 39%, respectively (log-rank, P = 0.01). The strong prognostic relevance of MDM2 amplification for both DFS and OS was confirmed in multivariate analysis (P < 0.01 for both comparisons). Our results suggest that MDM2 gene amplification analysis provides additional prognostic information in surgically treated NSCLC patients.

  2. Extensive gene amplification and concerted evolution within the CPR family of cuticular proteins in mosquitoes. (United States)

    Cornman, R Scott; Willis, Judith H


    Annotation of the Anopheles gambiae genome has revealed a large increase in the number of genes encoding cuticular proteins with the Rebers and Riddiford Consensus (the CPR gene family) relative to Drosophila melanogaster. This increase reflects an expansion of the RR-2 group of CPR genes, particularly the amplification of sets of highly similar paralogs. Patterns of nucleotide variation indicate that extensive concerted evolution is occurring within these clusters. The pattern of concerted evolution is complex, however, as sequence similarity within clusters is uncorrelated with gene order and orientation, and no comparable clusters occur within similarly compact arrays of the RR-1 group in mosquitoes or in either group in D. melanogaster. The dearth of pseudogenes suggests that sequence clusters are maintained by selection for high gene-copy number, perhaps due to selection for high expression rates. This hypothesis is consistent with the apparently parallel evolution of compact gene architectures within sequence clusters relative to single-copy genes. We show that RR-2 proteins from sequence-cluster genes have complex repeats and extreme amino-acid compositions relative to single-copy CPR proteins in An. gambiae, and that the amino-acid composition of the N-terminal and C-terminal sequence flanking the chitin-binding consensus region evolves in a correlated fashion.

  3. Selective Amplification of the Genome Surrounding Key Placental Genes in Trophoblast Giant Cells. (United States)

    Hannibal, Roberta L; Baker, Julie C


    While most cells maintain a diploid state, polyploid cells exist in many organisms and are particularly prevalent within the mammalian placenta [1], where they can generate more than 900 copies of the genome [2]. Polyploidy is thought to be an efficient method of increasing the content of the genome by avoiding the costly and slow process of cytokinesis [1, 3, 4]. Polyploidy can also affect gene regulation by amplifying a subset of genomic regions required for specific cellular function [1, 3, 4]. This mechanism is found in the fruit fly Drosophila melanogaster, where polyploid ovarian follicle cells amplify genomic regions containing chorion genes, which facilitate secretion of eggshell proteins [5]. Here, we report that genomic amplification also occurs in mammals at selective regions of the genome in parietal trophoblast giant cells (p-TGCs) of the mouse placenta. Using whole-genome sequencing (WGS) and digital droplet PCR (ddPCR) of mouse p-TGCs, we identified five amplified regions, each containing a gene family known to be involved in mammalian placentation: the prolactins (two clusters), serpins, cathepsins, and the natural killer (NK)/C-type lectin (CLEC) complex [6-12]. We report here the first description of amplification at selective genomic regions in mammals and present evidence that this is an important mode of genome regulation in placental TGCs.

  4. Clinicopathologic significance of HER-2/neu protein expression and gene amplification in gastric carcinoma

    Institute of Scientific and Technical Information of China (English)

    Shi-Yan Yan; Ying Hu; Jian-Gao Fan; Guo-Quan Tao; Yong-Ming Lu; Xu Cai; Bao-Hua Yu; Yi-Qun Du


    AIM: To study the HER-2/neu protein expression and gene amplification in gastric carcinoma and their relation. METHODS: One hundred and forty-five formalin-fixed and paraffin- embedded tumor tissue samples from Chinese gastric carcinoma patients were studied with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) methods. Clinicopathologic data about all patients were collected. RESULTS: The levels of HER-2 3+, HER-2 2+ and HER2 1+ were measurable in 6.9%, 8.3% and 17.2% of the samples, respectively. No HER-2 was stained in 67.6% of the samples. FISH showed that HER-2 gene was amplified in 18 samples, 10 HER-2 3+ samples, 5 HER-2 2+ samples, and 3 HER-2 1+ samples with IHC staining. HER-2 status was not correlated with the sex and age of patients, and tumor size, location or differentiation, but with the depth of invasion, TNM stage, lymph node and distant metastasis as well as histopathological classification of gastric cancer (P < 0.05). CONCLUSION: All samples with IHC as HER-2 expression should be analyzed with FISH. Detection of HER-2 gene amplification can assess the malignant biological behaviors and prognosis of gastric cancer.

  5. Optimization of laser capture microdissection and RNA amplification for gene expression profiling of prostate cancer

    Directory of Open Access Journals (Sweden)

    Vasmatzis George


    Full Text Available Abstract Background To discover prostate cancer biomarkers, we profiled gene expression in benign and malignant cells laser capture microdissected (LCM from prostate tissues and metastatic prostatic adenocarcinomas. Here we present methods developed, optimized, and validated to obtain high quality gene expression data. Results RNase inhibitor was included in solutions used to stain frozen tissue sections for LCM, which improved RNA quality significantly. Quantitative PCR assays, requiring minimal amounts of LCM RNA, were developed to determine RNA quality and concentration. SuperScript II™ reverse transcriptase was replaced with SuperScript III™, and SpeedVac concentration was eliminated to optimize linear amplification. The GeneChip® IVT labeling kit was used rather than the Enzo BioArray™ HighYield™ RNA transcript labeling kit since side-by-side comparisons indicated high-end signal saturation with the latter. We obtained 72 μg of labeled complementary RNA on average after linear amplification of about 2 ng of total RNA. Conclusion Unsupervised clustering placed 5/5 normal and 2/2 benign prostatic hyperplasia cases in one group, 5/7 Gleason pattern 3 cases in another group, and the remaining 2/7 pattern 3 cases in a third group with 8/8 Gleason pattern 5 cases and 3/3 metastatic prostatic adenocarcinomas. Differential expression of alpha-methylacyl coenzyme A racemase (AMACR and hepsin was confirmed using quantitative PCR.

  6. Oligonucleotide primers for targeted amplification of single-copy nuclear genes in apocritan Hymenoptera.

    Directory of Open Access Journals (Sweden)

    Gerrit Hartig

    Full Text Available BACKGROUND: Published nucleotide sequence data from the mega-diverse insect order Hymenoptera (sawflies, bees, wasps, and ants are taxonomically scattered and still inadequate for reconstructing a well-supported phylogenetic tree for the order. The analysis of comprehensive multiple gene data sets obtained via targeted PCR could provide a cost-effective solution to this problem. However, oligonucleotide primers for PCR amplification of nuclear genes across a wide range of hymenopteran species are still scarce. FINDINGS: Here we present a suite of degenerate oligonucleotide primer pairs for PCR amplification of 154 single-copy nuclear protein-coding genes from Hymenoptera. These primers were inferred from genome sequence data from nine Hymenoptera (seven species of ants, the honeybee, and the parasitoid wasp Nasonia vitripennis. We empirically tested a randomly chosen subset of these primer pairs for amplifying target genes from six Hymenoptera, representing the families Chrysididae, Crabronidae, Gasteruptiidae, Leucospidae, Pompilidae, and Stephanidae. Based on our results, we estimate that these primers are suitable for studying a large number of nuclear genes across a wide range of apocritan Hymenoptera (i.e., all hymenopterans with a wasp-waist and of aculeate Hymenoptera in particular (i.e., apocritan wasps with stingers. CONCLUSIONS: The amplified nucleotide sequences are (a with high probability from single-copy genes, (b easily generated at low financial costs, especially when compared to phylogenomic approaches, (c easily sequenced by means of an additionally provided set of sequencing primers, and (d suitable to address a wide range of phylogenetic questions and to aid rapid species identification via barcoding, as many amplicons contain both exonic and fast-evolving intronic nucleotides.

  7. Rapid PCR amplification protocols decrease the turn-around time for detection of antibiotic resistance genes in Gram-negative pathogens. (United States)

    Geyer, Chelsie N; Hanson, Nancy D


    A previously designed end-point multiplex PCR assay and singleplex assays used to detect β-lactamase genes were evaluated using rapid PCR amplification methodology. Amplification times were 16-18 minutes with an overall detection time of 1.5 hours. Rapid PCR amplifications could decrease the time required to identify resistance mechanisms in Gram-negative organisms.

  8. Isolation of epithelial cells from tooth brush and gender identification by amplification of SRY gene

    Directory of Open Access Journals (Sweden)

    A Vikram Simha Reddy


    Full Text Available Introduction: This study determines the importance of tooth brush from which DNA can be isolated and used for sex determination in forensic analysis. Materials and Methods: A total of 30 samples were collected and stored at room temperature for different periods of time interval. The epithelial cells adhered to the bristles of tooth brush were collected and genomic DNA was extracted and quantified using Nanodrop 1000 spectrophotometer. Results: Gender identification was done by amplification of sex determining region on Y chromosome (SRY gene using real-time polymerase chain reaction and minimal amount of DNA (in pico grams with 100% sensitivity and 73.3% specificity, i.e., all male samples showed positive results and out of 15 female samples 4 showed false positive results, i.e wrongly identified as males. Conclusion: With this study, we conclude that PCR is a valuable and sensitive procedure where minute contamination may cause alteration in the result, i.e, 4 females showed false positive result. Minute amount of DNA in picograms, which was collected at different intervals is enough for amplification of SRY gene and tooth brush can be used as one of the very valuable sources of gender identification.

  9. High-Resolution Genomic and Expression Profiling Reveals 105 Putative Amplification Target Genes in Pancreatic Cancer

    Directory of Open Access Journals (Sweden)

    Eija H. Mahlamaki


    Full Text Available Comparative genomic hybridization (CGH studies have provided a wealth of information on common copy number aberrations in pancreatic cancer, but the genes affected by these aberrations are largely unknown. To identify putative amplification target genes in pancreatic cancer, we performed a parallel copy number and expression survey in 13 pancreatic cancer cell lines using a 12,232-clone cDNA microarray, providing an average resolution of 300 kb throughout the human genome. CGH on cDNA microarray allowed highly accurate mapping of copy number increases and resulted in identification of 24 independent amplicons, ranging in size from 130 kb to 11 Mb. Statistical evaluation of gene copy number and expression data across all 13 cell lines revealed a set of 105 genes whose elevated expression levels were directly attributable to increased copy number. These included genes previously reported to be amplified in cancer as well as several novel targets for copy number alterations, such as p21-activated kinase 4 (PAK4, which was previously shown to be involved in cell migration, cell adhesion, and anchorage-independent growth. In conclusion, our results implicate a set of 105 genes that is likely to be actively involved in the development and progression of pancreatic cancer.


    Directory of Open Access Journals (Sweden)

    Karaneh Eftekhari


    Full Text Available Cancer is a disease that begins in the cells ofthe body which is characterized by uncontrolled, uncoordinated and undesirable cell division. If a cell accumulates critical mutations in five or six of the proto-oncogenes, tumour suppressor genes and DNA repair genes are likely to result in a fully malignant cell, capable of forming a tumour. In this work we described the isolation and amplification of the BRCA1 gene. Primers were designed and synthesised later used to amplify the BRCA1 gene. The total new workflow includes all steps from purified DNA to data analysis, and includes PCR for all amplicons covering the gene, PCR cleanup, cycle sequencing, electrophoresis, and data analysis. To simplify workflows and decrease the time-to-result, we focused on the method “one sample, one assay” approach. The success of this workflow was the 24-well plate design, which contained prespotted PCR primers covering the gene and also included multiplex nontemplate controls. The workflow was developed using a Genetic Analyzer and bands were observed.

  11. Amplification of the E2F1 transcription factor gene in the HEL erythroleukemia cell line

    DEFF Research Database (Denmark)

    Saito, M; Helin, K; Valentine, M B;


    and overexpressed in HEL erythroleukemia cells and translocated to other chromosomes in several established human leukemia cell lines. This study provides the first evidence of gene amplification involving a member of the E2F family of transcription factors. We propose that E2F1 overexpression in erythroid......The E2F transcription factor plays an important regulatory role in cell proliferation, mediating the expression of genes whose products are essential for inducing resting cells to enter the cell cycle and synthesize DNA. To investigate the possible involvement of E2F in hematopoietic malignancies...... progenitors may stimulate abnormal cell proliferation by overriding negative regulatory signals mediated by tumor suppressor proteins such as pRb....

  12. Monosomy of chromosome 17 in breast cancer during interpretation of HER2 gene amplification (United States)

    Brunelli, Matteo; Nottegar, Alessia; Bogina, Giuseppe; Caliò, Anna; Cima, Luca; Eccher, Albino; Vicentini, Caterina; Marcolini, Lisa; Scarpa, Aldo; Pedron, Serena; Brunello, Eleonora; Knuutila, Sakari; Sapino, Anna; Marchiò, Caterina; Bria, Emilio; Molino, Annamaria; Carbognin, Luisa; Tortora, Giampaolo; Jasani, Bharat; Miller, Keith; Merdol, Ibrahim; Zanatta, Lucia; Laurino, Licia; Wirtanen, Tiina; Zamboni, Giuseppe; Marconi, Marcella; Chilosi, Marco; Manfrin, Erminia; Martignoni, Guido; Bonetti, Franco


    Monosomy of chromosome 17 may affect the assessment of HER2 amplification. Notably, the prevalence ranges from 1% up to 49% due to lack of consensus in recognition. We sought to investigate the impact of monosomy of chromosome 17 to interpretation of HER2 gene status. 201 breast carcinoma were reviewed for HER2 gene amplification and chromosome 17 status. FISH analysis was performed by using double probes (LSI/CEP). Absolute gene copy number was also scored per each probe. HER2 FISH test was repeated on serial tissue sections, ranging in thickness from 3 to 20 µm. Ratio was scored and subsequently corrected by monosomy after gold control test using the aCGH method to overcome false interpretation due to artefactual nuclear truncation. HER2 immunotests was performed on all cases. 26/201 cases were amplified (13%). Single signals per CEP17 were revealed in 7/201 (3.5%) cases. Five out of 7 cases appeared monosomic with aCGH (overall, 5/201, 2.5%) and evidenced single signals in >60% of nuclei after second-look on FISH when matching both techniques. Among 5, one case showed amplification with a pattern 7/1 (HER2/CEP17>2) of copies (3+ at immunotest); three cases revealed single signals per both probes (LSI/CEP=1) and one case revealed a 3:1 ratio; all last 4 cases showed 0/1+ immunoscore. We concluded that: 1) monosomy of chromosome 17 may be observed in 2.5% of breast carcinoma; 2) monosomy of chromosome 17 due to biological reasons rather than nuclear truncation was observed when using the cut-off of 60% of nuclei harboring single signals; 3) the skewing of the ratio due to single centromeric 17 probe may lead to false positive evaluation; 4) breast carcinomas showing a 3:1 ratio (HER2/CEP17) usually show negative 0/1+ immunoscore and <6 gene copy number at FISH. PMID:26328251

  13. Sequencing the mouse Y chromosome reveals convergent gene acquisition and amplification on both sex chromosomes. (United States)

    Soh, Y Q Shirleen; Alföldi, Jessica; Pyntikova, Tatyana; Brown, Laura G; Graves, Tina; Minx, Patrick J; Fulton, Robert S; Kremitzki, Colin; Koutseva, Natalia; Mueller, Jacob L; Rozen, Steve; Hughes, Jennifer F; Owens, Elaine; Womack, James E; Murphy, William J; Cao, Qing; de Jong, Pieter; Warren, Wesley C; Wilson, Richard K; Skaletsky, Helen; Page, David C


    We sequenced the MSY (male-specific region of the Y chromosome) of the C57BL/6J strain of the laboratory mouse Mus musculus. In contrast to theories that Y chromosomes are heterochromatic and gene poor, the mouse MSY is 99.9% euchromatic and contains about 700 protein-coding genes. Only 2% of the MSY derives from the ancestral autosomes that gave rise to the mammalian sex chromosomes. Instead, all but 45 of the MSY's genes belong to three acquired, massively amplified gene families that have no homologs on primate MSYs but do have acquired, amplified homologs on the mouse X chromosome. The complete mouse MSY sequence brings to light dramatic forces in sex chromosome evolution: lineage-specific convergent acquisition and amplification of X-Y gene families, possibly fueled by antagonism between acquired X-Y homologs. The mouse MSY sequence presents opportunities for experimental studies of a sex-specific chromosome in its entirety, in a genetically tractable model organism.

  14. CASCADE, a platform for controlled gene amplification for high, tunable and selection-free gene expression in yeast (United States)

    Strucko, Tomas; Buron, Line Due; Jarczynska, Zofia Dorota; Nødvig, Christina Spuur; Mølgaard, Louise; Halkier, Barbara Ann; Mortensen, Uffe Hasbro


    Over-expression of a gene by increasing its copy number is often desirable in the model yeast Saccharomyces cerevisiae. It may facilitate elucidation of enzyme functions, and in cell factory design it is used to increase production of proteins and metabolites. Current methods are typically exploiting expression from the multicopy 2 μ-derived plasmid or by targeting genes repeatedly into sequences like Ty or rDNA; in both cases, high gene expression levels are often reached. However, with 2 μ-based plasmid expression, the population of cells is very heterogeneous with respect to protein production; and for integration into repeated sequences it is difficult to determine the genetic setup of the resulting strains and to achieve specific gene doses. For both types of systems, the strains often suffer from genetic instability if proper selection pressure is not applied. Here we present a gene amplification system, CASCADE, which enables construction of strains with defined gene copy numbers. One or more genes can be amplified simultaneously and the resulting strains can be stably propagated on selection-free medium. As proof-of-concept, we have successfully used CASCADE to increase heterologous production of two fluorescent proteins, the enzyme β-galactosidase the fungal polyketide 6-methyl salicylic acid and the plant metabolite vanillin glucoside. PMID:28134264

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

    Directory of Open Access Journals (Sweden)

    Wennuan Liu


    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.

  16. Specific amplification of iron receptor genes in Xylella fastidiosa strains from different hosts

    Directory of Open Access Journals (Sweden)

    Flávia Teresa Hansen Pacheco


    Full Text Available Bacterial production of siderophores may involve specific genes related to nonribosomal peptide and polyketide biosynthesis, which have not been fully identified in the genome of Xylella fastidiosa strain 9a5c. However, a search for siderophore-related genes in strain 9a5c indicated five membrane receptors, including siderophore, ferrichrome-iron and hemin receptors. All these biomolecules are thought to be associated with iron transport and utilization. Eighty isolates obtained from citrus orchards containing trees that developed citrus variegated chlorosis (CVC were screened for siderophore production. The results demonstrated that only 10 of the isolates did not produce siderophores. Additional strains obtained from coffee, almond, mulberry, elm, ragweed, periwinkle and grape also infected by X. fastidiosa were also shown by the chromeazurol bioassay to produce siderophores. In order to correlate siderophore production with the presence of siderophore-related genes, a polymerase chain reaction (PCR was developed using specific primers for the catechol-type ferric enterobactin receptor (pfeA and the hydroxamate-type ferrisiderophore receptor (fiuA genes of strain 9a5c. The PCR results confirmed our hypothesis by demonstrating that amplification products were detected in all strains except for those isolates that did not produce siderophores.

  17. Amplification of a Gene Related to Mammalian mdr Genes in Drug-Resistant Plasmodium falciparum (United States)

    Wilson, Craig M.; Serrano, Adelfa E.; Wasley, Annemarie; Bogenschutz, Michael P.; Shankar, Anuraj H.; Wirth, Dyann F.


    The malaria parasite Plasmodium falciparum contains at least two genes related to the mammalian multiple drug resistance genes, and at least one of the P. falciparum genes is expressed at a higher level and is present in higher copy number in a strain that is resistant to multiple drugs than in a strain that is sensitive to the drugs.

  18. Specific amplification by PCR of rearranged genomic variable regions of immunoglobulin genes from mouse hybridoma cells. (United States)

    Berdoz, J; Monath, T P; Kraehenbuhl, J P


    We have designed a novel strategy for the isolation of the rearranged genomic fragments encoding the L-VH-D-JH and L-V kappa/lambda-J kappa/lambda regions of mouse immunoglobulin genes. This strategy is based on the PCR amplification of genomic DNA from mouse hybridomas using multiple specific primers chosen in the 5'-untranslated region and in the intron downstream of the rearranged JH/J kappa/lambda sequences. Variable regions with intact coding sequences, including full-length leader peptides (L) can be obtained without previous DNA sequencing. Our strategy is based on a genomic template that produces fragments that do not need to be adapted for recombinant antibody expression, thus facilitating the generation of chimeric and isotype-switched immunoglobulins.

  19. Human-specific gene ARHGAP11B promotes basal progenitor amplification and neocortex expansion. (United States)

    Florio, Marta; Albert, Mareike; Taverna, Elena; Namba, Takashi; Brandl, Holger; Lewitus, Eric; Haffner, Christiane; Sykes, Alex; Wong, Fong Kuan; Peters, Jula; Guhr, Elaine; Klemroth, Sylvia; Prüfer, Kay; Kelso, Janet; Naumann, Ronald; Nüsslein, Ina; Dahl, Andreas; Lachmann, Robert; Pääbo, Svante; Huttner, Wieland B


    Evolutionary expansion of the human neocortex reflects increased amplification of basal progenitors in the subventricular zone, producing more neurons during fetal corticogenesis. In this work, we analyze the transcriptomes of distinct progenitor subpopulations isolated by a cell polarity-based approach from developing mouse and human neocortex. We identify 56 genes preferentially expressed in human apical and basal radial glia that lack mouse orthologs. Among these, ARHGAP11B has the highest degree of radial glia-specific expression. ARHGAP11B arose from partial duplication of ARHGAP11A (which encodes a Rho guanosine triphosphatase-activating protein) on the human lineage after separation from the chimpanzee lineage. Expression of ARHGAP11B in embryonic mouse neocortex promotes basal progenitor generation and self-renewal and can increase cortical plate area and induce gyrification. Hence, ARHGAP11B may have contributed to evolutionary expansion of human neocortex.

  20. Simultaneous amplification of two bacterial genes: more reliable method of Helicobacter pylori detection in microbial rich dental plaque samples. (United States)

    Chaudhry, Saima; Idrees, Muhammad; Izhar, Mateen; Butt, Arshad Kamal; Khan, Ayyaz Ali


    Polymerase Chain reaction (PCR) assay is considered superior to other methods for detection of Helicobacter pylori (H. pylori) in oral cavity; however, it also has limitations when sample under study is microbial rich dental plaque. The type of gene targeted and number of primers used for bacterial detection in dental plaque samples can have a significant effect on the results obtained as there are a number of closely related bacterial species residing in plaque biofilm. Also due to high recombination rate of H. pylori some of the genes might be down regulated or absent. The present study was conducted to determine the frequency of H. pylori colonization of dental plaque by simultaneously amplifying two genes of the bacterium. One hundred dental plaque specimens were collected from dyspeptic patients before their upper gastrointestinal endoscopy and presence of H. pylori was determined through PCR assay using primers targeting two different genes of the bacterium. Eighty-nine of the 100 samples were included in final analysis. With simultaneous amplification of two bacterial genes 51.6% of the dental plaque samples were positive for H. pylori while this prevalence increased to 73% when only one gene amplification was used for bacterial identification. Detection of H. pylori in dental plaque samples is more reliable when two genes of the bacterium are simultaneously amplified as compared to one gene amplification only.

  1. EPSPS gene amplification conferring resistance to glyphosate in windmill grass (Chloris truncata) in Australia. (United States)

    Ngo, The D; Malone, Jenna M; Boutsalis, Peter; Gill, Gurjeet; Preston, Christopher


    Five glyphosate-resistant populations of Chloris truncata originally collected from New South Wales were compared with one susceptible (S) population from South Australia to confirm glyphosate resistance and elucidate possible mechanisms of resistance. Based on the amounts of glyphosate required to kill 50% of treated plants (LD50 ), glyphosate resistance (GR) was confirmed in five populations of C. truncata (A536, A528, T27, A534 and A535.1). GR plants were 2.4-8.7-fold more resistant and accumulated less shikimate after glyphosate treatment than S plants. There was no difference in glyphosate absorption and translocation between GR and S plants. The EPSPS gene did not contain any point mutation that had previously been associated with resistance to glyphosate. The resistant plants (A528 and A536) contained up to 32-48 more copies of the EPSPS gene than the susceptible plants. This study has identified EPSPS gene amplification contributing to glyphosate resistance in C. truncata. In addition, a Glu-91-Ala mutation within EPSPS was identified that may contribute to glyphosate resistance in this species. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  2. mdm-2 gene amplification in 3T3-L1 preadipocytes. (United States)

    Berberich, S J; Litteral, V; Mayo, L D; Tabesh, D; Morris, D


    In this study the regulation of the murine double minute-2 (mdm-2) gene was examined in NIH 3T3-L1 preadipocytes. The 3T3-L1 cell line, under proper conditions, has the capacity to differentiate from fibroblasts into adipocytes [15]. A recent report demonstrated that mdm-2 overexpression could block myogenesis [12]. While examining the regulation of the mdm-2 gene during adipogenesis, it was discovered that 3T3-L1 cells possess a 36-fold elevation of mdm-2 mRNA relative to A31 cells, another immortalized Balb/c 3T3 fibroblast cell line that lacks the capacity to differentiate. Based on Southern blot analysis, the increase in mdm-2 mRNA was the result of a mdm-2 gene amplification. The level of Mdm-2 protein in undifferentiated 3T3-L1 cells was elevated relative to A31 fibroblasts and resulted from translation of mRNA transcripts initiating from the p53-independent P1 promoter. We also examined how mdm-2 and p53 levels changed as undifferentiated fibroblasts converted to adipocytes. While mdm-2 mRNA levels remained elevated, p53 mRNA, protein, and DNA-binding activity decreased. These results suggest that adipogenesis is unaffected by elevated Mdm-2 levels and that the overexpression of mdm-2 mRNA is predominantly p53 independent.

  3. Gene synthesis by integrated polymerase chain assembly and PCR amplification using a high-speed thermocycler (United States)

    TerMaat, Joel R.; Pienaar, Elsje; Whitney, Scott E.; Mamedov, Tarlan G.; Subramanian, Anuradha


    Polymerase chain assembly (PCA) is a technique used to synthesize genes ranging from a few hundred base pairs to many kilobase pairs in length. In traditional PCA, equimolar concentrations of single stranded DNA oligonucleotides are repeatedly hybridized and extended by a polymerase enzyme into longer dsDNA constructs, with relatively few full-length sequences being assembled. Thus, traditional PCA is followed by a second primer-mediated PCR reaction to amplify the desired full-length sequence to useful, detectable quantities. Integration of assembly and primer-mediated amplification steps into a single reaction using a high-speed thermocycler is shown to produce similar results. For the integrated technique, the effects of oligo concentration, primer concentration, and number of oligonucleotides are explored. The technique is successfully demonstrated for the synthesis of two genes encoding EPCR-1 (653 bp) and pUC19 β-lactamase (929 bp) in under 20 min. However, rapid integrated PCA–PCR was found to be problematic when attempted with the TM-1 gene (1509 bp). Partial oligonucleotide sets of TM-1 could be assembled and amplified simultaneously, indicating that the technique may be limited to a maximum number of oligonucleotides due to competitive annealing and competition for primers. PMID:19799938

  4. MLL gene amplification in acute myeloid leukemia and myelodysplastic syndromes is associated with characteristic clinicopathological findings and TP53 gene mutation. (United States)

    Tang, Guilin; DiNardo, Courtney; Zhang, Liping; Ravandi, Farhad; Khoury, Joseph D; Huh, Yang O; Muzzafar, Tariq; Medeiros, L Jeffrey; Wang, Sa A; Bueso-Ramos, Carlos E


    MLL gene rearrangements are well-recognized aberrations in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). In contrast, MLL gene amplification in AML/MDS remains poorly characterized. Here, we report a series of 21 patients with myeloid neoplasms associated with MLL gene amplification from 1 institution. This series included 13 men and 8 women, with a median age of 64 years. Eleven patients presented as AML with myelodysplasia-related changes, 6 as therapy-related AML, and 4 as therapy-related MDS. All patients had a highly complex karyotype, including frequent -5/del(5q), -18, and -17/del(17p) abnormalities; 16 patients were hypodiploid. TP53 mutations were detected in all 12 patients tested, and 3 patients showed TP53 mutation before MLL amplification. Morphologically, the leukemic cells frequently showed cytoplasmic vacuoles, bilobed nuclei, and were associated with background dyspoiesis. Immunophenotypically, 15 patients had a myeloid and 4 had myelomonocytic immunophenotype. Laboratory coagulopathies were common; 7 patients developed disseminated intravascular coagulopathy, and 3 died of intracranial bleeding. All patients were refractory to therapy; the median overall survival was 1 month, after MLL gene amplification was detected. We concluded that AML/MDS with MLL gene amplification is likely a subset of therapy-related AML/MDS or AML with myelodysplasia-related changes, associated with distinct clinicopathological features, frequent disseminated intravascular coagulopathy, a highly complex karyotype, TP53 deletion/mutation, and an aggressive clinical course.

  5. Development of a Loop-Mediated Isothermal Amplification Assay Targeting the mpb64 Gene for Diagnosis of Intraocular Tuberculosis (United States)

    Balne, Praveen Kumar; Barik, Manas Ranjan; Sharma, Savitri


    A loop-mediated isothermal amplification (LAMP) assay targeting the mpb64 gene for the diagnosis of intraocular tuberculosis was highly specific (100%), sensitive (85.7%), rapid, and easy to perform. The LAMP assay can be an alternative to conventional PCR for the diagnosis of ocular tuberculosis in resource-limited settings. PMID:23966513

  6. Molecular diagnosis of Kingella kingae pericarditis by amplification and sequencing of the 16S rRNA gene. (United States)

    Matta, Matta; Wermert, Delphine; Podglajen, Isabelle; Sanchez, Olivier; Buu-Hoï, Annie; Gutmann, Laurent; Meyer, Guy; Mainardi, Jean-Luc


    Kingella kingae is a fastidious gram-negative bacillus that is considered an emerging pathogen in pediatric settings but remains less common in adults. Here we describe a case of pericarditis in an immunocompetent adult host. The microorganism was identified directly from the clinical sample by molecular techniques, i.e., 16S rRNA gene amplification and sequencing.

  7. AIB1 gene amplification and the instability of polyQ encoding sequence in breast cancer cell lines

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    Clarke Robert


    Full Text Available Abstract Background The poly Q polymorphism in AIB1 (amplified in breast cancer gene is usually assessed by fragment length analysis which does not reveal the actual sequence variation. The purpose of this study is to investigate the sequence variation of poly Q encoding region in breast cancer cell lines at single molecule level, and to determine if the sequence variation is related to AIB1 gene amplification. Methods The polymorphic poly Q encoding region of AIB1 gene was investigated at the single molecule level by PCR cloning/sequencing. The amplification of AIB1 gene in various breast cancer cell lines were studied by real-time quantitative PCR. Results Significant amplifications (5–23 folds of AIB1 gene were found in 2 out of 9 (22% ER positive cell lines (in BT-474 and MCF-7 but not in BT-20, ZR-75-1, T47D, BT483, MDA-MB-361, MDA-MB-468 and MDA-MB-330. The AIB1 gene was not amplified in any of the ER negative cell lines. Different passages of MCF-7 cell lines and their derivatives maintained the feature of AIB1 amplification. When the cells were selected for hormone independence (LCC1 and resistance to 4-hydroxy tamoxifen (4-OH TAM (LCC2 and R27, ICI 182,780 (LCC9 or 4-OH TAM, KEO and LY 117018 (LY-2, AIB1 copy number decreased but still remained highly amplified. Sequencing analysis of poly Q encoding region of AIB1 gene did not reveal specific patterns that could be correlated with AIB1 gene amplification. However, about 72% of the breast cancer cell lines had at least one under represented (3CAA(CAG9(CAACAG3(CAACAGCAG2CAA of the original cell line, a number of altered poly Q encoding sequences were found in the derivatives of MCF-7 cell lines. Conclusion These data suggest that poly Q encoding region of AIB1 gene is somatic unstable in breast cancer cell lines. The instability and the sequence characteristics, however, do not appear to be associated with the level of the gene amplification.

  8. Calcium Channel Genes Associated with Bipolar Disorder Modulate Lithium's Amplification of Circadian Rhythms (United States)

    McCarthy, Michael J.; LeRoux, Melissa; Wei, Heather; Beesley, Stephen; Kelsoe, John R.; Welsh, David K.


    Bipolar disorder (BD) is associated with mood episodes and low amplitude circadian rhythms. Previously, we demonstrated that fibroblasts grown from BD patients show weaker amplification of circadian rhythms by lithium compared to control cells. Since calcium signals impact upon the circadian clock, and L-type calcium channels (LTCC) have emerged as genetic risk factors for BD, we examined whether loss of function in LTCCs accounts for the attenuated response to lithium in BD cells. We used fluorescent dyes to measure Ca2+ changes in BD and control fibroblasts after lithium treatment, and bioluminescent reporters to measure Per2∷luc rhythms in fibroblasts from BD patients, human controls, and mice while pharmacologically or genetically manipulating calcium channels. Longitudinal expression of LTCC genes (CACNA1C, CACNA1D and CACNB3) was then measured over 12-24 hr in BD and control cells. Our results indicate that independently of LTCCs, lithium stimulated intracellular Ca2+ less effectively in BD vs. control fibroblasts. In longitudinal studies, pharmacological inhibition of LTCCs or knockdown of CACNA1A, CACNA1C, CACNA1D and CACNB3 altered circadian rhythm amplitude. Diltiazem and knockdown of CACNA1C or CACNA1D eliminated lithium's ability to amplify rhythms. Knockdown of CACNA1A or CACNB3 altered baseline rhythms, but did not affect rhythm amplification by lithium. In human fibroblasts, CACNA1C genotype predicted the amplitude response to lithium, and the expression profiles of CACNA1C, CACNA1D and CACNB3 were altered in BD vs. controls. We conclude that in cells from BD patients, calcium signaling is abnormal, and that LTCCs underlie the failure of lithium to amplify circadian rhythms. PMID:26476274

  9. Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. (United States)

    Brakstad, O G; Aasbakk, K; Maeland, J A


    Synthetic oligonucleotide primers of 21 and 24 bases, respectively, were used in the polymerase chain reaction (PCR) to amplify a sequence of the nuc gene, which encodes the thermostable nuclease of Staphylococcus aureus. A DNA fragment of approximately 270 bp was amplified from lysed S. aureus cells or isolated DNA. The PCR product was detected by agarose gel electrophoresis or Southern blot analysis by using a 33-mer internal nuc gene hybridization probe. With S. aureus cells the lower detection limit was less than 10 CFU, and with the isolated target the lower detection limit was 0.69 pg of DNA. The primers recognized 90 of 90 reference or clinical S. aureus strains. Amplification was not recorded when 80 strains representing 16 other staphylococcal species were tested or when 20 strains representing 9 different nonstaphylococcal species were tested. Some of the non-S. aureus staphylococci produced thermostable nucleases but were PCR negative. The PCR product was generated when in vitro-cultured S. aureus was used to prepare simulated clinical specimens of blood, urine, cerebrospinal fluid, or synovial fluid. No PCR product was generated when the sterile body fluids were tested. However, the sensitivity of the PCR was reduced when S. aureus in blood or urine was tested in comparison with that when bacteria in saline were tested. With the bacteria in blood, the detection limit of the PCR was 10(3) CFU. A positive PCR result was recorded when a limited number of clinical samples from wounds verified to be infected with S. aureus were tested, while the PCR product was not detected in materials from infections caused by other bacteria. Generation of PCR products was not affected by exposure of S. aureus to bactericidal agents, including cloxacillin and gentamicin, prior to testing, but was affected by exposure to UV radiation. The PCR for amplification of the nuc gene has potential for the rapid diagnosis of S. aureus infections by direct testing of clinical

  10. Polymerase chain reaction (PCR) amplification of a nucleoprotein gene sequence of infectious hematopoietic necrosis virus (United States)

    Arakawa, C.K.; Deering, R.E.; Higman, K.H.; Oshima, K.H.; O'Hara, P.J.; Winton, J.R.


    The polymerase chain reaction [PCR) was used to amplify a portion of the nucleoprotein [NI gene of infectious hematopoietic necrosis virus (IHNV). Using a published sequence for the Round Butte isolate of IHNV, a pair of PCR pnmers was synthesized that spanned a 252 nucleotide region of the N gene from residue 319 to residue 570 of the open reading frame. This region included a 30 nucleotide target sequence for a synthetic oligonucleotide probe developed for detection of IHNV N gene messenger RNA. After 25 cycles of amplification of either messenger or genomic RNA, the PCR product (DNA) of the expected size was easily visible on agarose gels stained with ethidium bromide. The specificity of the amplified DNA was confirmed by Southern and dot-blot analysis using the biotinylated oligonucleotide probe. The PCR was able to amplify the N gene sequence of purified genomic RNA from isolates of IHNV representing 5 different electropherotypes. Using the IHNV primer set, no PCR product was obtained from viral hemorrhagic septicemia virus RNA, but 2 higher molecular weight products were synthesized from hirame rhabdovirus RNA that did not hybridize with the biotinylated probe. The PCR could be efficiently performed with all IHNV genomic RNA template concentrations tested (1 ng to 1 pg). The lowest level of sensitivity was not determined. The PCR was used to amplify RNA extracted from infected cell cultures and selected tissues of Infected rainbow trout. The combination of PCR and nucleic acid probe promises to provide a detection method for IHNV that is rapid, h~ghly specific, and sensitive.

  11. Lager yeasts possess dynamic genomes that undergo rearrangements and gene amplification in response to stress. (United States)

    James, Tharappel C; Usher, Jane; Campbell, Susan; Bond, Ursula


    A long-term goal of the brewing industry is to identify yeast strains with increased tolerance to the stresses experienced during the brewing process. We have characterised the genomes of a number of stress-tolerant mutants, derived from the lager yeast strain CMBS-33, that were selected for tolerance to high temperatures and to growth in high specific gravity wort. Our results indicate that the heat-tolerant strains have undergone a number of gross chromosomal rearrangements when compared to the parental strain. To determine if such rearrangements can spontaneously arise in response to exposure to stress conditions experienced during the brewing process, we examined the chromosome integrity of both the stress-tolerant strains and their parent during a single round of fermentation under a variety of environmental stresses. Our results show that the lager yeast genome shows tremendous plasticity during fermentation, especially when fermentations are carried out in high specific gravity wort and at higher than normal temperatures. Many localised regions of gene amplification were observed especially at the telomeres and at the rRNA gene locus on chromosome XII, and general chromosomal instability was evident. However, gross chromosomal rearrangements were not detected, indicating that continued selection in the stress conditions are required to obtain clonal isolates with stable rearrangements. Taken together, the data suggest that lager yeasts display a high degree of genomic plasticity and undergo genomic changes in response to environmental stress.

  12. Widespread hypomethylation occurs early and synergizes with gene amplification during esophageal carcinogenesis.

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    Hector Alvarez


    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.

  13. Preparation and Amplification of Colony of Goat Transgenic Fetal Fibroblast and Mammary Gland Epithelial Cell with Human Lactoferrin Gene

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yu-ling; LIU Feng-jun; ZHANG Yong


    [Objective] The aim was to explore technical system of making single transgenic positive cells become colony cells by amplification culture. [Method] Fetal fibroblasts and mammary gland epithelial cells of single goat fetus of pBLM-C1 which specifically expressed human lactoferrin were cloned. Single cell colony of single transfection cell was prepared with 3 concentrations of 0%, 50% and 100% conditioned culture media. Transfection cell and non-transfection cell were carried out amplification culture by con-culture, neo gene was as screened gene, genome DNA of transfection cell was detected by PCR method. Chromosome karyotype analysis of single colony cell was tested. [Result] Compared with non-conditioned culture medium, 100% conditioned culture medium could greatly increase survived rate of single colony cells (FF: 53.33% vs. 10.00%; MGE: 33.33% vs. 6.67%). Compared with control, con-culture of transfection cell and non-transfection cell could greatly increase rate of transfection cell single colony after amplification culture (FF: 53.33% vs. 10.00%;MGE: 33.33% vs. 6.67%), confluence time of amplification culture was significantly decreased (20-30 d). The result of PCR showed that the colony cell obtained by above method contained hLF target gene. The result of karyotype analysis showed that most cloned cell chromosomes were normal. [Conclusion] The study provides a reliable method for separating transgenic cell, inserting and diagnosing ideal vector, and can save expense and time for transgenic animal production.

  14. Amplification and expression of genes from the 17q11 approximately q12 amplicon in breast cancer cells. (United States)

    Luoh, Shiuh-Wen


    Amplification of a portion of 17q11 approximately q12 involving the human epidermal growth factor receptor-2 (HER-2/neu) gene is common in solid tumors. HER-2/neu amplification in breast cancer is associated with a poor prognosis and may predict response to therapeutic interventions. The molecular complexity and informative content of the 17q11 approximately q12 amplified DNA remain largely unknown. Study of available sequence information in public database allowed us to construct a contig in bacterial artificial chromosome that covers this region. We have identified a several hundred kilobase core segment from this region that is amplified in three out of three breast cancer cell lines with HER-2/neu amplification. There is striking correlation between amplification and overexpression of genes from this region. Amplified and overexpressed genes, in addition to HER-2/neu, may play functional roles in the pathogenesis of breast cancer and may serve as additional targets for therapy. Published by Elsevier Science Inc.

  15. Distinct phenotypic differences associated with differential amplification of receptor tyrosine kinase genes at 4q12 in glioblastoma.

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    Anna Burford

    Full Text Available Gene amplification at chromosome 4q12 is a common alteration in human high grade gliomas including glioblastoma, a CNS tumour with consistently poor prognosis. This locus harbours the known oncogenes encoding the receptor tyrosine kinases PDGFRA, KIT, and VEGFR2. These receptors are potential targets for novel therapeutic intervention in these diseases, with expression noted in tumour cells and/or associated vasculature. Despite this, a detailed assessment of their relative contributions to different high grade glioma histologies and the underlying heterogeneity within glioblastoma has been lacking. We studied 342 primary high grade gliomas for individual gene amplification using specific FISH probes, as well as receptor expression in the tumour and endothelial cells by immunohistochemistry, and correlated our findings with specific tumour cell morphological types and patterns of vasculature. We identified amplicons which encompassed PDGFRA only, PDGFRA/KIT, and PDGFRA/KIT/VEGFR2, with distinct phenotypic correlates. Within glioblastoma specimens, PDGFRA amplification alone was linked to oligodendroglial, small cell and sarcomatous tumour cell morphologies, and rare MGMT promoter methylation. A younger age at diagnosis and better clinical outcome in glioblastoma patients is only seen when PDGFRA and KIT are co-amplified. IDH1 mutation was only found when all three genes are amplified; this is a subgroup which also harbours extensive MGMT promoter methylation. Whilst PDGFRA amplification was tightly linked to tumour expression of the receptor, this was not the case for KIT or VEGFR2. Thus we have identified differential patterns of gene amplification and expression of RTKs at the 4q12 locus to be associated with specific phenotypes which may reflect their distinct underlying mechanisms.


    Institute of Scientific and Technical Information of China (English)

    周中军; 罗贤懋; 林晨; 陈凤


    P-glycoprotein plays an important role in highly drug resistant cells. But its high expression cannot be acheived by chemotherapy. In order to study the effect of P-glycoprotein on clinical tumors, wo ostablished a low ADM resistant colon cancer ceil line HR/ADM and determined the amplification and expression of mdr-1 gene. The GLC/ADM showed a resistant pattern similar to classical MDR and the transcription of mdr-1 gene determined by RT-PCR increased. The immunocytcchemical analysis showed strong positive staining with monoelonal antibozly. The gene amplification of mdr-l was dearly demonstrated by southern blot. Our results suggested that moderate expression of P-glycoprotein might be enough for a high resistant pattern.

  17. MYEOV : A candidate gene for DNA amplification events occurring centromeric to CCNDI in breast cancer

    NARCIS (Netherlands)

    Janssen, JWG; Cuny, M; Orsetti, B; Rodriguez, C; Valles, H; Bartram, CR; Schuuring, E; Theillet, C


    Rearrangements of chromosome 11q13 are frequently observed in human cancer. The 11q13 region harbors several chromosomal breakpoint clusters found in hematologic malignancies and exhibits frequent DNA amplification in carcinomas. DNA amplification patterns in breast tumors are consistent with the ex

  18. Detection of MYCN Gene Amplification in Neuroblastoma by Fluorescence In Situ Hybridization: A Pediatric Oncology Group Study

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    Prasad Mathew


    Full Text Available To assess the utility of fluorescence in situ hybridization (FISH for analysis of MYCN gene amplification in neuroblastoma, we compared this assay with Southern blot analysis using tumor specimens collected from 232 patients with presenting characteristics typical of this disease. The FISH technique identified MYCN amplification in 47 cases, compared with 39 by Southern blotting, thus increasing the total number of positive cases by 21%. The major cause of discordancy was a low fraction of tumor cells (≤30% replacement in clinical specimens, which prevented an accurate estimate of MYCN copy number by Southern blotting. With FISH, by contrast, it was possible to analyze multiple interphase nuclei of tumor cells, regardless of the proportion of normal peripheral blood, bone marrow, or stromal cells in clinical samples. Thus, FISH could be performed accurately with very small numbers of tumor cells from touch preparations of needle biopsies. Moreover, this procedure allowed us to discern the heterogeneous pattern of MYCN amplification that is characteristic of neuroblastoma. We conclude that FISH improves the detection of MYCN gene amplification in childhood neuroblastomas in a clinical setting, thus facilitating therapeutic decisions based on the presence or absence of this prognostically important biologic marker.

  19. DNA sequence templates adjacent nucleosome and ORC sites at gene amplification origins in Drosophila. (United States)

    Liu, Jun; Zimmer, Kurt; Rusch, Douglas B; Paranjape, Neha; Podicheti, Ram; Tang, Haixu; Calvi, Brian R


    Eukaryotic origins of DNA replication are bound by the origin recognition complex (ORC), which scaffolds assembly of a pre-replicative complex (pre-RC) that is then activated to initiate replication. Both pre-RC assembly and activation are strongly influenced by developmental changes to the epigenome, but molecular mechanisms remain incompletely defined. We have been examining the activation of origins responsible for developmental gene amplification in Drosophila. At a specific time in oogenesis, somatic follicle cells transition from genomic replication to a locus-specific replication from six amplicon origins. Previous evidence indicated that these amplicon origins are activated by nucleosome acetylation, but how this affects origin chromatin is unknown. Here, we examine nucleosome position in follicle cells using micrococcal nuclease digestion with Ilumina sequencing. The results indicate that ORC binding sites and other essential origin sequences are nucleosome-depleted regions (NDRs). Nucleosome position at the amplicons was highly similar among developmental stages during which ORC is or is not bound, indicating that being an NDR is not sufficient to specify ORC binding. Importantly, the data suggest that nucleosomes and ORC have opposite preferences for DNA sequence and structure. We propose that nucleosome hyperacetylation promotes pre-RC assembly onto adjacent DNA sequences that are disfavored by nucleosomes but favored by ORC.

  20. Complex oncogenic translocations with gene amplification are initiated by specific DNA breaks in lymphocytes. (United States)

    Wright, Sarah M; Woo, Yong H; Alley, Travis L; Shirley, Bobbi-Jo; Akeson, Ellen C; Snow, Kathy J; Maas, Sarah A; Elwell, Rachel L; Foreman, Oded; Mills, Kevin D


    Chromosomal instability is a hallmark of many tumor types. Complex chromosomal rearrangements with associated gene amplification, known as complicons, characterize many hematologic and solid cancers. Whereas chromosomal aberrations, including complicons, are useful diagnostic and prognostic cancer markers, their molecular origins are not known. Although accumulating evidence has implicated DNA double-strand break repair in suppression of oncogenic genome instability, the genomic elements required for chromosome rearrangements, especially complex lesions, have not been elucidated. Using a mouse model of B-lineage lymphoma, characterized by complicon formation involving the immunoglobulin heavy chain (Igh) locus and the c-myc oncogene, we have now investigated the requirement for specific genomic segments as donors for complex rearrangements. We now show that specific DNA double-strand breaks, occurring within a narrow segment of Igh, are necessary to initiate complicon formation. By contrast, neither specific DNA breaks nor the powerful intronic enhancer Emu are required for complicon-independent oncogenesis. This study is the first to delineate mechanisms of complex versus simple instability and the first to identify specific chromosomal elements required for complex chromosomal aberrations. These findings will illuminate genomic cancer susceptibility and risk factors.

  1. BHK cell lines with increased rates of gene amplification are hypersensitive to ultraviolet light

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    Giulotto, E.; Bertoni, L.; Attolini, C.; Rainaldi, G.; Anglana, M. (Dipartimento di Genetica e Microbiologia Adriano Buzzati-Traverso, Pavia (Italy))


    Four cell lines (MP1, -4, -5, -7), isolated from baby hamster kidney cells after simultaneous selection with N-(phosphonacetyl)-L-aspartate and methotrexate, have previously been shown to amplify their DNA at an increased rate. We now show that all four lines are hypersensitive to killing by UV light and mitomycin C. At high doses of UV light or mitomycin C, the MP lines survived less than 10% or less than 5% as well as parental cells, respectively. After UV irradiation, inhibition of DNA and RNA synthesis was greater in MP than in parental cells, and recovery was slower or absent. A 2- to 3.5-fold increase in the frequency of UV-induced sister chromatid exchange was also seen in the four cell lines. In MP5, unscheduled DNA replication after treatment with UV light was only approximately 70% as great as in parental cells and the other MP lines. In MP4 and MP7 cells S phase was elongated. Although their individual properties confirm that the four cell lines are independent, their common properties suggest a relationship between tolerance of DNA damage and gene amplification.

  2. Amplification of hTERT and hTERC genes in leukemic cells with high expression and activity of telomerase. (United States)

    Nowak, Tomasz; Januszkiewicz, Danuta; Zawada, Mariola; Pernak, Monika; Lewandowski, Krzysztof; Rembowska, Jolanta; Nowicka, Karina; Mankowski, Przemyslaw; Nowak, Jerzy


    Reactivation of telomerase plays an important role in carcinogenesis. Malignant cells almost always possess high activity and expression of telomerase. The aim of this study was to see whether there is any relationship between telomerase activity and expression and hTERT and hTERC gene amplification in acute lymphoblastic leukemia (ALL) and non-lymphoblastic leukemia (ANLL) cells. In addition telomere length was tested in leukemic cells at the time of diagnosis and during remission. Expression of the three components of telomerase (hTERT, hTERC and TP1) as well as telomerase activity was found both in ALL and ANLL cells. Telomerase activity was diminished in patients in remission. The leukemic cells showed considerable heterogeneity of terminal restriction fragments, that is telomere length. ALL cells showed a variable pattern of telomere length in contrast to ANLL cells which produced a predominantly short telomere pattern. Telomere length in the lymphocytes of leukemia patients was shorter in remission as compared to the time of diagnosis. FISH analysis revealed amplification of hTERT and hTERC genes in ALL and ANLL cells. Quantitative analysis showed that leukemic cells possess higher number of hTERT and hTERC copies than the normal PBL. Our results suggest that the activation of telomerase in leukemic cells is connected with amplification of hTERT and hTERC genes. The high expression and activity of telomerase found in leukemic cells may be partially explained by amplified hTERT and hTERC genes. Amplification of the telomerase genes seems to be a common event in carcinogenesis and may play a role in telomerase reactivation leading to cell immortalization.

  3. Comparison of the c-MET gene amplification between primary tumor and metastatic lymph nodes in non-small cell lung cancer. (United States)

    Xu, Chun-Wei; Wang, Wen-Xian; Wu, Mei-Juan; Zhu, You-Cai; Zhuang, Wu; Lin, Gen; Du, Kai-Qi; Huang, Yun-Jian; Chen, Yan-Ping; Chen, Gang; Fang, Mei-Yu


    c-MET has recently been identified as a promising novel target in non-small cell lung cancer (NSCLC). We detected the consistency of c-MET gene amplification in metastatic lymph nodes and tumor tissues of NSCLC patients and discuss the clinical application value of c-MET gene amplification in metastatic lymph nodes. Real-time fluorescent quantitative PCR was used to test tumor tissues in 368 NSCLC patients and 178 paired metastatic lymph node samples. The amplification consistency in metastatic lymph nodes and tissue samples were compared and the correlation between c-MET gene amplification and the clinical characteristics of patients was analyzed. The c-MET gene amplification rate was 8.97% (33/368) in tumor tissues. Of the 178 paired cases, c-MET gene amplification was positive in 7.95% (15/178) of cancerous tissues and 18.54% (33/178) of metastatic lymph nodes. c-MET gene amplification was detected more frequently in metastatic lymph nodes than in primary cancerous tissue. When metastatic lymph nodes were used as surrogate samples of primary cancerous tissues, the sensitivity was 86.67% (13/15) and the specificity was 87.69% (143/163). Screening for c-MET gene amplification in lymph node metastases could determine which patients are eligible for tyrosine kinase inhibitor therapy. Lymph node metastasis can predict c-MET gene amplification in a primary tumor and guide the clinical use of c-MET gene targeted drugs. © 2017 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

  4. [Evaluation of c-myc and CCNE2 amplification in breast cancer with quantitative multi-gene fluorescence in-situ hybridization]. (United States)

    Li, Zhishuang; Meng, Qingyong; Yu, Qiong; Zhou, Zhiqiang; Li, Li


    To investigate c-myc and CCNE2 gene amplifications and their relationship in breast cancer. Sixty-six infiltrating ductal breast carcinomas with foci of ductal carcinoma in situ components collected from January 2005 to December 2007 were selected for tissue microarray and quantitative multi-gene FISH for c-myc and CCNE2 gene amplification, and the relationship with the clinicopathologic features was analyzed. Of the 66 cases, 18 (27.3%) showed c-myc amplification and 23 (34.8%) showed CCNE2 amplification. A strong correlation was found between c-myc and CCNE2 amplification (P c-myc and CCNE2 amplifications were all aneuploidy, and were HER2 positive (P c-myc amplification also showed higher Ki-67 index (P C-myc and CCNE2 amplifications are common events in breast cancer, and they often coexist. C-myc and CCNE2 genes may play critical roles in the pathogenesis and development of breast cancer through unique and overlapping signaling pathways.

  5. Amplification of Whole Tumor Genomes and Gene-by-Gene Mapping of Genomic Aberrations from Limited Sources of Fresh-Frozen and Paraffin-Embedded DNA (United States)

    Bredel, Markus; Bredel, Claudia; Juric, Dejan; Kim, Young; Vogel, Hannes; Harsh, Griffith R.; Recht, Lawrence D.; Pollack, Jonathan R.; Sikic, Branimir I.


    Sufficient quantity of genomic DNA can be a bottleneck in genome-wide analysis of clinical tissue samples. DNA polymerase Phi29 can be used for the random-primed amplification of whole genomes, although the amplification may introduce bias in gene dosage. We have performed a detailed investigation of this technique in archival fresh-frozen and formalin-fixed/paraffin-embedded tumor DNA by using cDNA microarray-based comparative genomic hybridization. Phi29 amplified DNA from matched pairs of fresh-frozen and formalin-fixed/paraffin-embedded tumor samples with similar efficiency. The distortion in gene dosage representation in the amplified DNA was nonrandom and reproducibly involved distinct genomic loci. Regional amplification efficiency was significantly linked to regional GC content of the template genome. The biased gene representation in amplified tumor DNA could be effectively normalized by using amplified reference DNA. Our data suggest that genome-wide gene dosage alterations in clinical tumor samples can be reliably assessed from a few hundred tumor cells. Therefore, this amplification method should lend itself to high-throughput genetic analyses of limited sources of tumor, such as fine-needle biopsies, laser-microdissected tissue, and small paraffin-embedded specimens. PMID:15858140

  6. The SRY-HMG box gene, SOX4, is a target of gene amplification at chromosome 6p in lung cancer

    NARCIS (Netherlands)

    Medina, P.P.; Castillo, S.D.; Blanco, S.; Sanz-Garcia, M.; Largo, C.; Alvarez, S.; Yokota, J.; Gonzalez-Neira, A.; Benitez, J.; Clevers, H.; Cigudosa, J.C.; Lazo, P.A.; Sanchez-Cespedes, M.


    The search for oncogenes is becoming increasingly important in cancer genetics because they are suitable targets for therapeutic intervention. To identify novel oncogenes, activated by gene amplification, we analyzed cDNA microarrays by high-resolution comparative genome hybridization and compared D

  7. Human identification from forensic materials by amplification of a human-specific sequence in the myoglobin gene.


    Ono T; Miyaishi S; Yamamoto Y; Yoshitome K; Ishikawa T.; Ishizu H


    We developed a method for human identification of forensic biological materials by PCR-based detection of a human-specific sequence in exon 3 of the myoglobin gene. This human-specific DNA sequence was deduced from differences in the amino acid sequences of myoglobins between humans and other animal species. The new method enabled amplification of the target DNA fragment from 30 samples of human DNA, and the amplified sequences were identical with that already reported. Using this method, we ...

  8. Human telomerase gene amplification and high-risk human papillomavirus infection in women with cervical intra-epithelial neoplasia. (United States)

    Takac, Iztok; Arko, D; Kodric, T; Poljak, M; Zagorac, A; Erjavec-Skerget, A; Kokalj-Vokac, N


    This study was designed to investigate whether a correlation exists between amplification of the human telomerase gene (human telomerase RNA component [TERC]) and high-risk human papillomavirus (HR-HPV) infection in 101 women with cervical intra-epithelial neoplasia (CIN). Eight patients (7.9%) had CIN 1, 24 (23.8%) had CIN 2 and 69 (68.3%) had CIN 3. TERC was amplified in 31.7% of all CIN patients. The difference in frequency of TERC amplification between patients with low-grade CIN (CIN 1) and those with high-grade CIN (CIN 2 and CIN 3) was not significant. HR-HPV infection was detected in 88.1% of all CIN cases and was significantly more frequent in patients with CIN 2 and CIN 3 than in patients with CIN 1. There was no significant difference in the frequency of HR-HPV infection between groups of patients with and without TERC amplification. In conclusion, this study found no correlation between TERC amplification and HR-HPV infection in patients with CIN.

  9. Clinical significance of hTERC and C-Myc genes amplification in a group of Egyptian patients with cancer cervix. (United States)

    Eid, M M; Nossair, H M; Ismael, M T; Amira, G; Hosney, M M; Abdul Rahman, R


    Cervical cancer is the second most common cancer in women worldwide after breast cancer. Cervical cancer is a preventable disease. The implementation of cervical cancer screening programs has greatly decreased the morbidity and mortality, as precancerous lesions and early invasive cervical cancer could be detected and treated effectively. The detection of hTERC gene amplification was suggested as a possible diagnostic marker for use in routine cytological screening. The present study was designed to detect genomic gains of the hTERC and C-MYC genes using FISH technique and to investigate the relationship between genes amplification and the clinical data of the patients. The current study was carried out on twelve cases with cervical cancer at different grades (three cases were grade I, six cases were grade II and three cases were grade III). Interphase FISH analysis using LSI probe, Cervical Cancer probe hTERC (3q26) & C-MYC (8q24), was successfully performed on 12 patients with cancer cervix. Interphase FISH analysis revealed positive hTERC gene amplification in all cases of cancer cervix (100%). However C-MYC gene amplification was detected in four cases only (33.3%). Statistical analysis of the data revealed significant correlation between hTERC amplification and grading. Also, there was significant correlation between C-MYC amplification and grading and highly significant correlation between C-MYC amplification and hTERC amplification. On the other hand hTERC and C-MYC genes amplification showed an inverse correlation with the ages of the patients. The present study highlights the importance of using hTERC and C-MYC genes FISH probes for cases with cancer cervix or pre-malignant lesions as a sensitive technique. This method provides an easy and effective applicable approach which helps in the diagnosis and prognosis, as an increased copy number is associated with a more advanced grade that could be detected in the early stages of the disease.

  10. Comparison of different protocols for DNA preparation and PCR amplification of mitochondrial genes of tardigrades

    Directory of Open Access Journals (Sweden)

    Ralph O. SCHILL


    Full Text Available Phylogenetic relationships and molecular taxonomy within the Tardigrada have been given a lot of attention in recent years. Here I present the first comparison of different protocols for DNA preparation by investigating six commercial available DNA extraction kits and the CTAB method. Successful extraction of DNA from tardigrades depends strongly on the life-stage (embryo, adult, and on the condition of the specimens, respectively on the preservation (anhydrobiotic, ethanol. Although the extraction kits showed differences in the amount of extracted DNA, in all cases fresh tissue of live animals or embryos resulted in the best quality and quantity of DNA. A lesser amount of DNA was extractable from anhydrobiotic animals and embryos and the results of specimens fixed in ethanol were unsatisfactory. All used commercially available DNA extraction kits and PCR cocktails have been focused on vertebrate tissues, blood, cultured cells, bacteria and yeast. However, I used successfully the kits according to the manufacturer’s instruction without changes in the protocols for DNA extraction of tardigrades. Commercial kits provide a simple and convenient way to isolate pure genomic DNA of high-quality from tardigrades. Furthermore I tested eight different Taq polymerase enzymes for PCR amplification of mitochondrial genes of tardigrades. Each of the enzymes resulted in a PCR product, and even if the amount of the PCR products was quite different, it was possible to use it successful for direct sequencing. Summarizing, the successful PCR of the target DNA depends on the purity and quality of the DNA template and for this the species preservation is more critical than the extraction method or the PCR cocktail which can be optimized.

  11. Human telomerase RNA component (hTERC gene amplification detected by FISH in precancerous lesions and carcinoma of the larynx

    Directory of Open Access Journals (Sweden)

    Liu Yu


    Full Text Available Abstract Background Gain of 3q26 is frequently observed in squamous cell carcinomas of mucosal origin, including those originating in the head and neck region. The human telomerase RNA component (hTERC gene, which is located on chromosome 3q26, encodes for an RNA subunit of telomerase that maintains the length of telomeres through cellular divisions, and is activated in malignant diseases. The present study was designed to detect hTERC amplification in laryngeal lesions and evaluate whether this might serve as a supportive biomarker in histopathological analysis for in the diagnosis of laryngeal lesions. Methods Fluorescent in situ hybridization (FISH was applied on formalin-fixed paraffin-embedded blocks of 93 laryngeal specimens, including 14 normal epithelium (NE, 15 mild dysplasia (Md, 18 moderate dysplasia (MD, 16 severe dysplasia (SD, 9 carcinoma in situ (CIS, and 21 invasive carcinoma (IC. Results By histopathologic examination, hTERC amplification rates in NE, Md, MD, SD, CIS and IC cases were 0% (0/14, 13.33% (2/15, 72.22% (13/18, 81.25% (13/16, 100% (9/9 and 100% (21/21, respectively. Amplification of hTERC was significantly associated with histopathologic diagnosis (P Conclusions The hTERC amplification is important in the development of laryngeal squamous cell carcinoma (LSCC. FISH detection of hTERC amplification may provide an effective approach in conjunction with histopathologic evaluation for differential diagnosis of laryngeal lesions. Virtual Slides The virtual slide(s for this article can be found here:

  12. mRNA Expression of Vimentin Gene in Lens of Transgenic Mouse and DNA Amplification in Human Cataracts

    Institute of Scientific and Technical Information of China (English)

    YanLi; XienpingLiu; 等


    Purpose:To investigate the role of vimentin gene in cataractogenesis.Methods:The12.7kb chicken vimentin genes were microinjected into the male pronuclei of 918 fertilized mice eggs.841injected embryos were transferred into oviducts of pseudopregnant recipient females.of which 12pregnant mice gave birth to 49offsping mice.The integration and expression of exogenous gene in the offsping were analysed by Southern and Northern blot byhridizations,In the human senile cataract,the lens vimentin gene was analyzed with the chicken vi-mentin gene probe.Results:It showed that four of F1offspring were transgenic mice in which the chicken vimenttin gene was integrated in their genomes.The transgenic band was12kb,similar to the12.7kb chicken vimentin fragment injected.One2kbvi-mentin mRNAwas visualized on E2 mouse lens blot.which revealed that the chicken vimentin gene was efficiently expressed in this transgenic mouse.In the humansenile cataract lens,12kb BamHI-restricted vimentin fragments displayed a stronger hybridization signal than that of the control lens in Southern blot anal-ysis,It implies that the Formation of human senile cataract may be associated with the amplification of vimentin gene.Conclusions:We have successfully developed four transgenic mice bearing chicken vimentin gene and having mRNA expression which can be used for further study.It is to be observed if the normal lens cell function is affected by the expressed product and cataract occurs in our transgenic mice.The cause of the gene ampli-fication in human ctaract remains for further investigation.Eye Science 1995;11:113-116.

  13. Use of aiiA gene amplification for AHL-lactonase production from endophytic bacterium Enterobacter species. (United States)

    Rajesh, P S; Rai, V Ravishankar


    AHL-lactonase has gained renewed interest due to biotechnological applications such as antiquorum sensing, antibiofilm strategies, biofouling, etc. In our study, the production of AHL-lactonase from endophytic bacteria Enterobacter aerogenes VT66 was optimized by response surface methodology (RSM) using central composite design (CCD) for four different cultural conditions. The relative activity of AHL-lactonase was correlated with amplification of aiiA homologous gene amplification with respect to cultural conditions. Statistical analysis by ANOVA of the quadratic regression model showed that the RSM model constructed is highly significant, as indicated by F-test with a low probability value (p(model) < 0.0001) and high regression coefficient (0.9997) as well as lower coefficient of variation (1.86%) indicate that suitability of variable parameters. The quadratic regression model of AHL-lactonase production in terms of relative activity was built and the optimal cultural conditions for maximum enzyme production were determined as 32.5 °C temperature, pH 7.0, 350 μM of substrate concentration and 33 h of incubation time. The enhanced AHL-lactonase yielded 1.33 fold increases in relative activity and it positively correlated with the amplification of aiiA gene.

  14. EPSPS Gene Amplification in Glyphosate-Resistant Italian Ryegrass (Lolium perenne ssp. multiflorum) Populations from Arkansas (United States). (United States)

    Salas, Reiofeli A; Scott, Robert C; Dayan, Franck E; Burgos, Nilda R


    Glyphosate-resistant Italian ryegrass was detected in Arkansas (United States) in 2007. In 2014, 45 populations were confirmed resistant in eight counties across the state. The level of resistance and resistance mechanisms in six populations were studied to assess the severity of the problem and identify alternative management approaches. Dose-response bioassays, glyphosate absorption and translocation experiments, herbicide target (EPSPS) gene sequence analysis, and gene amplification assays were conducted. The dose causing 50% growth reduction (GR50) was 7-19 times higher for the resistant population than for the susceptible standard. Uptake and translocation of (14)C-glyphosate were similar in resistant and susceptible plants, and no mutation in the EPSPS gene known to be associated with resistance to glyphosate was detected. Resistant plants contained from 11- to >100-fold more copies of the EPSPS gene than the susceptible plants, whereas the susceptible plants had only one copy of EPSPS. Plants surviving the recommended dose of glyphosate contained at least 10 copies. The EPSPS copy number was positively related to glyphosate resistance level (r = 80). Therefore, resistance to glyphosate in these populations is due to multiplication of the target site. Resistance mechanisms could be location-specific. Suppressing the mechanism for gene amplification may overcome resistance.

  15. Isolation of epithelial cells from acrylic removable dentures and gender identification by amplification of SRY gene using real time PCR

    Directory of Open Access Journals (Sweden)

    Renjith George


    Full Text Available This study evaluates the usefulness of acrylic dentures as the source of DNA for forensic analysis. Thirty-eight samples (21 males and 17 females were collected and stored for different time periods. The epithelial cells adhered to the dentures were retrieved and the genomic DNA was extracted. All the samples yielded sufficient amount of DNA for analysis irrespective of the storage time. Gender determination was done by amplification of the sex determining region on the Y chromosome (SRY using real-time polymerase chain reaction with 100% accuracy, within minimal time. With this study, we conclude that saliva-stained acrylic dentures can act as a source of forensic DNA and co-amplification of SRY gene with other routine sex typing markers will give unambiguous gender identification.

  16. HIV-1包膜单基因Nest-PCR体系优化及包膜基因准种扩增%Optimization of HIV-1 envelope gene PCR amplification system and amplification of envelope gene quasispecies

    Institute of Scientific and Technical Information of China (English)

    全红; 刘松; 张昊天; 任彩云; 庄敏; 凌虹; 李妍


    目的 确定HIV-1包膜(envelope,env)单基因扩增(single genome amplification,SGA)的最优反应体系,获得env单基因扩增产物.方法 采用SGA及Nest-PCR扩增HIV-1 env全长基因,并对Nest-PCR反应体系中各成分量进行优化;在此基础上,适当稀释HIV-1感染者cDNA模板进行Nest-PCR,以获得PCR扩增产物阳性率不超过30%的模板稀释度.结果 SGA Nest-PCR扩增HIV-1 env全长基因,引物浓度为0.3 μmol/L,dNTP浓度为0.25 μmol/L时可获得特异性高的env全长基因;凝胶回收纯化及稀释第一轮PCR产物可有效提高特异性条带的产量.采用优化反应体系成功从一名HIV-1感染者体内扩增出18株env单基因序列.结论 优化了HIV-1 env基因SGA Nest-PCR的反应体系,并从HIV-1感染者获得了病毒包膜基因准种,为后续进行准种分析奠定了基础.%Objective To optimize the PCR conditions for single genome amplification (SGA) of HIV-1 envelope (env) gene. Methods The efficiency of the SGA-PCR was assessed based on the amplification results of env gene using the non-diluted cDNA template, the optimal dilution of cDNA template that may result in no more than 30% positive amplification was predicted. Results The elements of SGA and Nested-PCR such as the amounts of primers were 0. 3 μmol/L, dNTP were 0. 25 μmol/L, purification of products of the first-round PCR reactions and diluting cDNA templates increased the amount of interest products effectively. One HIV-1 infected individual after the env gene SGA, 18 interest products confirmed by sequencing env sequence. Conclusion Optimization of the HIV-1 env gene the SGA Nest-PCR reaction system, and the viral envelope gene quasispecies from HIV-1 infection, laid the foundation for subsequent analysis of the quasispecies.

  17. A phase II study of Epirubicin in oxaliplatin-resistant patients with metastatic colorectal cancer and TOP2A gene amplification

    DEFF Research Database (Denmark)

    Tarpgaard, Line S.; Qvortrup, Camilla; Nygård, Sune Boris;


    A gene amplification in their tumor cells. BACKGROUND: Epirubicin is an anthracycline that targets DNA topoisomerase 2-α enzyme encoded by the TOP2A gene. It is used for treatment of several malignancies, but currently not in CRC. TOP2A gene amplifications predict improved efficacy of epirubicin...... normal tissue. The TOP2A gene is located on chromosome 17 and when the TOP2A/CEN-17 ratio was applied to identify tumors with gene loss or amplifications, 10.5 % had a ratio ≥ 1.5 consistent with gene amplification and 2.6 % had a ratio ≤ 0.8 suggesting gene deletions. Based on these observations......, investigating the efficacy of epirubicin in patients with oxaliplatin refractory mCRC and with a cancer cell TOP2A/CEN-17 ratio ≥ 1.5. TOP2A gene amplification measured by fluorescence in situ hybridization. A total of 25 evaluable patients (15 + 10 in two steps) will be included (Simon's two-stage minimax...

  18. Amplification of TLO Mediator Subunit Genes Facilitate Filamentous Growth in Candida Spp. (United States)

    Liu, Zhongle; Moran, Gary P.; Myers, Lawrence C.


    Filamentous growth is a hallmark of C. albicans pathogenicity compared to less-virulent ascomycetes. A multitude of transcription factors regulate filamentous growth in response to specific environmental cues. Our work, however, suggests the evolutionary history of C. albicans that resulted in its filamentous growth plasticity may be tied to a change in the general transcription machinery rather than transcription factors and their specific targets. A key genomic difference between C. albicans and its less-virulent relatives, including its closest relative C. dubliniensis, is the unique expansion of the TLO (TeLOmere-associated) gene family in C. albicans. Individual Tlo proteins are fungal-specific subunits of Mediator, a large multi-subunit eukaryotic transcriptional co-activator complex. This amplification results in a large pool of ‘free,’ non-Mediator associated, Tlo protein present in C. albicans, but not in C. dubliniensis or other ascomycetes with attenuated virulence. We show that engineering a large ‘free’ pool of the C. dubliniensis Tlo2 (CdTlo2) protein in C. dubliniensis, through overexpression, results in a number of filamentation phenotypes typically associated only with C. albicans. The amplitude of these phenotypes is proportional to the amount of overexpressed CdTlo2 protein. Overexpression of other C. dubliniensis and C. albicans Tlo proteins do result in these phenotypes. Tlo proteins and their orthologs contain a Mediator interaction domain, and a potent transcriptional activation domain. Nuclear localization of the CdTlo2 activation domain, facilitated naturally by the Tlo Mediator binding domain or artificially through an appended nuclear localization signal, is sufficient for the CdTlo2 overexpression phenotypes. A C. albicans med3 null mutant causes multiple defects including the inability to localize Tlo proteins to the nucleus and reduced virulence in a murine systemic infection model. Our data supports a model in which the

  19. PCR-based amplification and heterologous expression of Pseudomonas alcohol dehydrogenase genes from the soil metagenome for biocatalysis. (United States)

    Itoh, Nobuya; Isotani, Kentaro; Makino, Yoshihide; Kato, Masaki; Kitayama, Kouta; Ishimota, Tuyoshi


    The amplification of useful genes from metagenomes offers great biotechnological potential. We employed this approach to isolate alcohol dehydrogenase (adh) genes from Pseudomonas to aid in the synthesis of optically pure alcohols from various ketones. A PCR primer combination synthesized by reference to the adh sequences of known Pseudomonas genes was used to amplify full-length adh genes directly from 17 samples of DNA extracted from soil. Three such adh preparations were used to construct Escherichia coli plasmid libraries. Of the approximately 2800 colonies obtained, 240 putative adh-positive clones were identified by colony-PCR. Next, 23 functional adh genes named using the descriptors HBadh and HPadh were analyzed. The adh genes obtained via this metagenomic approach varied in their DNA and amino acid sequences. Expression of the gene products in E. coli indicated varying substrate specificity. Two representative genes, HBadh-1 and HPadh-24, expressed in E. coli and Pseudomonas putida, respectively, were purified and characterized in detail. The enzyme products of these genes were confirmed to be useful for producing anti-Prelog chiral alcohols.

  20. Heterogeneous HER2 gene amplification: impact on patient outcome and a clinically relevant definition. (United States)

    Bartlett, Alastair I; Starcyznski, Jane; Robson, Tammy; Maclellan, Alex; Campbell, Fiona M; van de Velde, Cornelis J H; Hasenburg, Annette; Markopoulos, Christos; Seynaeve, Caroline; Rea, Daniel; Bartlett, John M S


    Heterogeneous expression or amplification is a challenge to HER2 diagnostics. A guideline defines heterogeneity as the presence of between 5% and 50% cells with HER2/CEP17 ratios of more than 2.20. We audited the frequency of such cells and their clinical impact in the results from 2 pathology laboratories combined with data from the TEAM [Tamoxifen vs Exemestane Adjuvant Multicentre] pathology study. HER2 reports were scanned and the percentages of amplified cells reported. Of 6,461 eligible cases, 754 (11.7%) exhibited 50% or more cells with ratios of more than 2.20, which is "amplified" by College of American Pathologists guidelines. Of the cases, 2,166 (33.5%) exhibited more than 5% but less than 50% of cells with HER2/CEP17 ratios of more than 2.20, or "heterogeneous amplification." No prognostic impact was observed when fewer than 30% of cells exhibited ratios of more than 2.20. All amplified cases with 30% to 50% of cells with ratios more than 2.20 were identified as such by United Kingdom guidelines. The percentage of tumor cells with HER2/CEP17 ratios more than 2.20 does not identify cases with heterogeneous amplification or poor outcome. A modified approach for identification of true heterogeneous amplification is suggested.

  1. Investigation of Gene Expression Correlating With Centrosome Amplification in Development and Progression of Breast Cancer (United States)


    Endocrinology 142, 4006-4014. 9. Colditz, G. A., Stampfer , M. J., Willett, W. C., Hennekens, C. H., Rosner, B. 37. Li, J. J., Hou, X., Banerjee, S. K...overexpression induces centrosome amplification, mitotic spindle abnormalities, and aneuploidy Christopher J. Nelsen,Ŗ Ryoko Kuriyama,3𔃾 Betsy Hirsch,4𔃿

  2. SMAD5 Gene Expression, Rearrangements, Copy Number, Amplification at Fragile Site FRA5C in Human Hepatocellular Carcinoma

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    Drazen B. Zimonjic


    Full Text Available Signaling by the transforming growth factor (TGFfamily members is transduced from the cell surface to the nucleus by the Smad group of intracellular proteins. Because we detected alterations on the long arm of chromosome 5, we examined the status of the SMAD5 gene in human hepatocellular carcinoma (HCC cell lines and primary HCC. In 16 cell lines, chromosome alterations of chromosome 5 were observed in nine cell lines by fluorescence in situ hybridization (FISH, an increase in SMAD5 gene copy number relative to the ploidy level was found in eight lines. The breakpoints in unbalanced translocations and deletions frequently occurred near the SMAD5 locus, but apparently did not cause loss of SMAD5. In one cell line, where comparative genomic hybridization showed DNA copy number gain confined to the region 5831, we detected by FISH high-level amplification of the SMAD5 gene located within the fragile site FRA5C. Semiquantitative polymerase chain reaction did not reveal changes in SMAD5 DNA levels in 15 of 17 primary HCC specimens. In 17 HCC cell lines, SMAD5 mRNA levels were either maintained or upregulated by an increase in gene dosage or another mechanism. Collectively, our results show that SMAD5 undergoes copy number gain and increased expression, rather than loss of expression, therefore suggest that this gene does not act as a tumorsuppressor gene in HCC. The Hep-40 HCC cell line with high-level amplification and significant overexpression of SMAD5 may be useful in studying the interaction of SMAD5 with other genes.

  3. Clinical significance of fluorescence in situ hybridization for detection of hTERC gene amplification in cervical cancer and precancerous tissues cases

    Directory of Open Access Journals (Sweden)

    Shuang LIU


    Full Text Available Objective  To detect the human telomerase RNA gene (hTERC amplification in cervical lesions, and explore its clinical significance. Methods  The tissues of the cervical lesions were collected from 195 patients, including 33 of chronic cervicitis, 34 of CINⅠ, 37 of CIN Ⅱ-Ⅲ, 30 of cervical squamous cell carcinoma, and 61 of cervica1 adenocarcinoma, and abnormal hTERC was detected with amplification of fluorescence in situhybridization (FISH. The relationship between hTERC gene amplification and clinicopathological parameters was analyzed. Results  Among the 195 patients, the positive rate of hTERC gene amplification was 3.03% (1/33, 29.41% (10/34, 72.97% (27/37, 100% (30/30, 91.8% (56/61 in chronic cervicitis, CINⅠ, CIN Ⅱ-Ⅲ, cervical squamous cell carcinoma and cervica1 adenocarcinoma respectively, and the results showed that hTERC amplification rate was significantly higher in group CIN Ⅱ-Ⅲthan in group CINⅠ(P 0.05. Conclusion  Detection of gene amplification by FISH technology can be used as a means for accurate diagnosis and prediction of the histologically difficult-to-diagnose lesion and for risk assessment after treatment of cervical precancerous lesions.

  4. Whole-genome amplification: a useful approach to characterize new genes in unculturable protozoan parasites such as Bonamia exitiosa. (United States)

    Prado-Alvarez, Maria; Couraleau, Yann; Chollet, Bruno; Tourbiez, Delphine; Arzul, Isabelle


    Bonamia exitiosa is an intracellular parasite (Haplosporidia) that has been associated with mass mortalities in oyster populations in the Southern hemisphere. This parasite was recently detected in the Northern hemisphere including Europe. Some representatives of the Bonamia genus have not been well categorized yet due to the lack of genomic information. In the present work, we have applied Whole-Genome Amplification (WGA) technique in order to characterize the actin gene in the unculturable protozoan B. exitiosa. This is the first protein coding gene described in this species. Molecular analysis revealed that B. exitiosa actin is more similar to Bonamia ostreae actin gene-1. Actin phylogeny placed the Bonamia sp. infected oysters in the same clade where the herein described B. exitiosa actin resolved, offering novel information about the classification of the genus. Our results showed that WGA methodology is a promising and valuable technique to be applied to unculturable protozoans whose genomic material is limited.

  5. Amplification of a cytochrome P450 gene is associated with resistance to neonicotinoid insecticides in the aphid Myzus persicae.

    Directory of Open Access Journals (Sweden)

    Alin M Puinean


    Full Text Available The aphid Myzus persicae is a globally significant crop pest that has evolved high levels of resistance to almost all classes of insecticide. To date, the neonicotinoids, an economically important class of insecticides that target nicotinic acetylcholine receptors (nAChRs, have remained an effective control measure; however, recent reports of resistance in M. persicae represent a threat to the long-term efficacy of this chemical class. In this study, the mechanisms underlying resistance to the neonicotinoid insecticides were investigated using biological, biochemical, and genomic approaches. Bioassays on a resistant M. persicae clone (5191A suggested that P450-mediated detoxification plays a primary role in resistance, although additional mechanism(s may also contribute. Microarray analysis, using an array populated with probes corresponding to all known detoxification genes in M. persicae, revealed constitutive over-expression (22-fold of a single P450 gene (CYP6CY3; and quantitative PCR showed that the over-expression is due, at least in part, to gene amplification. This is the first report of a P450 gene amplification event associated with insecticide resistance in an agriculturally important insect pest. The microarray analysis also showed over-expression of several gene sequences that encode cuticular proteins (2-16-fold, and artificial feeding assays and in vivo penetration assays using radiolabeled insecticide provided direct evidence of a role for reduced cuticular penetration in neonicotinoid resistance. Conversely, receptor radioligand binding studies and nucleotide sequencing of nAChR subunit genes suggest that target-site changes are unlikely to contribute to resistance to neonicotinoid insecticides in M. persicae.

  6. Quantification and assessment of viability of Cryptococcus neoformans by LightCycler amplification of capsule gene mRNA. (United States)

    Amjad, Muhammad; Kfoury, Najla; Cha, Raymond; Mobarak, Reem


    Cryptococcus neoformans is an opportunistic fungal pathogen. It infects the central nervous system causing meningitis, which is fatal if untreated, especially in AIDS and immunosuppressed patients. In this study a method of quantification and assessment of viability of C. neoformans by LightCycler RT-PCR amplification of the capsule gene mRNA is established. The sequence of primers and probes were derived from C. neoformans capsular CAP10 gene mRNA (GenBank accession number AF144574), and were species specific. Agarose gel electrophoresis analysis of LightCycler RT-PCR product showed a single band of 223 bp in length. In order to develop an internal control a 223 bp exon fragment of capsule mRNA was cloned in the pCR2.1 plasmid vector and RNA was generated by in vitro transcription. To determine the sensitivity of the assay, serial dilutions of in vitro-transcribed RNA with known concentrations and copy numbers, and serially diluted cultures of viable and nonviable C. neoformans were used. Under optimal conditions as little as 0.472 fg of capsule mRNA could be detected, corresponding to 1-10 c.f.u. ml(-1) of the sample. No amplification was observed from up to 10(5) heat/UV radiation-killed yeast cells and RNA of other bacterial and fungal pathogens and human genomic DNA or RNA. The amplification of capsule mRNA represents a sensitive, specific and quantitative means of detection of viable C. neoformans in clinical specimens and can be useful in the evaluation of the therapeutic efficacy of antifungal drugs in the treatment of C. neoformans meningitis.

  7. Altered regulation of DNA ligase IV activity by aberrant promoter DNA methylation and gene amplification in colorectal cancer. (United States)

    Kuhmann, Christine; Li, Carmen; Kloor, Matthias; Salou, Mariam; Weigel, Christoph; Schmidt, Christopher R; Ng, Linda W C; Tsui, Wendy W Y; Leung, Suet Y; Yuen, Siu T; Becker, Natalia; Weichenhan, Dieter; Plass, Christoph; Schmezer, Peter; Chan, Tsun L; Popanda, Odilia


    Colorectal cancer (CRC) presents as a very heterogeneous disease which cannot sufficiently be characterized with the currently known genetic and epigenetic markers. To identify new markers for CRC we scrutinized the methylation status of 231 DNA repair-related genes by methyl-CpG immunoprecipitation followed by global methylation profiling on a CpG island microarray, as altered expression of these genes could drive genomic and chromosomal instability observed in these tumors. We show for the first time hypermethylation of MMP9, DNMT3A and LIG4 in CRC which was confirmed in two CRC patient groups with different ethnicity. DNA ligase IV (LIG4) showed strong differential promoter methylation (up to 60%) which coincided with downregulation of mRNA in 51% of cases. This functional association of LIG4 methylation and gene expression was supported by LIG4 re-expression in 5-aza-2'-deoxycytidine-treated colon cancer cell lines, and reduced ligase IV amounts and end-joining activity in extracts of tumors with hypermethylation. Methylation of LIG4 was not associated with other genetic and epigenetic markers of CRC in our study. As LIG4 is located on chromosome 13 which is frequently amplified in CRC, two loci were tested for gene amplification in a subset of 47 cases. Comparison of amplification, methylation and expression data revealed that, in 30% of samples, the LIG4 gene was amplified and methylated, but expression was not changed. In conclusion, hypermethylation of the LIG4 promoter is a new mechanism to control ligase IV expression. It may represent a new epigenetic marker for CRC independent of known markers.

  8. Amplification and expression of a salivary gland DNA puff gene in the prothoracic gland of Bradysia hygida (Diptera: Sciaridae). (United States)

    Candido-Silva, Juliana Aparecida; Machado, Maiaro Cabral Rosa; Hartfelder, Klaus Hartmann; de Almeida, Jorge Cury; Paçó-Larson, Maria Luisa; Monesi, Nadia


    The DNA puff BhC4-1 gene, located in DNA puff C4 of Bradysiahygida, is amplified and expressed in the salivary gland at the end of the fourth larval instar as a late response to the increase in 20-hydroxyecdysone titer that triggers metamorphosis. Functional studies revealed that the mechanisms that regulate BhC4-1 expression in the salivary gland are conserved in transgenic Drosophila. These studies also led to the identification of a cis-regulatory module that drives developmentally regulated expression of BhC4-1-lacZ in the prothoracic gland cells of the ring gland, a compound organ which in Drosophila results from the fusion of the prothoracic glands, the corpus allatum and the corpus cardiacum. Here we have investigated the occurrence of BhC4-1 expression in B. hygida prothoracic glands. We report the identification of the B. hygida prothoracic gland and demonstrate that it releases ecdysone. Using RT-qPCR, western blots and immunolocalization experiments, we demonstrate that the BhC4-1 mRNA and the BhC4-1 protein are both expressed in the B. hygida prothoracic glands at the same time that DNA puff C4 is formed in the salivary gland. We also show that BhC4-1 is concomitantly amplified 4.8-fold in the prothoracic gland and 23-fold in the salivary gland. Our results reveal the occurrence of stage specific expression of a DNA puff gene in the prothoracic glands of B. hygida, and extend previous studies that have shown that DNA puff genes expression is not restricted to the salivary gland. In addition, the description of stage specific gene amplification in the prothoracic glands of B. hygida constitutes the first demonstration that gene amplification in Diptera might occur concomitantly in two different tissues in the same developmental stage.

  9. Rapid and specific detection of the thermostable direct hemolysin gene in Vibrio parahaemolyticus by loop-mediated isothermal amplification. (United States)

    Nemoto, Jiro; Sugawara, Chiyo; Akahane, Kenji; Hashimoto, Keiji; Kojima, Tadashi; Ikedo, Masanari; Konuma, Hirotaka; Hara-Kudo, Yukiko


    Several investigators have reported that thermostable direct hemolysin (TDH) and TDH-related hemolysin are important virulence factors of Vibrio parahaemolyticus, but it has been difficult to detect these factors rapidly in seafood and other environmental samples. A novel nucleic acid amplification method, termed the loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity and rapidity under isothermal conditions, was applied. In this study, we designed tdh gene-specific LAMP primers for detection of TDH-producing V. parahaemolyticus. The specificity of this assay was evaluated with 32 strains of TDH-producing V. parahaemolyticus, one strain of TDH-producing Grimontia hollisae, 10 strains of TDH-nonproducing V. parahaemolyticus, and 94 strains of TDH-nonproducing bacteria, and the sensitivity was high enough to detect one cell per test. Moreover, to investigate the detection of TDH-producing V. parahaemolyticus in oysters, the LAMP assay was performed with enrichment culture in alkaline peptone water of oyster samples inoculated with TDH-producing V. parahaemolyticus and TDH-nonproducing V. parahaemolyticus and V. alginolyticus after enrichment in alkaline peptone water. These results suggest that the LAMP assay targeting tdh gene has high sensitivity and specificity and is useful to detect TDH-producing V. parahaemolyticus in oyster after enrichment.

  10. Rapid and sensitive detection of Listeria ivanovii by loop-mediated isothermal amplification of the smcL gene.

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    Yi Wang

    Full Text Available A loop-mediated isothermal amplification (LAMP assay for rapid and sensitive detection of the L. ivanovii strains had been developed and evaluated in this study. Oligonucleotide primers specific for L. ivanovii species were designed corresponding to smcL gene sequences. The primers set comprise six primers targeting eight regions on the species-specific gene smcL. The LAMP assay could be completed within 1 h at 64°C in a water bath. Amplification products were directly observed by the Loopamp Fluorescent Detection Reagent (FD or detected by agarose gel electrophoresis. Moreover, the LAMP reactions were also detected by real-time measurement of turbidity. The exclusivity of 77 non-L. ivanovii and the inclusivity of 17 L. ivanovii were both 100% in the assay. Sensitivity of the LAMP assay was 250 fg DNA and 16 CFU per reaction for detection of L. ivanovii in pure cultures and simulated human stool. The LAMP assay was 10 and 100-fold more sensitive than quantitative PCR (qPCR and conventional PCR assays,respectively. When applied to human stool samples spiked with low level (8 CFU/0.5 g of L. ivanovii strains, the new LAMP assay described here achieved positive detection after 6 hours enrichment. In conclusion, the new LAMP assay in this study can be used as a valuable, rapid and sensitive detection tool for the detection of L. ivanovii in field, medical and veterinary laboratories.

  11. Loop-mediated amplification of the Clavibacter michiganensis subsp. michiganensis micA gene is highly specific. (United States)

    Yasuhara-Bell, Jarred; Kubota, Ryo; Jenkins, Daniel M; Alvarez, Anne M


    Loop-mediated amplification (LAMP) was used to specifically identify Clavibacter michiganensis subsp. michiganensis, causal agent of bacterial canker of tomato. LAMP primers were developed to detect micA, a chromosomally stable gene that encodes a type II lantibiotic, michiganin A, which inhibits growth of other C. michiganensis subspecies. In all, 409 bacterial strains (351 C. michiganensis subsp. michiganensis and 58 non-C. michiganensis subsp. michiganensis) from a worldwide collection were tested with LAMP to determine its specificity. LAMP results were compared with genetic profiles established using polymerase chain reaction (PCR) amplification of seven genes (dnaA, ppaJ, pat-1, chpC, tomA, ppaA, and ppaC). C. michiganensis subsp. michiganensis strains produced eight distinct profiles. The LAMP reaction identified all C. michiganensis subsp. michiganensis strains and discriminated them from other C. michiganensis subspecies and non-Clavibacter bacteria. LAMP has advantages over immunodiagnostic and other molecular detection methods because of its specificity and isothermal nature, which allows for easy field application. The LAMP reaction is also not affected by as many inhibitors as PCR. This diagnostic tool has potential to provide an easy, one-step test for rapid identification of C. michiganensis subsp. michiganensis.

  12. Development of a rapid and specific loop-mediated isothermal amplification detection method that targets Marek's disease virus meq gene. (United States)

    Wei, Xiuying; Shi, Xingming; Zhao, Yan; Zhang, Jing; Wang, Mei; Liu, Changjun; Cui, Hongyu; Hu, Shunlei; Quan, Yanming; Chen, Hongyan; Wang, Yunfeng


    A rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) method was developed and evaluated for the detection of Marek's disease virus (MDV) by amplification of conserved MDV meq gene sequences. LAMP is an innovative technique that allows the rapid detection of targeted nucleic acid sequences under isothermal conditions without the need for complex instrumentation. In this study, meq gene sequences were amplified successfully from different MDV strains by LAMP within 60min and no cross-reactivity was observed in a panel of related viruses that were associated with diseases of chickens. The detection limit of LAMP was 3.2 copies/million cells compared with 320 copies/million cells required for conventional PCR. Positive detection rates were assessed using either LAMP or PCR by examination of feather follicles that were collected from chickens infected experimentally with either strain J-1 (n=20) or strain Md5 (n=17), In addition to these samples, three isolates that were suspected to have been infected in the clinic were also tested. Results showed that the positive detection rate for LAMP was 95% (38/40), compared with 87.5% (35/40) and 90% (38/40) for strains J-1 and Md5 by PCR, respectively. These results indicated that the LAMP assay was more sensitive, rapid and specific than conventional PCR for the detection of MDV. This easy-to-perform technique will be useful for the detection of MDV and will aid in the establishment of disease control protocols.

  13. A titratable two-step transcriptional amplification strategy for targeted gene therapy based on ligand-induced intramolecular folding of a mutant human estrogen receptor

    DEFF Research Database (Denmark)

    Chen, Ian Y; Paulmurugan, Ramasamy; Nielsen, Carsten Haagen


    PURPOSE: The efficacy and safety of cardiac gene therapy depend critically on the level and the distribution of therapeutic gene expression following vector administration. We aimed to develop a titratable two-step transcriptional amplification (tTSTA) vector strategy, which allows modulation of ...


    Directory of Open Access Journals (Sweden)

    Stalis Norma Ethica


    Full Text Available Primaclade and In Silico web-based tools were used as a strategy to obtain the correct-size PCR amplicon targeting a fragment of gene encoding glycerol-3-phosphate dehydrogenase (glpD of Azospirillum sp. JG3. The bacterial strains are soil, Gram-negative PGPR (Plant-Growth Promoting Rhizobacteria isolated from an agricultural land in Purwokerto, Central Java, Indonesia, which have ability to produce several commercial enzymes. The aim is to obtain a pair of reliable degenerate primers from a limited number of glpD sequences from other Azospirilla retrieved in GenBank using bioinformatics approach. We demonstrated degenerate primer design that led to successful PCR amplification corresponding to the targeted DNA fragment. Homology analysis showed that the obtained DNA fragment is 61% and 99% similar to sn-glycerol-3-phosphate dehydrogenase genes of Azospirillum brasilense and Stenotrophomonas maltophili respectively.

  15. Adaptive gene amplification as an intermediate step in the expansion of virus host range.

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    Greg Brennan


    Full Text Available The majority of recently emerging infectious diseases in humans is due to cross-species pathogen transmissions from animals. To establish a productive infection in new host species, viruses must overcome barriers to replication mediated by diverse and rapidly evolving host restriction factors such as protein kinase R (PKR. Many viral antagonists of these restriction factors are species specific. For example, the rhesus cytomegalovirus PKR antagonist, RhTRS1, inhibits PKR in some African green monkey (AGM cells, but does not inhibit human or rhesus macaque PKR. To model the evolutionary changes necessary for cross-species transmission, we generated a recombinant vaccinia virus that expresses RhTRS1 in a strain that lacks PKR inhibitors E3L and K3L (VVΔEΔK+RhTRS1. Serially passaging VVΔEΔK+RhTRS1 in minimally-permissive AGM cells increased viral replication 10- to 100-fold. Notably, adaptation in these AGM cells also improved virus replication 1000- to 10,000-fold in human and rhesus cells. Genetic analyses including deep sequencing revealed amplification of the rhtrs1 locus in the adapted viruses. Supplying additional rhtrs1 in trans confirmed that amplification alone was sufficient to improve VVΔEΔK+RhTRS1 replication. Viruses with amplified rhtrs1 completely blocked AGM PKR, but only partially blocked human PKR, consistent with the replication properties of these viruses in AGM and human cells. Finally, in contrast to AGM-adapted viruses, which could be serially propagated in human cells, VVΔEΔK+RhTRS1 yielded no progeny virus after only three passages in human cells. Thus, rhtrs1 amplification in a minimally permissive intermediate host was a necessary step, enabling expansion of the virus range to previously nonpermissive hosts. These data support the hypothesis that amplification of a weak viral antagonist may be a general evolutionary mechanism to permit replication in otherwise resistant host species, providing a molecular foothold

  16. From amplification to gene in thyroid cancer: A high-resolution mapped bacterial-artificial-chromosome resource for cancer chromosome aberrations guides gene discovery after comparative genome hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Chen, X.N.; Gonsky, R.; Korenberg, J.R. [UCLA School of Medicine, Los Angeles, CA (United States). Cedars-Sinai Research Inst.; Knauf, J.A.; Fagin, J.A. [Univ. of Cincinnati, OH (United States). Div. of Endocrinology/Metabolism; Wang, M.; Lai, E.H. [Univ. of North Carolina, Chapel Hill, NC (United States). Dept. of Pharmacology; Chissoe, S. [Washington Univ. School of Medicine, St. Louis, MO (United States). Genome Sequencing


    Chromosome rearrangements associated with neoplasms provide a rich resource for definition of the pathways of tumorigenesis. The power of comparative genome hybridization (CGH) to identify novel genes depends on the existence of suitable markers, which are lacking throughout most of the genome. The authors now report a general approach that translates CGH data into higher-resolution genomic-clone data that are then used to define the genes located in aneuploid regions. They used CGH to study 33 thyroid-tumor DNAs and two tumor-cell-line DNAs. The results revealed amplifications of chromosome band 2p21, with less-intense amplification on 2p13, 19q13.1, and 1p36 and with least-intense amplification on 1p34, 1q42, 5q31, 5q33-34, 9q32-34, and 14q32. To define the 2p21 region amplified, a dense array of 373 FISH-mapped chromosome 2 bacterial artificial chromosomes (BACs) was constructed, and 87 of these were hybridized to a tumor-cell line. Four BACs carried genomic DNA that was amplified in these cells. The maximum amplified region was narrowed to 3--6 Mb by multicolor FISH with the flanking BACs, and the minimum amplicon size was defined by a contig of 420 kb. Sequence analysis of the amplified BAC 1D9 revealed a fragment of the gene, encoding protein kinase C epsilon (PKC{epsilon}), that was then shown to be amplified and rearranged in tumor cells. In summary, CGH combined with a dense mapped resource of BACs and large-scale sequencing has led directly to the definition of PKC{epsilon} as a previously unmapped candidate gene involved in thyroid tumorigenesis.

  17. Amplification of the cap20 pathogenicity gene and genetic characterization using different markers molecular in Colletotrichum gloeosporioides isolates

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    Danielli Barreto Maciel


    Full Text Available Studies were performed to analyze the genetic characterization using RFLP-ITS and Intron (primer EI1 markers and the amplification of the cap20 pathogenicity gene by PCR in Colletotrichum gloeosporioides isolates of different hosts plant. The genetic variability was accessed using RFLP-ITS and Intron markers and grouping by UPGMA method. Primers to cap20 gene were constructed using selected sequences of the GenBank (National Center of Biotechnology Information, with the Primer 3 program. The dendrograms analysis showed that the RFLP-ITS marker was more informative to separate the Colletotrichum sp, and that primer EI1 demonstrated greater genetic diversity. The amplification of the DNA of the Colletotrichum isolates to the cap20 gene with primers P1 and P2 indicated that this gene could present variations into C. gloeosporioides related with the host, and also that it was present in other Colletotrichum sp.Estudos foram realizados para analisar a caracterização genética usando marcadores de RFLP-ITS e ISSP e a amplicação do gene de patogenicidade cap20 por PCR em isolados de Colletotrichum gloeosporioides de diferentes hospedeiros. Primers para o gene cap20 foram construídos a partir de seqüências selecionadas do GenBank (National Center of Biotechnology Information, com o programa Primer 3. A análise dos dendrogramas revelou que o marcador RFLP-ITS foi mais informativo em separar as espécies de Colletotrichum, e que o primer EI1 evidenciou maior diversidade genética. A amplificação do DNA dos isolados de Colletotrichum para o gene cap20 com os primers P1 e P2 indicou que este gene pode apresentar variações dentro de C. gloeosporioides relacionada ao hospedeiro, e que também está presente em outras espécies de Colletotrichum.

  18. Improved gene amplification by cell-cycle engineering combined with the Cre-loxP system in Chinese hamster ovary cells. (United States)

    Matsuyama, Rima; Tsutsui, Tomomi; Lee, Kyoung Ho; Onitsuka, Masayoshi; Omasa, Takeshi


    The dihydrofolate reductase gene amplification system is widely used in Chinese hamster ovary (CHO) cells for the industrial production of therapeutic proteins. To enhance the efficiency of conventional gene amplification systems, we previously presented a novel method using cell-cycle checkpoint engineering. Here, we constructed high-producing and stable cells by the conditional expression of mutant cell division cycle 25 homolog B (CDC25B) using the Cre-loxP system. A bispecific antibody-producing CHO DG44-derived cell line was transfected with floxed mutant CDC25B. After inducing gene amplification in the presence of 250 nM methotrexate, mutant CDC25B sequence was removed by Cre recombinase protein expression. Overexpression of the floxed mutant CDC25B significantly enhanced the efficiency of transgene amplification and productivity. Moreover, the specific production rate of the isolated clone CHO Cre-1 and Cre-2 were approximately 11-fold and 15-fold higher than that of mock-transfected clone CHO Mock-S. Chromosomal aneuploidy was increased by mutant CDC25B overexpression, but Cre-1 and Cre-2 did not show any changes in chromosome number during long-term cultivation, as is the case with CHO Mock-S. Our results suggest that high-producing and stable cells can be constructed by conditionally controlling a cell-cycle checkpoint integrated in conventional gene amplification systems.

  19. HER2/neu (c-erbB-2) gene amplification and protein expression are rare in uterine cervical neoplasia: a tissue microarray study of 814 archival specimens

    DEFF Research Database (Denmark)

    Lesnikova, Iana; Lidang, Marianne; Hamilton-Dutoit, Stephen;


    Published studies have reported widely variable incidence of HER2/neu (c-erbB-2) protein expression and HER2/neu (c-erbB-2) gene amplification in cervical carcinoma. We examined tissue microarrays (TMAs) constructed from 814 formaldehyde-fixed paraffin-embedded archival specimens of cervical...... and invasive cervical carcinoma specimens. When present, Her-2/neu positivity is more commonly seen in higher grades of cervical dysplasia and in carcinoma. However, this large TMA study shows that HER2/neu oncoprotein expression and HER2/neu gene amplification overall are uncommon events in cervical neoplasia...... intraepithelial neoplasia (CIN)1 (n = 262), CIN2 (n = 230), CIN3 (n = 186) and invasive carcinoma (n = 136), for HER2/neu protein expression by immunohistochemistry (IHC) and for HER2/neu gene amplification by chromogenic in situ hybridization (CISH). We found moderate or strong immunohistochemical positivity...

  20. Gene expression profiling of RNA extracted from FFPE tissues: NuGEN technologies' whole-transcriptome amplification system. (United States)

    Turner, Leah; Heath, Joe Don; Kurn, Nurith


    Gene expression profiling of RNA isolated from formalin fixed, paraffin-embedded (FFPE) tissue samples has been historically challenging. Yet FFPE samples are sought-after because of the in-depth retrospective records typically associated with them rendering these samples a valuable resource for translational medicine studies. Extensive degradation, chemical modifications, and cross-linking have made it difficult to isolate RNA of sufficient quality required for large-scale gene expression profiling studies. NuGEN Technologies' WT-Ovation™ FFPE System linearly amplifies RNA from FFPE samples through a robust and simple whole-transcriptome approach using as little as 50 ng total RNA isolated from FFPE samples. The amplified material may be labeled with validated kits and/or protocols from NuGEN for analysis on any of the major gene expression microarray platforms, including: Affymetrix, Agilent, and Illumina gene expression arrays. Results compare well with those obtained using RNA from fresh-frozen samples. RNA quality from FFPE samples varies significantly and neither sample age nor sample size analysis via gel electrophoresis or the Agilent Bioanalyzer system accurately predict materials suitable for amplification. Therefore, NuGEN has validated a correlative qPCR-based analytical method for the RNA derived from FFPE samples which effectively predicts array results. The NuGEN approach enables fast and successful analysis of samples previously thought to be too degraded for gene expression analysis.

  1. Combinatorial codon scrambling enables scalable gene synthesis and amplification of repetitive proteins (United States)

    Tang, Nicholas C.; Chilkoti, Ashutosh


    Most genes are synthesized using seamless assembly methods that rely on the polymerase chain reaction (PCR). However, PCR of genes encoding repetitive proteins either fails or generates nonspecific products. Motivated by the need to efficiently generate new protein polymers through high-throughput gene synthesis, here we report a codon-scrambling algorithm that enables the PCR-based gene synthesis of repetitive proteins by exploiting the codon redundancy of amino acids and finding the least-repetitive synonymous gene sequence. We also show that the codon-scrambling problem is analogous to the well-known travelling salesman problem, and obtain an exact solution to it by using De Bruijn graphs and a modern mixed integer linear programme solver. As experimental proof of the utility of this approach, we use it to optimize the synthetic genes for 19 repetitive proteins, and show that the gene fragments are amenable to PCR-based gene assembly and recombinant expression.

  2. Combinatorial codon scrambling enables scalable gene synthesis and amplification of repetitive proteins. (United States)

    Tang, Nicholas C; Chilkoti, Ashutosh


    Most genes are synthesized using seamless assembly methods that rely on the polymerase chain reaction (PCR). However, PCR of genes encoding repetitive proteins either fails or generates nonspecific products. Motivated by the need to efficiently generate new protein polymers through high-throughput gene synthesis, here we report a codon-scrambling algorithm that enables the PCR-based gene synthesis of repetitive proteins by exploiting the codon redundancy of amino acids and finding the least-repetitive synonymous gene sequence. We also show that the codon-scrambling problem is analogous to the well-known travelling salesman problem, and obtain an exact solution to it by using De Bruijn graphs and a modern mixed integer linear programme solver. As experimental proof of the utility of this approach, we use it to optimize the synthetic genes for 19 repetitive proteins, and show that the gene fragments are amenable to PCR-based gene assembly and recombinant expression.

  3. Assignment of genes encoding metallothioneins I and II to Chinese hamster chromosomes 3. Evidence for the role of chromosome rearrangement in gene amplification

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    Stallings, R.L.; Munk, A.C.; Longmire, J.L.; Hildebrand, C.E.; Crawford, B.D.


    Cadmium resistant (Cd/sup r/) variants with coordinately amplified metallothionein I and II (MTI and MTII) genes have been derived from both Chinese hamster ovary and near-euploid Chinese hamster cell lines. Cytogenetic analyses of Cd/sup r/ variants consistently revealed breakage and rearrangement involving chromosome 3p. In situ hybridization with Chinese hamster MT-encoding cDNA probe localized amplified MT gene sequences near the translocation breakpoint involving chromosome 3p. These observations suggested that both functionally related, isometallothionein loci are linked on Chinese hamster chromosome 3. Southern blot analyses of DNAs isolated from a panel of Chinese hamster x mouse somatic cell hybrids which segregate hamster chromosomes confirmed that both MTI and MTII are located on chromosome 3. The authors speculate that rearrangement of chromosome 3p could be causally involved with the amplification of MT genes in Cd/sup r/ hamster cell lines. 34 references, 3 figures, 1 table.

  4. Rapid screening of innate immune gene expression in zebrafish using reverse transcription - multiplex ligation-dependent probe amplification

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    Spaink Herman P


    Full Text Available Abstract Background With the zebrafish increasingly being used in immunology and infectious disease research, there is a need for efficient molecular tools to evaluate immune gene expression in this model species. RT-MLPA (reverse transcription - multiplex ligation-dependent probe amplification provides a sensitive and reproducible method, in which fluorescently labelled amplification products of unique lengths are produced for a defined set of target transcripts. The method employs oligonucleotide probes that anneal to adjacent sites on a target sequence and are then joined by a heat-stable ligase. Subsequently, multiplex PCR with universal primers gives rise to amplicons that can be analyzed with standard sequencing equipment and relative quantification software. Allowing the simultaneous quantification of around 40 selected markers in a one-tube assay, RT-MLPA is highly useful for high-throughput screening applications. Findings We employed a dual-colour RT-MLPA probe design for chemical synthesis of probe pairs for 34 genes involved in Toll-like receptor signalling, transcriptional activation of the immune response, cytokine and chemokine production, and antimicrobial defence. In addition, six probe pairs were included for reference genes unaffected by infections in zebrafish. First, we established assay conditions for adult zebrafish infected with different strains of Mycobacterium marinum causing acute and chronic disease. Addition of competitor oligonucleotides was required to achieve peak heights in a similar range for genes with different expression levels. For subsequent analysis of embryonic samples it was necessary to adjust the amounts of competitor oligonucleotides, as the expression levels of several genes differed to a large extent between adult and embryonic tissues. Assay conditions established for one-day-old Salmonella typhimurium-infected embryos could be transferred without further adjustment to five-day-old M. marinum

  5. Loop-mediated isothermal amplification assay for 16S rRNA methylase genes in Gram-negative bacteria. (United States)

    Nagasawa, Mitsuaki; Kaku, Mitsuo; Kamachi, Kazunari; Shibayama, Keigo; Arakawa, Yoshichika; Yamaguchi, Keizo; Ishii, Yoshikazu


    Using the loop-mediated isothermal amplification (LAMP) method, we developed a rapid assay for detection of 16S rRNA methylase genes (rmtA, rmtB, and armA), and investigated 16S rRNA methylase-producing strains among clinical isolates. Primer Explorer V3 software was used to design the LAMP primers. LAMP primers were prepared for each gene, including two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (LF and LB). Detection was performed with the Loopamp DNA amplification kit. For all three genes (rmtA, rmtB, and armA), 10(2) copies/tube could be detected with a reaction time of 60 min. When nine bacterial species (65 strains saved in National Institute of Infectious Diseases) were tested, which had been confirmed to possess rmtA, rmtB, or armA by PCR and DNA sequencing, the genes were detected correctly in these bacteria with no false negative or false positive results. Among 8447 clinical isolates isolated at 36 medical institutions, the LAMP method was conducted for 191 strains that were resistant to aminoglycosides based on the results of antimicrobial susceptibility tests. Eight strains were found to produce 16S rRNA methylase (0.09%), with rmtB being identified in three strains (0.06%) of 4929 isolates of Enterobacteriaceae, rmtA in three strains (0.10%) of 3284 isolates of Pseudomonas aeruginosa, and armA in two strains (0.85%) of 234 isolates of Acinetobacter spp. At present, the incidence of strains possessing 16S rRNA methylase genes is very low in Japan. However, when Gram-negative bacteria showing high resistance to aminoglycosides are isolated by clinical laboratories, it seems very important to investigate the status of 16S rRNA methylase gene-harboring bacilli and monitor their trends among Japanese clinical settings.

  6. Molecular detection of field isolates of Turkey Eimeria by polymerase chain reaction amplification of the cytochrome c oxidase I gene. (United States)

    Rathinam, T; Gadde, U; Chapman, H D


    Oocysts of Eimeria spp. were isolated from litter samples obtained from 30 commercial turkey farms. Genomic DNA was extracted from clean oocysts, and polymerase chain amplification of the species-specific cytochrome c oxidase subunit I (COI) gene was performed for five species of turkey Eimeria. The species tested were Eimeria adenoeides, Eimeria meleagrimitis, Eimeria meleagridis, Eimeria dispersa, and Eimeria gallopavonis. All DNA samples were positive for E. meleagrimitis, nine were positive for E. adenoeides, two were positive for E. dispersa, and none for E. meleagridis and E. gallopavonis. E. meleagrimitis occurred as a single species in 21 (70 %) of the farms while 9 (30 %) farms had a mixed species with E. meleagrimitis and E. adenoeides and 2 (7 %) were triple positive with E. meleagrimitis, E. adenoeides, and E. dispersa. This is the first account of the field prevalence of turkey Eimeria species using molecular methods.

  7. Loop-Mediated Isothermal Amplification Assay Targeting the MOMP Gene for Rapid Detection of Chlamydia psittaci Abortus Strain

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    Guo-Zhen Lin, Fu-Ying Zheng, Ji-Zhang Zhou, Guang-Hua Wang, Xiao-An Cao, Xiao-Wei Gong and Chang-Qing Qiu*


    Full Text Available For rapid detection of the Chlamydia psittaci abortus strain, a loop-mediated isothermal amplification (LAMP assay was developed and evaluated in this study. The primers for the LAMP assay were designed on the basis of the main outer membrane protein (MOMP gene sequence of C. psittaci. Analysis showed that the assay could detect the abortus strain of C. psittaci with adequate specificity. The sensitivity of the test was the same as that of the nested-conventional PCR and higher than that of chick embryo isolation. Testing of 153 samples indicated that the LAMP assay could detect the genome of the C. psittaci abortus strain effectively in clinical samples. This assay is a useful tool for rapid diagnosis of C. psittaci infection in sheep, swine and cattle.

  8. Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella. (United States)

    Rahn, K; De Grandis, S A; Clarke, R C; McEwen, S A; Galán, J E; Ginocchio, C; Curtiss, R; Gyles, C L


    Amplification of nucleotide sequences within the invA gene of Salmonella typhimurium was evaluated as a means of detecting Salmonella. A collection of 630 strains of Salmonella comprising over 100 serovars, including the 20 most prevalent serovars isolated from animals and humans in Canada, was examined. Controls consisted of 142 non-Salmonella strains comprising 21 genera of bacteria. Cultures were screened by inoculating a single colony of bacteria directly into a polymerase chain reaction (PCR) mixture which contained a pair of primers specific for the invA gene. The specific PCR product was a 284 bp DNA fragment which was visualized in 2% agarose gels. With the exception of two S. litchfield and two S. senftenberg strains, all Salmonella strains were detected. In contrast, none of the non-Salmonella strains yielded the specific amplification product. Non-specific amplification of a few non-Salmonella strains resulted in a product that was distinctly different in size from the specific 284 bp product. Specificity of amplification was further confirmed by demonstration of hybridization of a 32P-labelled invA gene fragment only to the specific 284 bp product. The detection of 99.4% of Salmonella strains tested and the failure to specifically amplify DNA from non-Salmonella strains confirm that the invA gene contains sequences unique to Salmonella and demonstrate that this gene is a suitable PCR target, with potential diagnostic applications.

  9. Characterisation of amplification patterns and target genes at chromosome 11q13 in CCND1-amplified sporadic and familial breast tumours. (United States)

    Holm, Karolina; Staaf, Johan; Jönsson, Göran; Vallon-Christersson, Johan; Gunnarsson, Haukur; Arason, Adalgeir; Magnusson, Linda; Barkardottir, Rosa B; Hegardt, Cecilia; Ringnér, Markus; Borg, Ake


    Amplification of chromosomal region 11q13, containing the cell cycle regulatory gene CCND1, is frequently found in breast cancer and other malignancies. It is associated with the favourable oestrogen receptor (ER)-positive breast tumour phenotype, but also with poor prognosis and treatment failure. 11q13 spans almost 14 Mb and contains more than 200 genes and is affected by various patterns of copy number gains, suggesting complex mechanisms and selective pressure during tumour progression. In this study, we used 32 k tiling BAC array CGH to analyse 94 CCND1-amplified breast tumours from sporadic, hereditary, and familial breast cancers to fine map chromosome 11q13. A set containing 281 CCND1-non-amplified breast tumours was used for comparisons. We used gene expression data to further validate the functional effect of gene amplification. We identified six core regions covering 11q13.1-q14.1 that were amplified in different combinations. The major core contained CCND1, whereas two cores were found proximal of CCND1 and three distal. The majority of the CCND1-amplified tumours were ER-positive and classified as luminal B. Furthermore, we found that CCND1 amplification is associated with a more aggressive phenotype within histological grade 2 tumours and luminal A subtype tumours. Amplification was equally prevalent in familial and sporadic tumours, but strikingly rare in BRCA1- and BRCA2-mutated tumours. We conclude that 11q13 includes many potential target genes in addition to CCND1.

  10. Relationship of Amplification and Expression of the C-MYC Gene with Survival among Gastric Cancer Patients. (United States)

    Khaleghian, Malihea; Shakoori, Abbas; Razavi, Amirnader Emami; Azimi, Cyrus


    During the past decades, the incidence and mortality rate of stomach cancer has demonstrated a great decrease in the world, but it is still one of the most common and fatal cancers especially among men worldwide, including Iran. The MYC proto-oncogene, which is located at 8q24.1, regulates 15% of genes and is activated in 20% of all human tumors. MYC amplification and overexpression of its protein product has been reported in 15-30% of gastric neoplasias. The aim of this investigation was to find the relative efficacy of CISH (chromogenic in situ hybridization) or IHC (immunohistochemistry) in diagnosis and prognosis of gastric cancer, as well as the relationship of amplification and expression of C-MYC gene with patient survival. In this cross-sectional study, 102 samples of gastric cancer were collected from patients who had undergone primary surgical resection at the Cancer Institute Hospital, Tehran University of Medical Sciences, from July 2009 to March 2014. All samples were randomly selected from those who were diagnosed with gastric adenocarcinomas. CISH and IHC methods were performed on all of them. Patients were classified into two groups. The first consisted of stage I and II cases, and the second of stage III and IV. Survival tests for both groups was carried out with referrnce to CISH test reults. Group II (stage III and IV) with CISH+ featured lower survival than those with CISH- (p=0.233), but group I (stage I and II) patients demonstrated no significant variation with CISH+ or CISH- (p=0.630). Kaplan-Meier for both groups was carried out with IHC test findings and showed similar results. This data revealed that both diffuse and intestinal types of gastric cancer occurred significantly more in men than women. Our data also showed that CISH+ patients (43%) were more frequent in comparison with IHC+ patients (14.7%). For planning treatment of gastric cancer patients, by focusing on expanding tumors, which is the greatest concern of the surgeons and

  11. A phase II study of Epirubicin in oxaliplatin-resistant patients with metastatic colorectal cancer and TOP2A gene amplification

    DEFF Research Database (Denmark)

    Tarpgaard, Line S.; Qvortrup, Camilla; Nygård, Sune Boris


    UNLABELLED: ᅟ: The overall purpose of this study is to provide proof of concept for introducing the anthracycline epirubicin as an effective, biomarker-guided treatment for metastatic colorectal cancer (mCRC) patients who are refractory to treatment with oxaliplatin-based chemotherapy and have TOP2......A gene amplification in their tumor cells. BACKGROUND: Epirubicin is an anthracycline that targets DNA topoisomerase 2-α enzyme encoded by the TOP2A gene. It is used for treatment of several malignancies, but currently not in CRC. TOP2A gene amplifications predict improved efficacy of epirubicin...... in patients with breast cancer and thus could be an alternative option for patients with CRC and amplified TOP2A gene. We have previously analysed the frequency of TOP2A gene aberrations in CRC and found that 46.6 % of these tumors had TOP2A copy gain and 2.0 % had loss of TOP2A when compared to adjacent...

  12. Determination of variants in the 3'-region of the Tyrosinase gene requires locus specific amplification.

    NARCIS (Netherlands)

    Chaki, M.; Mukhopadhyay, A.; Ray, K.


    Mutations in the Tyrosinase gene (TYR, 11q14-q21) cause oculocutaneous albinism type 1 (OCA1). The 3'-region of the TYR shows 98.55% sequence identity with a pseudogene, known as Tyrosinase-Like Gene (TYRL, 11p11.2-cen). A large number of publicly available nucleotide variants of TYR in this region

  13. A new rapid amplification of cDNA ends method for extremely guanine plus cytosine-rich genes. (United States)

    Shi, Xianzong; Jarvis, Donald L


    Rapid amplification of cDNA ends (RACE) is widely used to determine the 5'- and 3'-terminal nucleotide sequences of genes. Many different RACE methods have been developed to meet various requirements, but none addresses the difficult problems that arise when trying to isolate the ends of extremely guanine plus cytosine (GC)-rich genes. In this study, we found that we were unable to isolate the correct 5' or 3' end of an insect gene, which appeared to include extremely GC-rich sequences, using current RACE methods. Thus, we developed a new RACE method that can be used for this purpose. This new method entails first-strand cDNA synthesis at 70 degrees C with Thermo-X reverse transcriptase in the presence of homoectoine, followed by a polymerase chain reaction with 98 degrees C denaturation steps and Phusion DNA polymerase in the presence of 1M betaine and 5% dimethyl sulfoxide (DMSO). The use of these conditions yielded 5'- and 3'-RACE products that were approximately 80% GC over 213 and 162bp, respectively, and included shorter internal regions of 82 to 89% GC.

  14. Amplification, enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumours of glial origin. (United States)

    Libermann, T A; Nusbaum, H R; Razon, N; Kris, R; Lax, I; Soreq, H; Whittle, N; Waterfield, M D; Ullrich, A; Schlessinger, J

    Epidermal growth factor (EGF), through interaction with specific cell surface receptors, generates a pleiotropic response that, by a poorly defined mechanism, can induce proliferation of target cells. Subversion of the EGF mitogenic signal through expression of a truncated receptor may be involved in transformation by the avian erythroblastosis virus (AEV) oncogene v-erb-B, suggesting that similar EGF receptor defects may be found in human neoplasias. Overexpression of EGF receptors has been reported on the epidermoid carcinoma cell line A431, in various primary brain tumours and in squamous carcinomas. In A431 cells the receptor gene is amplified. Here we show that 4 of 10 primary brain tumours of glial origin which express levels of EGF receptors that are higher than normal also have amplified EGF receptor genes. Amplified receptor genes were not detected in the other brain tumours examined. Further analysis of EGF receptor defects may show that such altered expression and amplification is a particular feature of certain human tumours.

  15. Genomic amplification of the human telomerase gene (hTERC associated with human papillomavirus is related to the progression of uterine cervical dysplasia to invasive cancer

    Directory of Open Access Journals (Sweden)

    Liu Hongqian


    Full Text Available Abstract Background Human papillomavirus (HPV infection plays an etiological role in the development of cervical dysplasia and cancer. Amplification of human telomerase gene (hTERC and over expression of telomerase were found to be associated with cervical tumorigenesis. This study was performed to analyze genomic amplification of hTERC gene, telomerase activity in association with HPV infection in different stages of cervical intraepithelial neoplasia (CIN and cervical cancer. We were studying the role of hTERC in the progression of uterine cervical dysplasia to invasive cancer, and proposed an adjunct method for cervical cancer screening. Methods Exfoliated cervical cells were collected from 114 patients with non neoplastic lesion (NNL, n=27, cervical intraepithelial neoplasia (CIN1, n=26, CIN2, n=16, CIN3, n=24 and cervical carcinoma (CA, n=21, and analyzed for amplification of hTERC with two-color fluorescence in situ hybridization (FISH probe and HPV-DNA with Hybrid Capture 2. From these patients, 53 were taken biopsy to analyze telomerase activity by telomeric repeat amplification protocol (TRAP and expression of human telomerase reverse transcriptase (hTERT, with immunohistochemistry (IHC. All biopsies were clinically confirmed by phathologists. Results Amplification of hTERC was significantly associated with the histologic diagnoses (p Conclusions hTERC ampliffication can be detected with FISH technique on exfoliated cervical cells. Amplification of hTERC and HPV infection are associated with more progressive CIN3 and CA. The testing of hTERC amplification might be a supplementary to cytology screening and HPV test, especially high-risk patients. Virtual slides The virtual slide(s for this article can be found here:

  16. Detection of denitrification genes by in situ rolling circle amplification - fluorescence in situ hybridization (in situ RCA-FISH) to link metabolic potential with identity inside bacterial cells

    DEFF Research Database (Denmark)

    Hoshino, Tatsuhiko; Schramm, Andreas


    A target-primed in situ rolling circle amplification (in situ RCA) protocol was developed for detection of single-copy genes inside bacterial cells and optimized with Pseudomonas stutzeri, targeting nitrite and nitrous oxide reductase genes (nirS and nosZ). Two padlock probes were designed per gene......). Nevertheless, multiple genes (nirS and nosZ; nirS and the 16S rRNA gene) could be detected simultaneously in P. stutzeri. Environmental application of in situ RCA-FISH was demonstrated on activated sludge by the differential detection of two types of nirS-defined denitrifiers; one of them was identified...

  17. Amplification of the telomerase RNA component gene as a new genetic marker for disease progression and prognosis in esophageal squamous cell carcinoma. (United States)

    Wang, J-D; Ma, J; Wang, F-Y; Peng, L-B; Wang, X; Shi, S-S; Ma, H-H; Lu, Z-F; Lu, G-M; Zhou, X-J


    Amplification of the human telomerase RNA component (TERC) gene was found in esophageal squamous cell carcinoma (ESCC). However, its roles in the progression and prognosis of ESCC have not been well understood. The amplification of TERC in normal mucosa, low-grade and high-grade intraepithelial neoplasia, and invasive ESCC samples were evaluated using a fluorescence in situ hybridization assay. The amplification of TERC invariably occurred in high-grade intraepithelial neoplasia and invasive ESCC, partially occurred in low-grade intraepithelial neoplasia specimens, and seldom occurred in normal mucosa. The average signal ratio of TERC to chromosome 3 centromere-specific probe (TERC/CSP3) was 1.00 ± 0.01 (average ± standard deviation) in normal mucosas, 1.01 ± 0.08 in low-grade intraepithelial neoplasias, 1.39 ± 0.26 in high-grade intraepithelial neoplasias, and 1.56 ± 0.41 in invasive ESCC. High TERC/CSP3 ratio was positively associated with lymph node metastasis (P = 0.005) and advanced tumor stage (P = 0.045). Patients with high amplification of TERC had poor survival (P = 0.01). The amplification of TERC could be used as a new genomic marker for disease progression and prognosis of ESCC. The amplified TERC gene may be a potential therapeutic target for ESCC.

  18. Rapid detection of the Klebsiella pneumoniae carbapenemase (KPC) gene by loop-mediated isothermal amplification (LAMP). (United States)

    Nakano, Ryuichi; Nakano, Akiyo; Ishii, Yoshikazu; Ubagai, Tsuneyuki; Kikuchi-Ueda, Takane; Kikuchi, Hirotoshi; Tansho-Nagakawa, Shigeru; Kamoshida, Go; Mu, Xiaoqin; Ono, Yasuo


    Klebsiella pneumoniae carbapenemases (KPC), which are associated with resistance to carbapenem, have recently spread worldwide and have become a global concern. It is necessary to detect KPC-producing organisms in clinical settings to be able to control the spread of this resistance. We have developed a loop-mediated isothermal amplification (LAMP) method for rapid detection of KPC producers. LAMP primer sets were designed to recognize the homologous regions of blaKPC-2 to blaKPC-17 and could amplify blaKPC rapidly. The specificity and sensitivity of the primers in the LAMP reactions for blaKPC detection were determined. This LAMP assay was able to specifically detect KPC producers at 68 °C, and no cross-reactivity was observed for other types of β-lactamase (class A, B, C, or D) producers. The detection limit for this assay was found to be 10(0) CFU per tube, in 25 min, which was 10-fold more sensitive than a PCR assay for blaKPC detection. Then, the sensitivity of the LAMP reactions for blaKPC detection in human specimens (sputum samples, urine samples, fecal samples and blood samples) was analyzed; it was observed that the LAMP assay had almost the same sensitivity in these samples as when using purified DNA. The LAMP assay is easy to perform and rapid. It may therefore be routinely applied for detection of KPC producers in the clinical laboratory.

  19. CAT基因突变热点区域的PCR扩增方法%Optimization of PCR Amplification Parameters for Mutational Hotspots in the Human Catalase Gene

    Institute of Scientific and Technical Information of China (English)

    李毅; 赵华; 赵红宇; 章锦才


    Objective To study the speciality and sensitivity of PCR for mutational hotspots in the human catalase gene. Methods Blood samples were taken from volunteers for genomic DNA preparation. Polymerase chain reaction (PCR) amplification of sixteen gene segments encompassing mutational hotspots in the human catalase was carried out. Touchdown and Hot-start PCR was imple-mented to improve the efficiency of gene amplification. Results High and constant amplification effi-ciency is obtained using touchdown and hot-start PCR. Conclusion The stable and reproducible PCR amplification parameters for mutational hotspots in the human catalase gene were optimized.%目的 探讨过氧化氢酶基因突变热点区域PCR扩增方法 ,提高PCR反应的特异性和灵敏度,有助于快速检测CAT基因相关疾病.方法 从人静脉血液标本提取人血液基因组DNA,设计引物扩增特定的CAT基因片段,联合应用热启动PCR和降落PCR技术.结果 建立了重复性好,分辨率高的PCR反应体系.结论 建立了适用于CAT基因突变热点区域的PCR反应体系,有助于快速检测CAT基因相关痰病.

  20. Regulation of Pattern Formation and Gene Amplification During Drosophila Oogenesis by the miR-318 microRNA

    DEFF Research Database (Denmark)

    Ge, Wanzhong; Deng, Qiannan; Guo, Ting


    and laid eggs with abnormal morphology. Removal of miR-318 disrupted the dorsal-anterior follicle cell patterning, resulting in abnormal dorsal appendages. miR-318 mutant females also produced thin and fragile eggshells, due to impaired chorion gene amplification. We provide evidence that the ecdysone...

  1. Amplification of chromosome 2:Lq22.3 harboring trefoil factor family genes in liver fluke related cholangiocarcinoma is associated with poor prognosis

    Institute of Scientific and Technical Information of China (English)

    Kanuengnuch Muenphon; Temduang Limpaiboon; Patcharee Jearanaikoon; Chawalit Pairojkul; Banchob Sripa; Vajarabhongsa Bhudhisawasdi


    AIM: To determine allelic imbalance on chromosomal region 21q22-qter including trefoil factor family genes (TFF) in cholangiocarcinoma (CCA) patients and analyze the correlation between allelic imbalances and clinicopathological parameters.METHODS: Quantitative PCR amplification was performed on four microsatellite markers and trefoil factor family genes (TFF1, TFF2, and TFF3) using a standard curve and SYBR Green Ⅰ dye method. The relative copy number was determined by DNA copy number of tested locus to reference locus. The relative copy number was interpreted as deletion or amplification by comparison with normal reference range. Associations between allelic imbalance and clinicopathological parameters of CCA patients were evaluated by x2-tests.Kaplan-Meier method was used to analyze survival.RESULTS: The frequencies of amplification at D21S1890,D21S1893, and TFF3 were 32.5%, 30.0%, and 28.7%,respectively. Patients who had amplification at regions covering D21S1893, D21S1890, and TFF showed poor prognosis, whereas patients who had deletion showed favorable prognosis (mean: 51.7 wk vs 124.82 wk,P = 0.012). Multivariate Cox regression analysis revealed that amplification of D21S1893, D21S1890 and TFF,blood vessel invasion, and staging were associated with poor prognosis.CONCLUSION: D21S1893-D21S1890 region may harbor candidate genes especially TFF and serine protease family, which might be involved in tumor invasion and metastasis contributing to poor survival. The amplification in this region may be used as a prognostic marker in the treatment of CCA patients.

  2. Fusion of the Dhfr/Mtx and IR/MAR gene amplification methods produces a rapid and efficient method for stable recombinant protein production.

    Directory of Open Access Journals (Sweden)

    Chiemi Noguchi

    Full Text Available Amplification of the dihydrofolate reductase gene (Dhfr by methotrexate (Mtx exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR and a matrix attachment region (MAR, which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone.

  3. Fusion of the Dhfr/Mtx and IR/MAR gene amplification methods produces a rapid and efficient method for stable recombinant protein production. (United States)

    Noguchi, Chiemi; Araki, Yoshio; Miki, Daisuke; Shimizu, Noriaki


    Amplification of the dihydrofolate reductase gene (Dhfr) by methotrexate (Mtx) exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO) cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR), which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone.

  4. Modified PCR methods for 3' end amplification from serial analysis of gene expression (SAGE) tags. (United States)

    Xu, Wang-Jie; Wang, Zhao-Xia; Qiao, Zhong-Dong


    Serial analysis of gene expression (SAGE) is a powerful technique to study gene expression at the genome level. However, a disadvantage of the shortness of SAGE tags is that it prevents further study of SAGE library data, thus limiting extensive application of the SAGE method in gene expression studies. However, this problem can be solved by extension of the SAGE tags to 3' cDNAs. Therefore, several methods based on PCR have been developed to generate a 3' longer fragment cDNA corresponding to a SAGE tag. The list of modified methods is extensive, and includes rapid RT-PCR analysis of unknown SAGE tags (RAST-PCR), generation of longer cDNA fragments from SAGE tags for gene identification (GLGI), a high-throughput GLGI procedure, reverse SAGE (rSAGE), two-step analysis of unknown SAGE tags (TSAT-PCR), etc. These procedures are constantly being updated because they have characteristics and advantages that can be shared. Development of these methods has promoted the widespread use of the SAGE technique, and has accelerated the speed of studies of large-scale gene expression.

  5. Gene identification using exon amplification on human chromosome 18q21: implications for bipolar disorder. (United States)

    Chen, H; Huo, Y; Patel, S; Zhu, X; Swift-Scanlan, T; Reeves, R H; DePaulo, R; Ross, C A; McInnis, M G


    We previously reported linkage between bipolar disorder and a region on human chromosome (HC) 18q21. To identify genes in this region, exon trapping was performed on cosmids isolated from an HC18-specific cosmid library (LL18NC02) using 47 sequence tagged site (STS) markers from 18q21 as hybridization probes. A total of 285 unique sequences (exons) were obtained from 850 sequenced clones. Homology searching of the databases using NCBI's BLAST algorithms revealed that 31 exons have identity to known genes and/or ESTs, seven are identical to regions of finished genomic sequences in the 18q21 region, 20 have significant similarity (>30% sequence identity) to genes from human and/or other species, 19 were repetitive sequences, and 208 sequences (72%) are novel. Seventy per cent of the trapped sequences were predicted to be derived from genes using library screening and RT-PCR analyses. This represents an initial stage in characterizing genes in a susceptibility region for further study in bipolar disorder or other diseases that map to this region.

  6. Rapid, highly sensitive and highly specific gene detection by combining enzymatic amplification and DNA chip detection simultaneously

    Directory of Open Access Journals (Sweden)

    Koji Hashimoto


    Full Text Available We have developed a novel gene detection method based on the loop-mediated isothermal amplification (LAMP reaction and the DNA dissociation reaction on the same DNA chip surface to achieve a lower detection limit, broader dynamic range and faster detection time than are attainable with a conventional DNA chip. Both FAM- and thiol-labeled DNA probe bound to the complementary sequence accompanying Dabcyl was immobilized on the gold surface via Au/thiol bond. The LAMP reaction was carried out on the DNA probe fixed gold surface. At first, Dabcyl molecules quenched the FAM fluorescence. According to the LAMP reaction, the complementary sequence with Dabcyl was competitively reacted with the amplified targeted sequence. As a result, the FAM fluorescence increased owing to dissociation of the complementary sequence from the DNA probe. The simultaneous reaction of LAMP and DNA chip detection was achieved, and 103 copies of the targeted gene were detected within an hour by measuring fluorescence intensity of the DNA probe.

  7. Multiplex ligation-dependent probe amplification for rapid detection of deletions and duplications in the dystrophin gene

    Institute of Scientific and Technical Information of China (English)


    Objective:Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked disorders caused by mutations in the dystrophin gene. The majority of recognized mutations are copy number changes of individual exons. The objective of the present study was to assess the multiplex ligation-dependent probe amplification (MLPA) effects of detection of gene mutations. Methods: Samples of 20 control males and 80 males and their mothers referred to our diagnostic facility on the clinical suspicion of DMD or BMD were tested by MLPA and multiplex PCR. Results: The mean DQs for all peak of 20 control male samples was 1.02 (range from 0.83 to 1.21) by MLPA. Deletions or duplications were identified in 6 out of 31 families that had been previously tested as negative by multiplex PCR. One case of complex rearrangement involving a duplication of two regions: dupEX3-9 and dupEX 17-41 were found by MLPA. Conclusions: MLPA is a highly sensitive method and rapid alternative to multiplex PCR for detection of DMD and BMD.

  8. Mtp-40 and alpha antigen gene fragment amplification for the detection of Mycobacterium tuberculosis in Colombian clinical specimens

    Directory of Open Access Journals (Sweden)

    Rosalba Alfonso


    Full Text Available In this study, the use of Mtp-40 and alpha antigen polymerase chain reaction (PCR amplification fragments for the precise tuberculosis (TB diagnosis was evaluated. One hundred and ninety two different samples were obtained from 113 patients with suspected TB. Mtp-40 and alpha antigen protein genes were amplified by the PCR technique and compared to both the "gold standard" (culture test, as well as the clinical parameters (including a clinical record and X-ray film exam in 113 patients. Thirty-eight of the 113 patients had a presumptive clinical diagnosis of TB; 74% being detected by PCR technique, 58% by culture and 44% by direct microscopic visualization. Weconclude that it is possible to use PCR as a suitable technique for the detection of any mycobacteria by means of the alpha antigen product, or the specific infection of Mycobacterium tuberculosis by means of the mtp-40 gene. This might be a good supporting tool in difficult clinical TB diagnosis and pauci-bacillary cases.

  9. Loop-mediated isothermal amplification of specific endoglucanase gene sequence for detection of the bacterial wilt pathogen Ralstonia solanacearum.

    Directory of Open Access Journals (Sweden)

    Rok Lenarčič

    Full Text Available The increased globalization of crops production and processing industries also promotes the side-effects of more rapid and efficient spread of plant pathogens. To prevent the associated economic losses, and particularly those related to bacterial diseases where their management relies on removal of the infected material from production, simple, easy-to-perform, rapid and cost-effective tests are needed. Loop-mediated isothermal amplification (LAMP assays that target 16S rRNA, fliC and egl genes were compared and evaluated as on-site applications. The assay with the best performance was that targeted to the egl gene, which shows high analytical specificity for diverse strains of the betaproteobacterium Ralstonia solanacearum, including its non-European and non-race 3 biovar 2 strains. The additional melting curve analysis provides confirmation of the test results. According to our extensive assessment, the egl LAMP assay requires minimum sample preparation (a few minutes of boiling for the identification of pure cultures and ooze from symptomatic material, and it can also be used in a high-throughput format in the laboratory. This provides sensitive and reliable detection of R. solanacearum strains of different phylotypes.

  10. Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples

    Energy Technology Data Exchange (ETDEWEB)

    Braker, G.; Witzel, K.P. [Max-Planck-Inst. fuer Limnologie, Ploen (Germany); Fesefeldt, A. [Univ. Kiel (Germany). Inst. fuer Allgemeine Mikrobiologie


    A system was developed for the detection of denitrifying bacteria by the application of specific nitrite reductase gene fragments with PCR. Primer sequences were found for the amplification of fragments from both nitrite reductase genes (nirK and nirS) after comparative sequence analysis. Whenever amplification was tried with these primers, the known nir type of denitrifying laboratory cultures could be confirmed. Likewise, the method allowed a determination of the nir type of five laboratory strains. The nirK gene could be amplified from Blastobacter denitrificans, Alcaligenes xylosoxidans, and Alcaligenes sp. (DSM 30128); the nirS gene was amplified from Alcaligenes eutrophus DSM 530 and from the denitrifying isolate IFAM 3698. For each of the two genes, at least one primer combination amplified successfully for all of the test strains. Specific amplification products were not obtained wit h nondenitrifying bacteria or with strains of the other nir type. The specificity of the amplified products was confirmed by subsequent sequencing. These results suggest the suitability of the method for the qualitative detection of denitrifying bacteria in environmental samples. This was shown by applying the generally amplifying primer combination for each nir gene developed in this study to total DNA preparations from aquatic habitats.

  11. Amplification and sequencing of a sulfur-rich 10kd prolamin gene from rice seeds

    Institute of Scientific and Technical Information of China (English)

    YUJingjuan; AOGuangming


    Nutritious value of seed storage protein is low due to deficiency in essential amino acid contents. Cereals are mainly deficient in lysine and legumes in sulfur-containing amino acids ( methionine and cysteine). So far, several sufur-rich seed protein genes have been isolated and the essential amino acid contents of seed proteins were increased in transgenic tobacco and Brassica napus.

  12. Gene Amplification Is A Mechanism For Rapid Weed Evolution To Herbicide Resistance (United States)

    The herbicide glyphosate became widely used in the U.S. and other parts of the world following the introduction of glyphosate-resistant crops. These crops were created by introduction of a modified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, the herbicide target site. Increased use of ...

  13. Amplification, expression, and packaging of a foreign gene by giardiavirus in Giardia lamblia. (United States)

    Yu, D C; Wang, A L; Wang, C C


    Giardia lamblia is an intestinal protozoan parasite and one of the earliest eukaryotic divergents. The trophozoite multiplies via asexual binary fission and lacks all natural means of lateral gene transfer. A system is developed here for long-term expression of a foreign gene in this organism by exploiting recombinant virions derived from the giardiavirus (GLV), a double-stranded RNA virus that infects many Giardia isolates. An in vitro transcript of the cloned GLV cDNA, comprising the firefly luciferase-encoding region flanked by 5' and 3' fragments of GLV positive-strand RNA, was electroporated into GLV-infected trophozoites. Luciferase activity in electroporated cells peaked on day 2 at levels 6 orders of magnitude above background. Expression of this foreign gene remained at 80% of its peak level after 30 days in the absence of selective pressure. The chimeric RNA was replicated as double-stranded RNA and packaged into virus-like particles. The recombinant virions were partially purified from the wild-type helper virus by CsCl equilibrium density-gradient centrifugation and used to superinfect Giardia trophozoites. At multiplicities of infection of 100 or higher, these chimeric virions were able to initiate new rounds of expression of luciferase activity in the superinfected cells. Thus, the engineered virion can be successfully used to introduce and efficiently express a heterologous gene in this eukaryotic microorganism.

  14. Novel subtractive transcription-based amplification of mRNA (STAR method and its application in search of rare and differentially expressed genes in AD brains

    Directory of Open Access Journals (Sweden)

    Walker P Roy


    Full Text Available Abstract Background Alzheimer's disease (AD is a complex disorder that involves multiple biological processes. Many genes implicated in these processes may be present in low abundance in the human brain. DNA microarray analysis identifies changed genes that are expressed at high or moderate levels. Complementary to this approach, we described here a novel technology designed specifically to isolate rare and novel genes previously undetectable by other methods. We have used this method to identify differentially expressed genes in brains affected by AD. Our method, termed Subtractive Transcription-based Amplification of mRNA (STAR, is a combination of subtractive RNA/DNA hybridization and RNA amplification, which allows the removal of non-differentially expressed transcripts and the linear amplification of the differentially expressed genes. Results Using the STAR technology we have identified over 800 differentially expressed sequences in AD brains, both up- and down- regulated, compared to age-matched controls. Over 55% of the sequences represent genes of unknown function and roughly half of them were novel and rare discoveries in the human brain. The expression changes of nearly 80 unique genes were further confirmed by qRT-PCR and the association of additional genes with AD and/or neurodegeneration was established using an in-house literature mining tool (LitMiner. Conclusion The STAR process significantly amplifies unique and rare sequences relative to abundant housekeeping genes and, as a consequence, identifies genes not previously linked to AD. This method also offers new opportunities to study the subtle changes in gene expression that potentially contribute to the development and/or progression of AD.

  15. 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

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    Yanagihara Kazuyoshi


    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

  16. Amplification of the flgE gene provides evidence for the existence of a Brazilian borreliosis

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    Elenice Mantovani


    Full Text Available INTRODUCTION: The symptoms of Brazilian borreliosis resemble the clinical manifestations of Lyme disease (LD. However, there are differences between the two in terms of epidemiological and laboratory findings. Primers usually employed to diagnose LD have failed to detect Borrelia strains in Brazil. OBJECTIVE: We aimed to identify the Brazilian Borrelia using a conserved gene that synthesizes the flagellar hook (flgE of Borrelia burgdorferi sensu lato. METHOD: Three patients presenting with erythema migrans and positive epidemiological histories were recruited for the study. Blood samples were collected, and the DNA was extracted by commercial kits. RESULTS: The gene flgE was amplified from DNA of all selected patients. Upon sequencing, these positive samples revealed 99% homology to B. burgdorferi flgE. CONCLUSION: These results support the existence of borreliosis in Brazil. However, it is unclear whether this borreliosis is caused by a genetically modified B. burgdorferi sensu stricto or by a new species of Borrelia spp.

  17. Coordinate amplification of metallothionein I and II genes in cadmium-resistant Chinese hamster cells: implications for mechanisms regulating metallothionein gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Crawford, B.D.; Enger, M.D.; Griffith, B.B.; Griffith, J.K.; Hanners, J.L.; Longmire, J.L.; Munk, A.C.; Stallings, R.L.; Tesmer, J.G.; Walters, R.A.; Hildebrand, C.E.


    The authors describe here the derivation, characterization, and use of clonal cadmium-resistance (Cd/sup r) strains of the Chinese hamster cell line CHO which differ in their metallothionein (MT) induction capacity. By nondenaturing polyacrylaminde gel electrophoresis, the authors showed that the stable Cd/sup r/ phenotype is correlated with the augmented expression of both isometallothioneins (MTI and MTII). In cells resistant to concentrations of CdCl2 exceeding 20 M, coordinate amplifications of genes encoding both isometallothioneins was demonstrated by using cDNA MT-coding sequence probes and probes specific for 3'-noncoding regions of Chinese hamster MTI and MTII genes. Molecular and in situ hybridization analyses supported close linkage of Chinese hamster MTI and MTII genes, which the authors have mapped previously to Chinese hamster chromosome 3. This suggests the existence of a functionally related MT gene cluster in this species. Amplified Cd/sup r/ variants expressing abundant MT and their corresponding Cd/sup s/ parental CHO cells should be useful for future studies directed toward elucidating the mechanisms that regulate expressions of the isometallothioneins. 59 references, 8 figures.

  18. A phase II study of Epirubicin in oxaliplatin-resistant patients with metastatic colorectal cancer and TOP2A gene amplification. (United States)

    Tarpgaard, Line S; Qvortrup, Camilla; Nygård, Sune B; Nielsen, Signe L; Andersen, Diana R; Jensen, Niels Frank; Stenvang, Jan; Detlefsen, Sönke; Brünner, Nils; Pfeiffer, Per


    The overall purpose of this study is to provide proof of concept for introducing the anthracycline epirubicin as an effective, biomarker-guided treatment for metastatic colorectal cancer (mCRC) patients who are refractory to treatment with oxaliplatin-based chemotherapy and have TOP2A gene amplification in their tumor cells. Epirubicin is an anthracycline that targets DNA topoisomerase 2-α enzyme encoded by the TOP2A gene. It is used for treatment of several malignancies, but currently not in CRC. TOP2A gene amplifications predict improved efficacy of epirubicin in patients with breast cancer and thus could be an alternative option for patients with CRC and amplified TOP2A gene. We have previously analysed the frequency of TOP2A gene aberrations in CRC and found that 46.6% of these tumors had TOP2A copy gain and 2.0% had loss of TOP2A when compared to adjacent normal tissue. The TOP2A gene is located on chromosome 17 and when the TOP2A/CEN-17 ratio was applied to identify tumors with gene loss or amplifications, 10.5% had a ratio ≥ 1.5 consistent with gene amplification and 2.6% had a ratio ≤ 0.8 suggesting gene deletions. Based on these observations and the knowledge gained from treatment of breast cancer patients, we have initiated a prospective clinical, phase II protocol using epirubicin (90 mg/m2 iv q 3 weeks) in mCRC patients, who are refractory to treatment with oxaliplatin. The study is an open label, single arm, phase II study, investigating the efficacy of epirubicin in patients with oxaliplatin refractory mCRC and with a cancer cell TOP2A/CEN-17 ratio ≥ 1.5. TOP2A gene amplification measured by fluorescence in situ hybridization. A total of 25 evaluable patients (15 + 10 in two steps) will be included (Simon's two-stage minimax design). Every nine weeks, response is measured by computed tomography imaging and evaluated according to RECIST 1.1. The primary end-point of the study is progression-free survival. Eudract no. 2013-001648-79.

  19. Double-hairpin molecular-beacon-based amplification detection for gene diagnosis linked to cancer. (United States)

    Xu, Huo; Zhang, Rongbo; Li, Feng; Zhou, Yingying; Peng, Ting; Wang, Xuedong; Shen, Zhifa


    A powerful double-hairpin molecular beacon (DHMB) was developed for cancer-related KRAS gene detection based on the one-to-two stoichiometry. During target DNA detection, DHMB can execute signal transduction even if no any exogenous element is involved. Unlike the conventional molecular beacon based on the one-to-one interaction, one target DNA not only hybridizes with one DHMB and opens its hairpin but also promotes the interaction between two DHMBs, causing the separation of two fluorophores from quenchers. This leads to an enhanced fluorescence signal. As a result, the target KRAS gene is able to be detected within a wide dynamic range from 0.05 to 200 nM with the detection limit of 50 pM, indicating a dramatic improvement compared with traditional molecular beacons. Moreover, the point mutations existing in target DNAs can be easily screened. The potential application for target species in real samples was indicated by the analysis of PCR amplicons of DNAs from the DNA extracted from SW620 cell. Besides becoming a promising candidate probe for molecular biology research and clinical diagnosis of genetic diseases, the DHMB is expected to provide a significant insight into the design of DNA probe-based homogenous sensing systems. Graphical Abstract A powerful double-hairpin molecular beacon (DHMB) was developed for cancer-related gene KRAS detection based on the one-to-two stoichiometry. Without the help of any exogenous probe, the point mutation is easily screened, and the target DNA can be quantified down to 50 pM, indicating a dramatic improvement compared with traditional molecular beacons.

  20. Amplifications of NCOA3 gene in colorectal cancers in a Chinese population

    Institute of Scientific and Technical Information of China (English)

    Zhi Li; Zheng-Yu Fang; Yi Ding; Wan-Tong Yao; Yang Yang; Zhong-Qing Zhu; Wen Wang; Qin-xian Zhang


    AIM:To investigate the copy number variation of NAC03 gene in colorectal cancer (CRC) and its correlation with tumor progression.METHODS:A total of 142 samples of case-matched CRC tissues and adjacent normal tissues were obtained from patients undergoing bowel resection.Quantitative real-time polymerase chain reaction method was used to investigate the copy number variations of NCOA3 as well as gene expression in the collected tissues.RESULTS:Copy number gains of NCOA3 were detected in 39 CRC samples (27.5%) and were correlated with tumor progression (x2 =6.42,P =0.0112).Moreover,there was a positive correlation between copy number gain and mRNA over-expression of NCOA3 in CRCs (P =0.0023).Expression level of NCOA3 mRNA was also enhanced in the CRC samples with unaltered copy numbers (3.85 ± 1.23 vs 2.71 ± 0.64,P < 0.01).CONCLUSION:Sporadic colorectal cancers exhibit different mechanisms of NCOA3 regulation.

  1. Rapid and sensitive detection of Plesiomonas shigelloides by loop-mediated isothermal amplification of the hugA gene.

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    Shuang Meng

    Full Text Available Plesiomonas shigelloides is one of the causative agents of human gastroenteritis, with increasing number of reports describing such infections in recent years. In this study, the hugA gene was chosen as the target to design loop-mediated isothermal amplification (LAMP assays for the rapid, specific, and sensitive detection of P. shigelloides. The performance of the assay with reference plasmids and spiked human stools as samples was evaluated and compared with those of quantitative PCR (qPCR. No false-positive results were observed for the 32 non-P. shigelloides strains used to evaluate assay specificity. The limit of detection for P. shigelloides was approximately 20 copies per reaction in reference plasmids and 5×10(3 CFU per gram in spiked human stool, which were more sensitive than the results of qPCR. When applied in human stool samples spiked with 2 low levels of P. shigelloides, the LAMP assays achieved accurate detection after 6-h enrichment. In conclusion, the LAMP assay developed in this study is a valuable method for rapid, cost-effective, and simple detection of P. shigelloides in basic clinical and field laboratories in the rural areas of China.

  2. Human identification from forensic materials by amplification of a human-specific sequence in the myoglobin gene.

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    Ono T


    Full Text Available We developed a method for human identification of forensic biological materials by PCR-based detection of a human-specific sequence in exon 3 of the myoglobin gene. This human-specific DNA sequence was deduced from differences in the amino acid sequences of myoglobins between humans and other animal species. The new method enabled amplification of the target DNA fragment from 30 samples of human DNA, and the amplified sequences were identical with that already reported. Using this method, we were able to distinguish human samples from those of 21 kinds of animals: the crab-eating monkey, horse, cow, sheep, goat, pig, wild boar, dog, raccoon dog, cat, rabbit, guinea pig, hamster, rat, mouse, whale, chicken, pigeon, turtle, frog, and tuna. However, we were unable to distinguish between human and gorilla samples. This method enabled us to detect the target sequence from 25 pg of human DNA, and the target DNA fragment from blood stored at 37 degrees C for 6 months, and from bloodstains heated at 150 degrees C for 4 h or stored at room temperature for 26 years. Herein we also report a practical application of the method for human identification of a bone fragment.

  3. Gene changes in Duchenne muscular dystrophy: Comparison of multiplex PCR and multiplex ligation-dependent probe amplification techniques

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    Kohli Sudha


    Full Text Available Background: Duchenne muscular dystrophy (DMD is a common X-linked recessive neuromuscular disorder, affecting 1 in 3,500 live male births. About 65% of cases are caused by deletions; ~5% to 8%, by duplication; and the remaining, by point mutations of the dystrophin gene. The frequency of complex rearrangements (double-deletion and non-contiguous duplications is reported to be 4%. Aim: In this study, we examined the usefulness of multiplex ligation-dependent probe amplification (MLPA for screening of deletion and duplication mutations in a group of DMD/ BMD (Becker muscular dystrophy patients from India. Patients and Methods: We analyzed 180 patients referred from all over India, by both multiplex PCR technique (22 exons and MLPA (all 79 exons. Results and Conclusion: By multiplex PCR, deletions were detected in 90 (50% patients. MLPA studies in these cases detected 3 additional deletions, 16 (8.9% duplications and 2 point mutations. MLPA is useful to verify absence of deletions/ duplications in all 79 exons. This sets the stage to look for point mutations using RNA- or DNA-based tests because of the availability of the drug PTC124. Also, the extent of the deletions and duplications could be more accurately defined by MLPA. The delineation of the precise extent of deletion helps in deciding whether exon-skipping technique would be useful as therapy.

  4. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. (United States)

    Tanenbaum, Marvin E; Gilbert, Luke A; Qi, Lei S; Weissman, Jonathan S; Vale, Ronald D


    Signals in many biological processes can be amplified by recruiting multiple copies of regulatory proteins to a site of action. Harnessing this principle, we have developed a protein scaffold, a repeating peptide array termed SunTag, which can recruit multiple copies of an antibody-fusion protein. We show that the SunTag can recruit up to 24 copies of GFP, thereby enabling long-term imaging of single protein molecules in living cells. We also use the SunTag to create a potent synthetic transcription factor by recruiting multiple copies of a transcriptional activation domain to a nuclease-deficient CRISPR/Cas9 protein and demonstrate strong activation of endogenous gene expression and re-engineered cell behavior with this system. Thus, the SunTag provides a versatile platform for multimerizing proteins on a target protein scaffold and is likely to have many applications in imaging and controlling biological outputs.

  5. Sex identification based on AMEL gene PCR amplification from blue sheep (Pseudois nayaur) fecal DNA samples. (United States)

    Liu, X; Yang, Y Y; Wang, X M; Liu, Z S; Wang, Z H; Ding, Y Z


    The use of noninvasive genetic sampling to identify the sex of wild animals is an extremely valuable and important tool in molecular ecology and wildlife conservation. Sex determination using the amelogenin gene has been conducted in many species because only a single pair of primers is required to amplify both X- and Y-linked alleles. However, this method has not been used in field research with the feces of wildlife. In this study, we applied this method to 222 fecal samples from wild blue sheep (Pseudois nayaur) using amelogenin primers (SE47/SE48) after testing the effectiveness of sex determination using tissue samples and fecal samples from blue sheep of known sex. We found this method to be highly reliable (80.2%) for blue sheep. Amelogenin can be used to identify the sex of wild animals using fecal samples.

  6. KIT amplification and gene mutations in acral/mucosal melanoma in Korea. (United States)

    Yun, Jina; Lee, Jeeyun; Jang, Jiryeon; Lee, Eui Jin; Jang, Kee Taek; Kim, Jung Han; Kim, Kyoung-Mee


    Mucosal and acral melanomas have demonstrated different genetic alterations and biological behavior compared with more common cutaneous melanomas. It was recently reported that gain-of-function KIT mutations and/or copy number increases are more common in mucosal and acral melanomas. Thus, we studied the frequency and pattern of KIT aberrations in mucosal and acral melanomas in Korea. We analyzed 97 patients who were pathologically confirmed with mucosal or acral melanoma between 1997 and 2010 at Samsung Medical Center. Of the 97 melanoma patients, 92 were screened for mutations in KIT exons 11, 13, 17, and 18, BRAF and NRAS genes. KIT copy number was assessed by quantitative, real-time PCR. Of the 97 patients, 55 (56.7%) were mucosal, 40 (41.2%) were acral melanoma, and two were of unknown primary origin. Among seven cases with KIT mutation, five (60.0%) occurred in exon 11, one (20.0%) in exon 17, and one (20.0%) in exon 13. Point mutations were the most common, resulting in substitutions in exon 11 (K558R, T574A, L576P, and V559A), exon 13 (N655K), and exon 17 (N822K). A novel Thr574Ala (c.1720A>G) KIT mutation, which has not been reported in melanoma or other tumor types, was identified in one genital melanoma case. Of the 97 mucosal or acral melanoma specimens, 49 were tested for KIT gene copy number changes using quantitative PCR. Increased KIT copy number was identified in 15 patients: seven (40%) of 20 acral melanomas and eight (31%) of 26 mucosal melanomas. Our study implicates that a significant proportion of acral and mucosal melanomas have KIT mutations in Asian population.

  7. Two-step amplification of the human PPT sequence provides specific gene expression in an immunocompetent murine prostate cancer model. (United States)

    Dzojic, H; Cheng, W-S; Essand, M


    The recombinant prostate-specific PPT sequence comprises a prostate-specific antigen enhancer, a PSMA enhancer and a TARP promoter. It is transcriptionally active in human prostate cancer cells both in the presence and absence of testosterone. However, in experimental murine prostate cancer, it has no detectable transcriptional activity. Herein, we describe that the PPT sequence in combination with a two-step transcriptional amplification (TSTA) system becomes active also in murine prostate cancer cells. An adenovirus with TSTA-amplified PPT-controlled expression of the luciferase reporter gene, Ad[PPT/TSTA-Luc], has up to 100-fold higher prostate-specific transcriptional activity than a non-amplified PPT-based adenovirus, Ad[PPT-Luc], in human cells. In addition, Ad[PPT/TSTA-Luc] confers prostate-specific transgene expression in murine cells, with an activity that is approximately 23% of Ad[CMV-Luc] in the transgenic adenocarcinoma of the mouse prostate (TRAMP)-C2 cells. Moreover, to visualize luciferase expression in living mice a charge-coupled device camera was used. Ad[PPT/TSTA-Luc] yielded approximately 30-fold higher transgene expression than Ad[PPT-Luc] in LNCaP tumor xenografts. Importantly, Ad[PPT/TSTA-Luc] also showed activity in murine TRAMP-C2 tumors, whereas Ad[PPT-Luc] activity was undetectable. These results highlight that the recombinant PPT sequence is active in murine prostate cancer cells when augmented by a TSTA system. This finding opens up for preclinical studies with prostate-specific therapeutic gene expression in immunocompetent mice.

  8. Species identification of Asini Corii Collas (donkey glue) by PCR amplification of cytochrome b gene. (United States)

    Kumeta, Yukie; Maruyama, Takuro; Asama, Hiroshi; Yamamoto, Yutaka; Hakamatsuka, Takashi; Goda, Yukihiro


    Asini Corii Collas (ACC; donkey glue) is a crude drug used to promote hematopoiesis and arrest bleeding. Because adulteration of the drug with substances from other animals such as horses, cattle, and pigs has been found, we examined PCR methods based on the sequence of the cytochrome b gene for source species identification. Two strategies for extracting DNA from ACC were compared, and the ion-exchange resin procedure was revealed to be more suitable than the silica-based one. Using DNA extracted from ACC by the ion-exchange resin procedure, PCR methods for species-specific detection of donkey, horse, cattle, and pig substances were established. When these species-specific PCR methods were applied to ACC, amplicons were obtained only by the donkey-specific PCR. Cattle-specific PCR detected as little as 0.1% admixture of cattle glue in the ACC. These results suggest that the species-specific PCR methods established in this study would be useful for simple and easy detection of adulteration of ACC.

  9. Involvement of facultative apomixis in inheritance of EPSPS gene amplification in glyphosate-resistant Amaranthus palmeri. (United States)

    Ribeiro, Daniela N; Pan, Zhiqiang; Duke, Stephen O; Nandula, Vijay K; Baldwin, Brian S; Shaw, David R; Dayan, Franck E


    The inheritance of glyphosate resistance in two Amaranthus palmeri populations (R1 and R2) was examined in reciprocal crosses (RC) and second reciprocal crosses (2RC) between glyphosate-resistant (R) and -susceptible (S) parents of this dioecious species. R populations and Female-R × Male-S crosses contain higher 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene copy numbers than the S population. EPSPS expression, EPSPS enzyme activity, EPSPS protein quantity, and level of resistance to glyphosate correlated positively with genomic EPSPS relative copy number. Transfer of resistance was more influenced by the female than the male parent in spite of the fact that the multiple copies of EPSPS are amplified in the nuclear genome. This led us to hypothesize that this perplexing pattern of inheritance may result from apomictic seed production in A. palmeri. We confirmed that reproductively isolated R and S female plants produced seeds, indicating that A. palmeri can produce seeds both sexually and apomictically (facultative apomixis). This apomictic trait accounts for the low copy number inheritance in the Female-S × Male-R offsprings. Apomixis may also enhance the stability of the glyphosate resistance trait in the R populations in the absence of reproductive partners.

  10. Identification of endotrypanum species from a sloth, a squirrel and Lutzomyia sandflies in ecuador by PCR amplification and sequencing of the mini-exon gene. (United States)

    Katakura, Ken; Mimori, Tatsuyuki; Furuya, Masato; Uezato, Hiroshi; Nonaka, Shigeo; Okamoto, Munehiro; Gomez L, Eduardo A; Hashiguchi, Yoshihisa


    PCR amplification and nucleotide sequencing of the mini-exon gene revealed that four strains isolated from a sloth (Choloepus hoffmanni), a squirrel (Sciurus granatensis) and two sandflies (Lutzomyia hartmanni) in Ecuador were indistinguishable from Endotrypanum monterogeii. Another strain isolated from Lu. hartmanni showed the high sequence similarity to E. schaudinni. Since three of these strains have been previously identified as Leishmania (Viannia) equatorensis, the results demonstrate that L. (V.) equatorensis is genetically closely related to the genus Endotrypanum. The present study also indicates that Endotrypanum species are distributed in arboreal animals and sandflies in Ecuador, and that mini-exon gene amplification is useful for epidemiological studies of Leishmania and Endotrypanum in the New World.

  11. CASCADE, a platform for controlled gene amplification for high, tunable and selection-free gene expression in yeast

    DEFF Research Database (Denmark)

    Strucko, Tomas; Buron, Line Due; Jarczynska, Zofia Dorota


    Over-expression of a gene by increasing its copy number is often desirable in the model yeast Saccharomyces cerevisiae. It may facilitate elucidation of enzyme functions, and in cell factory design it is used to increase production of proteins and metabolites. Current methods are typically exploi...... production of two fluorescent proteins, the enzyme β-galactosidase the fungal polyketide 6-methyl salicylic acid and the plant metabolite vanillin glucoside....

  12. Rapid amplification of cDNA ends (RACE) improves the PCR-based isolation of immunoglobulin variable region genes from murine and human lymphoma cells and cell lines. (United States)

    Doenecke, A; Winnacker, E L; Hallek, M


    The isolation of rearranged immunoglobulin (Ig) variable region (V) genes is usually performed by PCR with consensus primers binding to conserved regions within the V sequences. However, the isolation of Ig genes by this method is hampered in 15-35% by technical difficulties, mostly mismatches of oligonucleotide primers to V sequences. In order to obtain DNA sequences from V heavy chain (VH) genes which could not be amplified with consensus primers, we used a modified PCR technique, the rapid amplification of cDNA ends (RACE) PCR in combination with new heavy chain constant region primers for the isolation of human and murine VH genes. In comparison, consensus primer PCR with different sets of previously published oligonucleotide primers was used. Both methods were applied to isolate VH genes from murine B cell lymphoma (A20 and BCL1), myeloma (NS1) and hybridoma (SP6) cell lines and from freshly isolated human chronic lymphocytic leukemia and lymphoma cells. RACE PCR allowed the amplification and subsequent cloning of the complete VH gene in all cases. In contrast, consensus primer PCR failed to isolate the VH sequence of the murine A20 cell line; this was explained by a mismatch of consensus primers with VH sequences. When both PCR methods amplified VH sequences, the DNA sequences obtained were identical. Taken together, RACE PCR represents a reliable and versatile method for the isolation of VH genes from human and murine lymphoma cells, in particular if consensus primer PCR fails.

  13. The clinical pathological characteristics and prognosis of FGFR1 gene amplification in non-small-cell lung cancer: a meta-analysis

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    Xie FJ


    Full Text Available Fa-Jun Xie,1,2 Hong-Yang Lu,1,3 Qiu-Qing Zheng,3 Jing Qin,1,3 Yun Gao,3 Yi-Ping Zhang,1,3 Xun Hu,2 Wei-Min Mao3,4 1Department of Medical Oncology, Zhejiang Cancer Hospital, 2Cancer Institute (Key Laboratory for Cancer Intervention and Prevention, China National Ministry of Education, Zhejiang Provincial Key Laboratory of Molecular Biology in Medical Sciences, Second Affiliated Hospital, Zhejiang University School of Medicine,3Zhejiang Key Laboratory of Diagnosis and Treatment Technology on Thoracic Oncology (Lung and Esophagus, Hangzhou, 4Department of Thoracic Surgery, Zhejiang Cancer Hospital, Hangzhou, People’s Republic of China Abstract: FGFR1 amplification is recognized as a novel therapy target for non-small-cell lung cancer (NSCLC, especially in squamous cell carcinoma (SCC. However, the association between FGFR1 amplification and the clinicopathological characteristics of NSCLC remains controversial. We performed a meta-analysis of 17 eligible studies to examine the correlation between FGFR1 gene amplification and clinicopathological characteristics. FGFR1 amplification was closely related to these clinicopathological features, including sex (odds ratio [OR] 2.05, 95% confidence interval [CI] 1.50–2.80, smoking (OR 3.31, 95% CI 2.02–5.44, and histology (OR 3.60, 95% CI 2.82–4.59. FGFR1 amplification was associated with shorter overall survival, and no significant heterogeneity existed between studies (I2=3.8%. We should note that publication bias may partly account for these results, but our findings remained significant after the trim-and-fill method (hazard ratio 1.22, 95% CI 1.06–1.40. However, no significant correlation was found with poor disease-free survival (hazard ratio 1.43, 95% CI 0.96–2.12. In conclusion, this study showed that FGFR1 amplification was significantly associated with sex, smoking, and histology. FGFR1 amplification could be a marker of poor prognosis in NSCLC patients, especially in SCC patients

  14. Simultaneous detection of human papillomavirus integration and c-MYC gene amplification in cervical lesions: an emerging marker for the risk to progression. (United States)

    Gimenes, Fabrícia; Souza, Raquel Pantarotto; de Abreu, André Luelsdorf Pimenta; Pereira, Monalisa Wolski; Consolaro, Marcia Edilaine Lopes; da Silva, Vânia Ramos Sela


    The persistence of high-risk oncogenic human papillomavirus (HR-HPV) infection and its integration into the host genome are key steps in the induction of malignant alterations. c-MYC chromosome region is a frequent localization for HPV insertion that has been observed in chromosome band 8q24 by fluorescence in situ hybridization (FISH). We report the HPV viral integration and amplification patterns of the c-MYC gene in cytological smears with FISH as a potential biomarker for the progression of squamous intraepithelial lesions (SIL). HPV detection and genotyping by polymerase chain reaction (PCR) and FISH analysis by "Vysis Cervical FISH Probe" kit (ABBOTT Molecular Inc.) were performed in 37 cervical samples including 8 NILM, 7 ASC-US, 7 LSIL, 3 ASC-H, 7 HSIL and 5 SCC. The results show concordance between FISH and PCR techniques for HPV detection. The majority of the samples contained HR-HPV, the majority being -16 and -18 genotypes. HPV integration as determined by FISH was most frequent in high-risk lesions. The c-MYC gene amplification was found only in HPV-positive samples and was detected primarily in high-risk lesions and in cells with an integrated form of HPV. HPV integration and c-MYC gene amplification detected by FISH could be an important biomarker for use in clinical practice to determine SIL with a risk of progression.

  15. Rapid, simple and sensitive detection of Q fever by loop-mediated isothermal amplification of the htpAB gene.

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    Lei Pan

    Full Text Available BACKGROUND: Q fever is the most widespread zoonosis, and domestic animals are the most common sources of transmission. It is not only difficult to distinguish from other febrile diseases because of the lack of specific clinical manifestations in humans, but it is also difficult to identify the disease in C. burnetii-carrying animals because of the lack of identifiable features. Conventional serodiagnosis requires sera from the acute and convalescent stages of infection, which are unavailable at early diagnosis. Nested PCR and real-time PCR require equipment. In this study, we developed a Loop-Mediated Isothermal Amplification (LAMP assay to identify C. burnetii rapidly and sensitively. METHODS: A universal LAMP primer set was designed to detect the repeated sequence IS1111a of the htpAB gene of C. burnetii using PrimerExplorer V4 software. The sensitivity of the LAMP assay was evaluated using known quantities of recombined reference plasmids containing the targeted genes. The specificity of the developed LAMP assay was determined using 26 members of order Rickettsiae and 18 other common pathogens. The utility of the LAMP assay was further compared with real time PCR by the examination 24 blood samples including 6 confirmed and 18 probable Q fever cases, which diagnosed by IFA serological assessment and real time PCR. In addition, 126 animal samples from 4 provinces including 97 goats, 7 cattle, 18 horses, 3 marmots and 1 deer were compared by these two methods. RESULTS: The limits of detection of the LAMP assay for the htpAB gene were 1 copy per reaction. The specificity of the LAMP assay was 100%, and no cross-reaction was observed among the bacteria used in the study. The positive rate of unknown febrile patients was 33.3%(95%CI 30.2%-36.4% for the LAMP assay and 8.3%(95%CI 7.4%-9.2% for the real time PCR(P<0.05. Similarly, the total positive rate of animals was 7.9%(95%CI 7.1%-8.7% for the LAMP assay and 0.8%(95%CI 0.7%-0.9%for the real time

  16. Characterization of Abcc4 gene amplification in stepwise-selected mouse J774 macrophages resistant to the topoisomerase II inhibitor ciprofloxacin. (United States)

    Marquez, Béatrice; Ameye, Geneviève; Vallet, Coralie M; Tulkens, Paul M; Poirel, Hélène A; Van Bambeke, Françoise


    Exposure of J774 mouse macrophages to stepwise increasing concentrations of ciprofloxacin, an antibiotic inhibiting bacterial topoisomerases, selects for resistant cells that overexpress the efflux transporter Abcc4 (Marquez et al. [2009] Antimicrob. Agents Chemother. 53: 2410-2416), encoded by the Abcc4 gene located on Chromosome 14qE4. In this study, we report the genomic alterations occurring along the selection process. Abcc4 expression progressively increased upon selection rounds, with exponential changes observed between cells exposed to 150 and 200 µM of ciprofloxacin, accompanied by a commensurate decrease in ciprofloxacin accumulation. Molecular cytogenetics experiments showed that this overexpression is linked to Abcc4 gene overrepresentation, grading from a partial trisomy of Chr 14 at the first step of selection (cells exposed to 100 µM ciprofloxacin), to low-level amplifications (around three copies) of Abcc4 locus on 1 or 2 Chr 14 (cells exposed to 150 µM ciprofloxacin), followed by high-level amplification of Abcc4 as homogeneous staining region (hsr), inserted on 3 different derivative Chromosomes (cells exposed to 200 µM ciprofloxacin). In revertant cells obtained after more than 60 passages of culture without drug, the Abcc4 hsr amplification was lost in approx. 70% of the population. These data suggest that exposing cells to sufficient concentrations of an antibiotic with low affinity for eukaryotic topoisomerases can cause major genomic alterations that may lead to the overexpression of the transporter responsible for its efflux. Gene amplification appears therefore as a mechanism of resistance that can be triggered by non-anticancer agents but contribute to cross-resistance, and is partially and slowly reversible.

  17. Amplification of the Full-Length PAMP Gene and Difference of the mRNA Expression Among Three Lean Pig Breeds

    Institute of Scientific and Technical Information of China (English)


    To understand the function of porcine adipocyte-special membrane protein (PAMP) gene and the difference of fat deposition ability among various lean pig breeds, a full-length porcine adipocyte-special membrane protein (PAMP) gene was successfully amplified using reverse transcription polymerase chain reaction (RT-PCR) and 5'-rapid amplification of cDNA end (5'-RACE). The open reading frame was 1 587 bp encoding 529 amino acids. The nucleotide sequence of the full-length PAMP gene was deposited in the GenBank under the accession number EF433431. The PAMP gene mRNA expression was analyzed on three lean pig breeds by quantitative reverse transcription polymerase chain reaction (QRT-PCR). The PAMP gene mRNA levels in YHM (Yorkshire × Hampshire × Meishan) pig and DLY (Duroc×Landrance× Yorkshire) pig were about 0.82 and 0.38 times of that in SW (Shanxi-White) pig, respectively.

  18. The correlation between dual-color chromogenic in situ hybridization and fluorescence in situ hybridization in assessing HER2 gene amplification in breast cancer

    DEFF Research Database (Denmark)

    Pedersen, Marianne; Rasmussen, Birgitte Bruun


    obtained by the 2 methods showed excellent correlation (correlation coefficient 0.95). In conclusion, it is possible by dual-color CISH method to demonstrate HER2 genes and chromosome 17 genes, in the same tissue section and reliably assess HER2 status. The CISH method is a very promising alternative...... invasive breast carcinomas, both FISH and CISH detected HER2 amplification in 24 cases and nonamplification was detected in 47 cases. One case showed a discrepancy between FISH and CISH. The concordance between CISH and FISH was found to be almost perfect (98.6%). The correlation between the HER2 ratios...

  19. Amplification of the Insulin-Like Growth Factor 1 Receptor Gene Is a Rare Event in Adrenocortical Adenocarcinomas: Searching for Potential Mechanisms of Overexpression

    Directory of Open Access Journals (Sweden)

    Tamaya Castro Ribeiro


    Full Text Available Context. IGF1R overexpression appears to be a prognostic biomarker of metastatic pediatric adrenocortical tumors. However, the molecular mechanisms that are implicated in its upregulation remain unknown. Aim. To investigate the potential mechanisms involved in IGF1R overexpression. Patients and Methods. We studied 64 adrenocortical tumors. IGF1R copy number variation was determined in all patients using MLPA and confirmed using real time PCR. In a subgroup of 32 patients, automatic sequencing was used to identify IGF1R allelic variants and the expression of microRNAs involved in IGF1R regulation by real time PCR. Results. IGF1R amplification was detected in an adrenocortical carcinoma that was diagnosed in a 46-year-old woman with Cushing’s syndrome and virilization. IGF1R overexpression was demonstrated in this case. In addition, gene amplification of other loci was identified in this adrenocortical malignant tumor, but no IGF1R copy number variation was evidenced in the remaining cases. Automatic sequencing revealed three known polymorphisms but they did not correlate with its expression. Expression of miR-100, miR-145, miR-375, and miR-126 did not correlate with IGF1R expression. Conclusion. We demonstrated amplification and overexpression of IGF1R gene in only one adrenocortical carcinoma, suggesting that these combined events are uncommon. In addition, IGF1R polymorphisms and abnormal microRNA expression did not correlate with IGF1R upregulation in adrenocortical tumors.

  20. Development and application of loop-mediated isothermal amplification methods targeting the seM gene for detection of Streptococcus equi subsp. equi. (United States)

    Hobo, Seiji; Niwa, Hidekazu; Oku, Kazuomi


    Loop-mediated isothermal amplification (LAMP) constitutes a potentially valuable diagnostic tool for rapid diagnosis of contagious diseases. In this study, we developed a novel LAMP method (seM-LAMP) to detect the seM gene of Streptococcus equi subsp. equi (S. equi), the causative agent of strangles in equids. The seM-LAMP successfully amplified the target sequence of the seM gene at 63°C within 60 min. The sensitivity of the seM-LAMP was slightly lower than the 2nd reaction of the seM semi-nested PCR. To evaluate the species specificity of the seM-LAMP, we tested 100 S. equi and 189 non-S. equi strains. Significant amplification of the DNA originating from S. equi was observed within 60 min incubation, but no amplification of non-S. equi DNA occurred. The results were identical to those of seM semi-nested PCR. To investigate the clinical usefulness of the methods, the seM-LAMP and the seM semi-nested PCR were used to screen 590 nasal swabs obtained during an outbreak of strangles. Both methods showed that 79 and 511 swabs were S. equi positive and negative, respectively, and the results were identical to those of the culture examination. These results indicate that the seM-LAMP is potentially useful for the reliable routine diagnosis of Streptococcus equi subsp. equi infections.

  1. Multiplexed real-time PCR amplification of tlh, tdh and trh genes in Vibrio parahaemolyticus and its rapid detection in shellfish and Gulf of Mexico water. (United States)

    Rizvi, Amy V; Bej, Asim K


    In this study, we have developed a SYBR Green I-based real-time multiplexed PCR assay for the detection of Vibrio parahaemolyticus in Gulf of Mexico water (gulf water), artificially seeded and natural oysters targeting three hemolysin genes, tlh, tdh and trh in a single reaction. Post-amplification melt-temperature analysis confirmed the amplification of all three targeted genes with high specificity. The detection sensitivity was 10 cfu (initial inoculum) in 1 ml of gulf water or oyster tissue homogenate, following 5 h enrichment. The results showed 58% of the oysters to be positive for tlh, indicating the presence of V. parahaemolyticus; of which 21% were positive for tdh; and 0.7% for trh, signifying the presence of pathogenic strains. The C(t) values showed that oyster tissue matrix had some level of inhibition, whereas the gulf water had negligible effect on PCR amplification. The assay was rapid (approximately 8 h), specific and sensitive, meeting the ISSC guidelines. Rapid detection using real-time multiplexed PCR will help reduce V. parahaemolyticus-related disease outbreaks, thereby increasing consumer confidence and economic success of the seafood industry.

  2. Detection of virus mRNA within infected host cells using an isothermal nucleic acid amplification assay: marine cyanophage gene expression within Synechococcus sp

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    Hall Matthew J


    Full Text Available Abstract Background Signal-Mediated Amplification of RNA Technology (SMART is an isothermal nucleic acid amplification technology, developed for the detection of specific target sequences, either RNA (for expression or DNA. Cyanophages are viruses that infect cyanobacteria. Marine cyanophages are ubiquitous in the surface layers of the ocean where they infect members of the globally important genus Synechococcus. Results Here we report that the SMART assay allowed us to differentiate between infected and non-infected host cultures. Expression of the cyanophage strain S-PM2 portal vertex gene (g20 was detected from infected host Synechococcus sp. WH7803 cells. Using the SMART assay, we demonstrated that g20 mRNA peaked 240 – 360 minutes post-infection, allowing us to characterise this as a mid to late transcript. g20 DNA was also detected, peaking 10 hours post-infection, coinciding with the onset of host lysis. Conclusion The SMART assay is based on isothermal nucleic acid amplification, allowing the detection of specific sequences of DNA or RNA. It was shown to be suitable for differentiating between virus-infected and non-infected host cultures and for the detection of virus gene expression: the first reported use of this technology for such applications.

  3. Development of Multiplex-Mismatch Amplification Mutation-PCR Assay for Simultaneous Detection of Campylobacter jejuni and Mutation in gyrA Gene Related to Fluoroquinolone Resistance. (United States)

    Cui, Mingquan; Wu, Chenbin; Zhang, Peng; Wu, Congming


    Campylobacter jejuni, a foodborne pathogen, is the major cause of enteritis in humans worldwide, however, its increasing resistance to fluoroquinolones reported recently is of a major concern. In the present study, multiplex-mismatch amplification mutation assay-polymerase chain reaction (MMAMA-PCR) was developed for the first time with the aim to quickly identify C. jejuni and to detect the single nucleotide mutation (C-257 to T) frequently observed in gyrA gene, associated with the acquisition of resistance to fluoroquinolones. In this assay, mismatch amplification mutation primers for the detection of gyrA mutation in C. jejuni were coupled with primers for the hip gene encoding for hippuricase and 16S rRNA gene of C. jejuni, respectively, in the multiplex PCR assay. The specificity and accuracy of this method were analyzed by the use of 78 C. jejuni strains with previously confirmed resistance phenotypes and the mutation (C-257 to T) in gyrA gene, as well as 107 clinical isolates of various bacterial species, including 29 C. jejuni isolates. This study indicates that MMAMA-PCR is a promising assay for the rapid identification of C. jejuni with a specific mutation in gyrA gene, responsible for the resistance to fluoroquinolones.

  4. Incidence of and factors associated with false positives in laboratory diagnosis of norovirus infection by amplification of the RNA-dependent RNA polymerase gene.

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    Fang-Ru Lin

    Full Text Available BACKGROUND: Conventional reverse transcription-polymerase chain reaction (RT-PCR amplification of the RNA-dependent RNA polymerase (RdRp gene remains a used method for the rapid detection of norovirus (NV in clinical laboratories. The incidence of and factors associated with false positives in this assay have not been previously evaluated. METHODS/PRINCIPAL FINDINGS: After an NV outbreak caused by the GII.4 Sydney strain in 2012, we reanalysed 250 stool samples positive for NV by RdRp gene detection. True positives were confirmed in 154 (61.6% samples by successful amplification and sequencing confirmation of the viral protein 1 gene. Of the remaining 96 samples that underwent RT-PCR for the RdRp gene, 34 samples yielded PCR products of the expected length. However, the sequences of the amplicons belonged to the human genome, with 91-97% matched nucleotide sequences, indicating false positives. Multivariate analysis of the clinical features of the patients identified a positive stool culture for bacteria (adjusted odds ratio [aOR] 9.07, 95% adjusted confidence interval [aCI] 2.17-37.92, P = .003 and the use of parenteral antibiotics (aOR 5.55, 95% aCI 1.21-24.73, P = .027 as significant and independent factors associated with false positives. CONCLUSION: Conventional RT-PCR targeting the RdRp gene of NV can lead to false positives in patients with bacterial enterocolitis by incidental amplification of DNA from a human source.

  5. Limitations to the development of recombinant human embryonic kidney 293E cells using glutamine synthetase-mediated gene amplification: Methionine sulfoximine resistance. (United States)

    Yu, Da Young; Noh, Soo Min; Lee, Gyun Min


    To investigate the feasibility of glutamine synthetase (GS)-mediated gene amplification in HEK293 cells for the high-level stable production of therapeutic proteins, HEK293E cells were transfected by the GS expression vector containing antibody genes and were selected at various methionine sulfoximine (MSX) concentrations in 96-well plates. For a comparison, CHOK1 cells were transfected by the same GS expression vector and selected at various MSX concentrations. Unlike CHOK1 cells, HEK293E cells producing high levels of antibodies were not selected at all. For HEK293E cells, the number of wells with the cell pool did not decrease with an increase in the concentration of MSX up to 500μM MSX. A q-RT-PCR analysis confirmed that the antibody genes in the HEK293E cells, unlike the CHOK1 cells, were not amplified after increasing the MSX concentration. It was found that the GS activity in HEK293E cells was much higher than that in CHOK1 cells (P<0.05). In a glutamine-free medium, the GS activity of HEK293E cells was approximately 4.8 times higher than that in CHOK1 cells. Accordingly, it is inferred that high GS activity of HEK293E cells results in elevated resistance to MSX and therefore hampers GS-mediated gene amplification by MSX. Thus, in order to apply the GS-mediated gene amplification system to HEK293 cells, the endogenous GS expression level in HEK293 cells needs to be minimized by knock-out or down-regulation methods.

  6. Loop-Mediated Isothermal Amplification (LAMP) for Rapid Detection and Quantification of Dehalococcoides Biomarker Genes in Commercial Reductive Dechlorinating Cultures KB-1 and SDC-9. (United States)

    Kanitkar, Yogendra H; Stedtfeld, Robert D; Steffan, Robert J; Hashsham, Syed A; Cupples, Alison M


    Real-time quantitative PCR (qPCR) protocols specific to the reductive dehalogenase (RDase) genes vcrA, bvcA, and tceA are commonly used to quantify Dehalococcoides spp. in groundwater from chlorinated solvent-contaminated sites. In this study, loop-mediated isothermal amplification (LAMP) was developed as an alternative approach for the quantification of these genes. LAMP does not require a real-time thermal cycler (i.e., amplification is isothermal), allowing the method to be performed using less-expensive and potentially field-deployable detection devices. Six LAMP primers were designed for each of three RDase genes (vcrA, bvcA, and tceA) using Primer Explorer V4. The LAMP assays were compared to conventional qPCR approaches using plasmid standards, two commercially available bioaugmentation cultures, KB-1 and SDC-9 (both contain Dehalococcoides species). DNA was extracted over a growth cycle from KB-1 and SDC-9 cultures amended with trichloroethene and vinyl chloride, respectively. All three genes were quantified for KB-1, whereas only vcrA was quantified for SDC-9. A comparison of LAMP and qPCR using standard plasmids indicated that quantification results were similar over a large range of gene concentrations. In addition, the quantitative increase in gene concentrations over one growth cycle of KB-1 and SDC-9 using LAMP was comparable to that of qPCR. The developed LAMP assays for vcrA and tceA genes were validated by comparing quantification on the Gene-Z handheld platform and a real-time thermal cycler using DNA isolated from eight groundwater samples obtained from an SDC-9-bioaugmented site (Tulsa, OK). These assays will be particularly useful at sites subject to bioaugmentation with these two commonly used Dehalococcoides species-containing cultures.

  7. Analyses of numerical aberrations of chromosome 17 and tp53 gene deletion/amplification in human oral squamous cell carcinoma using dual-color fluorescence in situ hybridization

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    Noemi MESZAROS


    Full Text Available In Romania, oral and facial cancers represent approximately 5% of all cancers. Deactivation and unregulated expression of oncogenes and tumor suppressor genes may be involved in the pathogenesis of oral squamous cell carcinoma. The genomic change results in numerical and structural chromosomal alterations, particularly in chromosomes 3, 9, 11 and 17. The aim of our study was to identify numerical aberrations of chromosome 17, deletion or amplification of p53 gene and to reveal correlations between abnormalities of chromosome 17and of p53 gene with TNM status and grading in 15 subjects with oral squamous cell carcinoma. 80 % of cases presented chromosome 17 polysomy and only 20% of cases had chromosome 17 monosomy. 46.6 % of samples revealed p53 gene amplification and 33.3 % of them p53 deletion. Polysomy of chromosome 17 was also detected in tumor-adjacent epithelia. The degree of the cytogenetic abnormality did not correlate with the stage of the disease, the histological differentiation of oral squamous cell carcinoma and lymph node metastasis. Molecular cytogenetic techniques, using fluorescence in situ hybridization with chromosome-specific DNA probes, facilitate the confirmation of presumed chromosomal aberrations with high sensitivity and specificity.

  8. Amplification of HER2 and TOP2A and deletion of TOP2A genes in breast cancer investigated by new FISH probes

    DEFF Research Database (Denmark)

    Olsen, Karen E; Knudsen, Helle; Rasmussen, Birgitte


    New strategies for improving treatment of patients with breast carcinoma have focused on the HER2 oncoprotein with regard to response to traditional therapy regimes and the effect of a new drug specifically directed against the protein. Furthermore, the status of the topoisomerase IIalpha (TOP2A......). In addition, the HercepTest was evaluated as a screening tool for choosing cases for FISH investigation of TOP2A gene aberrations. The HercepTest score 3+ identified HER2 gene amplification in 27 of 30 amplified tumours (sensitivity of 0.90) with a false-negative rate of 0.10 and a false-positive rate of 0.......06. TOP2A gene amplification or deletion was found in 20 cases. Sixteen (80%) of these carcinomas were in the HercepTest 3+ group, but four tumours had alterations in the TOP2A gene with normal HER2 status. Traditionally, in the FISH technique the result has been based on counting 60 cells. However, we...

  9. A novel gene trap method using terminator-REMI and 3' rapid amplification of cDNA ends (RACE) in Dictyostelium. (United States)

    Takeda, Kosuke; Saito, Tamao; Tanaka, Tomoyuki; Morio, Takahiro; Maeda, Mineko; Tanaka, Yoshimasa; Ochiai, Hiroshi


    We describe a novel restriction enzyme-mediated integration (REMI) method for gene trapping in Dictyostelium based on the use of a terminator-deficient vector. The vector has a blasticidin deaminase (bsr) gene as a selectable marker but lacks a terminator containing a poly(A) addition signal (AATAAA). Thus, the vector was expected to integrate into the coding region of a gene to create a fusion transcript flanked by the 3' proximal region of the trapped gene. The trapped gene can be identified by simply amplifying the fusion transcript by 3' rapid amplification of cDNA ends (3'-RACE). In the analysis of 35 integration events into known genes, the vectors were found to be integrated 20 times in close proximity to the 3' ends of the genes and in the direction of transcription. This strictly localized insertion seemed to be mediated by negative selection via the surveillance system referred to nonsense-mediated mRNA decay. In contrast, in 15 events the vector integrated in the opposite direction to transcription and at random positions throughout the coding sequence. Analysis of the trapped 3' sequences showed that the transcription of the fusion gene terminated prematurely without the apparent use of an endogenous terminator; nevertheless the transcript did exhibit a poly(A) tail. Based on these results, we designated the method terminator-REMI. Using this method, we have generated a library of tagged Dictyostelium clones from which we have thus far isolated 242 developmental mutants.

  10. Determination of the relative expression levels of rubisco small subunit genes in Arabidopsis by rapid amplification of cDNA ends. (United States)

    Yoon, M; Putterill, J J; Ross, G S; Laing, W A


    Multigene families are common in higher organisms. However, due to the close similarities between members, it is often difficult to assess the individual contribution of each gene to the overall expression of the family. In Arabidopsis thaliana, there are four genes encoding the small subunits (SSU) of ribulose-1.5-bisphosphate carboxylase oxygenase (rubisco) whose nucleotide sequences are up to 98.4% identical. In order to overcome the technical limitations associated with gene-specific probes (or primers) commonly used in existing methods, we developed a new gene expression assay based on the RACE (rapid amplification of cDNA ends) technique with a single pair of primers. With this RACE gene expression assay, we were able to determine the relative transcript levels between four Arabidopsis SSU genes. We found that the relative SSU gene expression differed significantly between plants grown at different temperatures. Our observation raises the possibility that an adaptation of rubisco to the environment may be achieved through the specific synthesis of the SSU proteins, which is determined by the relative expression levels between the SSU genes.

  11. Rapid detection of Candida albicans in clinical samples by DNA amplification of common regions from C. albicans-secreted aspartic proteinase genes. (United States)

    Flahaut, M; Sanglard, D; Monod, M; Bille, J; Rossier, M


    Laboratory diagnosis based on genomic amplification methods such as PCR may provide an alternative and more sensitive method than conventional culture for the early detection of deep-seated candidiasis, an increasing cause of morbidity and mortality among immunocompromised patients. A novel method of DNA extraction from clinical samples based on treatment with proteinase K and isolation of DNA on a silica membrane was developed. The targets used for DNA amplification were the Candida albicans-secreted aspartic proteinase (SAP) genes, a multiple-gene family of at least seven members in C. albicans. A single pair of primers was designed in order to detect six of these SAP genes and, subsequently, to increase the sensitivity of the test. Detection of the PCR product by enzyme-linked immunosorbent assay was found to be as sensitive as Southern blotting with an SAP-labeled probe. The sensitivity of the assay was 1 cell/ml from serially diluted Candida cultures and 1 to 4 cells/ml from seeded blood specimens. The sensitivity and specificity of the present assay were tested in a retrospective study performed blindly with 156 clinical samples and were 100 and 98%, respectively, compared with the results of culture. For the subset of blood culture samples (n = 124), the sensitivity and the specificity were 100%. The two false-positive PCR samples came from patients treated with azole antifungal agents, indicating that PCR was probably able to detect damaged organisms that could not be recovered by culture.

  12. Identification of goose (Anser anser) and mule duck (Anasplatyrhynchos x Cairina moschata) foie gras by multiplex polymerase chain reaction amplification of the 5S RDNA gene. (United States)

    Rodríguez, M A; García, T; González, I; Asensio, L; Fernández, A; Lobo, E; Hernández, P E; Martín, R


    Polymerase chain reaction (PCR) amplification of the nuclear 5S rDNA gene has been used for the identification of goose and mule duck foie gras. Two species-specific reverse primers were designed and used in a multiplex reaction, together with a forward universal primer, to amplify specific fragments of the 5S rDNA in each species. The different sizes of the species-specific amplicons, separated by agarose gel electrophoresis, allowed clear identification of goose and mule duck foie gras samples. This genetic marker can be useful for detecting fraudulent substitution of the duck liver for the more expensive goose liver.

  13. DNA Extraction from Formalin-fixed and Paraffin-embedded Tissues by Triton X-100 for Effective Amplification of EGFR Gene by Polymerase Chain Reaction

    Institute of Scientific and Technical Information of China (English)

    WANG Xiao-feng; DU Zhen-wu; WU Mei; ZHANG Yu-cheng; JIANG Yang; ZHANG Gui-zhen


    For first-line non-small-cell lung cancer(NSCLC) therapy,detecting mutation status of the epidermal growth factor receptor(EGFR) gene constitutes a prudent test to identify patients who are most likely to benefit from EGFR-tyrosine kinase inhibitor(TKI) therapy.Now,the material for detecting EGFR gene mutation status mainly comes from formalin-fixed and paraffin-embedded(FFPE) tissues.DNA extraction from FFPE and the amplification of EGFR gene by polymerase chain reaction(PCR) are two key steps for detecting EGFR gene mutation.We showed a simple method of DNA extraction from FFPE tissues for the effective amplification of EGFR gene.Extracting DNA from the FFPE tissues of NSCLC patients with 1% Triton X-100(pH=10.0) was performed by heating at 95 C for 30min.Meanwhile,a commercial kit was used to extract DNA from the same FFPE tissues of NSCLC patients for comparison.DNA extracted products were used as template for amplifying the exons 18,.19,20 and 21 of EGFR by PCR for different amplified fragments.Results show that DNA fragment size extracted from FFPE tissues with 1%Triton X was about 250-500 base pairs(bp).However,DNA fragment size extracted from FFPE tissues via commercial kit was about from several hundreds to several thousands bp.The DNA yield extracted from FFPE tissues with 1% Triton X was larger than that via commercial kit.For about 500 bp fragment,four exons of EGFR could not be amplified more efficiently from extracted DNA with 1% Triton X than with commercial kit.However,for about 200 bp fragment.This simple and non-laborious protocol could successfully be used to extract DNA from FFPE tissue for the amplification of EGFR gene by PCR,further screening of EGFR gene mutation and facilitating the molecular analysis of a large number of FFPE tissues from NSCLC patients.

  14. Prognostic significance of ESR1 gene amplification, mRNA/protein expression and functional profiles in high-risk early breast cancer: a translational study of the Hellenic Cooperative Oncology Group (HeCOG.

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    George Pentheroudakis

    Full Text Available BACKGROUND: Discrepant data have been published on the incidence and prognostic significance of ESR1 gene amplification in early breast cancer. PATIENTS AND METHODS: Formalin-fixed paraffin-embedded tumor blocks were collected from women with early breast cancer participating in two HeCOG adjuvant trials. Messenger RNA was studied by quantitative PCR, ER protein expression was centrally assessed using immunohistochemistry (IHC and ESR1 gene copy number by dual fluorescent in situ hybridization probes. RESULTS: In a total of 1010 women with resected node-positive early breast adenocarcinoma, the tumoral ESR1/CEP6 gene ratio was suggestive of deletion in 159 (15.7%, gene gain in 551 (54.6% and amplification in 42 cases (4.2%, with only 30 tumors (3% harboring five or more ESR1 copies. Gene copy number ratio showed a significant, though weak correlation to mRNA and protein expression (Spearman's Rho <0.23, p = 0.01. ESR1 clusters were observed in 9.5% (57 gain, 38 amplification of cases. In contrast to mRNA and protein expression, which were favorable prognosticators, gene copy number changes did not obtain prognostic significance. When ESR1/CEP6 gene ratio was combined with function (as defined by ER protein and mRNA expression in a molecular classifier, the Gene Functional profile, it was functional status that impacted on prognosis. In univariate analysis, patients with functional tumors (positive ER protein expression and gene ratio normal or gain/amplification fared better than those with non-functional tumors with ESR1 gain (HR for relapse or death 0.49-0.64, p = 0.003. Significant interactions were observed between gene gain/amplification and paclitaxel therapy (trend for DFS benefit from paclitaxel only in patients with ESR1 gain/amplification, p = 0.066 and Gene Functional profile with HER2 amplification (Gene Functional profile prognostic only in HER2-normal cases, p = 0.029. CONCLUSIONS: ESR1 gene deletion and

  15. Circular rapid amplification of cDNA ends for high-throughput extension cloning of partial genes. (United States)

    Fu, Glenn K; Wang, Jonathan T; Yang, Junming; Au-Young, Janice; Stuve, Laura L


    The rapid amplification of cDNA ends (RACE) procedure is a widely used PCR-based method to clone the cDNA ends of mRNA transcripts. Current RACE methods often produce a high background of nonspecific PCR products, which can exclude the identification of the target cDNA of interest. We describe here an improved RACE procedure using circular cDNA templates and demonstrate the successful extension cloning of 4406 cDNAs.

  16. Validation of whole genome amplification for analysis of the p53 tumor suppressor gene in limited amounts of tumor samples. (United States)

    Hasmats, Johanna; Green, Henrik; Solnestam, Beata Werne; Zajac, Pawel; Huss, Mikael; Orear, Cedric; Validire, Pierre; Bjursell, Magnus; Lundeberg, Joakim


    Personalized cancer treatment requires molecular characterization of individual tumor biopsies. These samples are frequently only available in limited quantities hampering genomic analysis. Several whole genome amplification (WGA) protocols have been developed with reported varying representation of genomic regions post amplification. In this study we investigate region dropout using a φ29 polymerase based WGA approach. DNA from 123 lung cancers specimens and corresponding normal tissue were used and evaluated by Sanger sequencing of the p53 exons 5-8. To enable comparative analysis of this scarce material, WGA samples were compared with unamplified material using a pooling strategy of the 123 samples. In addition, a more detailed analysis of exon 7 amplicons were performed followed by extensive cloning and Sanger sequencing. Interestingly, by comparing data from the pooled samples to the individually sequenced exon 7, we demonstrate that mutations are more easily recovered from WGA pools and this was also supported by simulations of different sequencing coverage. Overall this data indicate a limited random loss of genomic regions supporting the use of whole genome amplification for genomic analysis.

  17. The potential role of incorporating real-time PCR and DNA sequencing for amplification and detection of 16S rRNA gene signatures in neonatal sepsis. (United States)

    Midan, Dina A; Abo El Fotoh, Wafaa Moustafa M; El Shalakany, Abeer H


    This study aimed to explore whether 16S rRNA gene amplification by real time PCR and sequencing could serve as genetic-based methods in rapid and accurate diagnosis of neonatal sepsis. This case control study was conducted on 40 neonates suffering from sepsis like manifestations recruited from the neonatal intensive care unit of Menoufia university hospital over a period of 6 months. Their blood samples were used for paired analysis of bacterial growth using BACTEC 9050 instrument and real time PCR assay with subsequent DNA sequencing for bacterial species identification. The detection rate of culture proven sepsis was 70%. By using real time 16S r RNA PCR amplification method, the detection of bacteria was improved to 80%. Real time PCR revealed sensitivity, specificity, positive predictive value and negative predictive value of [100%, 66.7%, 87.5% and 100%] respectively. Compared to culture, the 16S rRNA real time PCR demonstrated a high negative value for ruling out neonatal sepsis. There was significant statistical difference between the PCR positive and negative cases as regards the hematological sepsis score. The results demonstrated the ability of DNA sequencing to recognize 4 pathogens which were negative by blood culture. The time consumed to detect sepsis using blood culture was up to 5 days while it took up to 16 h only by PCR and sequencing methods. 16S rRNA gene amplification by real time PCR and sequence analysis could be served as ideal and reliable genetic-based methods to diagnose and rule out sepsis with provision of additional data that cannot be obtained by routine laboratory tests with a shorter turnaround time than those with culture-based protocols.

  18. Impact of sample acquisition and linear amplification on gene expression profiling of lung adenocarcinoma: laser capture micro-dissection cell-sampling versus bulk tissue-sampling

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    Wigle Dennis A


    Full Text Available Abstract Background The methods used for sample selection and processing can have a strong influence on the expression values obtained through microarray profiling. Laser capture microdissection (LCM provides higher specificity in the selection of target cells compared to traditional bulk tissue selection methods, but at an increased processing cost. The benefit gained from the higher tissue specificity realized through LCM sampling is evaluated in this study through a comparison of microarray expression profiles obtained from same-samples using bulk and LCM processing. Methods Expression data from ten lung adenocarcinoma samples and six adjacent normal samples were acquired using LCM and bulk sampling methods. Expression values were evaluated for correlation between sample processing methods, as well as for bias introduced by the additional linear amplification required for LCM sample profiling. Results The direct comparison of expression values obtained from the bulk and LCM sampled datasets reveals a large number of probesets with significantly varied expression. Many of these variations were shown to be related to bias arising from the process of linear amplification, which is required for LCM sample preparation. A comparison of differentially expressed genes (cancer vs. normal selected in the bulk and LCM datasets also showed substantial differences. There were more than twice as many down-regulated probesets identified in the LCM data than identified in the bulk data. Controlling for the previously identified amplification bias did not have a substantial impact on the differences identified in the differentially expressed probesets found in the bulk and LCM samples. Conclusion LCM-coupled microarray expression profiling was shown to uniquely identify a large number of differentially expressed probesets not otherwise found using bulk tissue sampling. The information gain realized from the LCM sampling was limited to differential analysis

  19. Fluorescence immunophenotyping and interphase cytogenetics (FICTION) detects BCL6 abnormalities, including gene amplification, in most cases of nodular lymphocyte-predominant Hodgkin lymphoma. (United States)

    Bakhirev, Alexei G; Vasef, Mohammad A; Zhang, Qian-Yun; Reichard, Kaaren K; Czuchlewski, David R


    BCL6 translocations are a frequent finding in B-cell lymphomas of diverse subtypes, including some cases of nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL). However, reliable analysis of BCL6 rearrangements using fluorescence in situ hybridization is difficult in NLPHL because of the relative paucity of neoplastic cells. Combined immunofluorescence microscopy and fluorescence in situ hybridization, or fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms (FICTION), permits targeted analysis of neoplastic cells. To better define the spectrum of BCL6 abnormalities in NLPHL using FICTION analysis. We performed an optimized FICTION analysis of 24 lymph nodes, including 11 NLPHL, 5 follicular hyperplasia with prominent progressive transformation of germinal centers, and 8 follicular hyperplasia without progressive transformation of germinal centers. BCL6 rearrangement was identified in 5 of 11 cases of NLPHL (46%). In addition, BCL6 gene amplification, with large clusters of BCL6 signals in the absence of chromosome 3 aneuploidy, was detected in 3 of 11 cases of NLPHL (27%). One NLPHL showed extra copies of BCL6 present in conjunction with multiple copies of chromosome 3. Altogether, we detected BCL6 abnormalities in 9 of 11 cases of NLPHL (82%). None of the progressive transformation of germinal centers or follicular hyperplasia cases showed BCL6 abnormalities by FICTION. To our knowledge, this is the first report of BCL6 gene amplification in NLPHL. Our optimized protocol for FICTION permits detection of cytogenetic abnormalities in most NLPHL cases and may represent a useful ancillary diagnostic technique.

  20. Gene amplification, ABC transporters and cytochrome P450s: unraveling the molecular basis of pyrethroid resistance in the dengue vector, Aedes aegypti.

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    Vassiliki Bariami

    Full Text Available BACKGROUND: Pyrethroid insecticides are widely utilized in dengue control. However, the major vector, Aedes aegypti, is becoming increasingly resistant to these insecticides and this is impacting on the efficacy of control measures. The near complete transcriptome of two pyrethroid resistant populations from the Caribbean was examined to explore the molecular basis of this resistance. PRINCIPAL FINDINGS: Two previously described target site mutations, 1016I and 1534C were detected in pyrethroid resistant populations from Grand Cayman and Cuba. In addition between two and five per cent of the Ae. aegypti transcriptome was differentially expressed in the resistant populations compared to a laboratory susceptible population. Approximately 20 per cent of the genes over-expressed in resistant mosquitoes were up-regulated in both Caribbean populations (107 genes. Genes with putative monooxygenase activity were significantly over represented in the up-regulated subset, including five CYP9 P450 genes. Quantitative PCR was used to confirm the higher transcript levels of multiple cytochrome P450 genes from the CYP9J family and an ATP binding cassette transporter. Over expression of two genes, CYP9J26 and ABCB4, is due, at least in part, to gene amplification. SIGNIFICANCE: These results, and those from other studies, strongly suggest that increases in the amount of the CYP9J cytochrome P450s are an important mechanism of pyrethroid resistance in Ae. aegypti. The genetic redundancy resulting from the expansion of this gene family makes it unlikely that a single gene or mutation responsible for pyrethroid resistance will be identified in this mosquito species. However, the results from this study do pave the way for the development of new pyrethroid synergists and improved resistance diagnostics. The role of copy number polymorphisms in detoxification and transporter genes in providing protection against insecticide exposure requires further investigation.

  1. 番茄抗花叶病毒基因类似序列扩增研究%Studies on Gene Similar Sequence Amplification of Tomato Mosaic Virus

    Institute of Scientific and Technical Information of China (English)



    [目的]研究番茄抗花叶病毒基因类似序列扩增.[方法]以选育和生产上广泛使用的20个番茄品种为试材,对其进行了田间和实验室抗花叶病毒病(ToMV)筛选、基因类似序列扩增、RAPD聚类分析及特异引物PCR扩增.[结果]美味樱桃番茄、中杂9号、旱红宝、W2624、OH-2-2-11、黄圣果和美国番茄对ToMV具有较强的抗性;根据聚类分析,受试品种可分为3类,从中选出9个抗性和非抗性品种进行抗ToMV,基因类似序列分析,其中非抗性和购买无抗性番茄品种未扩增出任何谱带,而抗性品种扩增出约300 bp的谱带,初步认为该谱带为抗ToMV类似序列基因.[结论]为培育抗花叶病毒病的番茄品种提供了理论依据.%[ Objective ] The aim was to study the gene similar sequence amplification of tomato mosaic virus. [ Method ] Taking 20 tomato varieties cultivating in breeding and production widely as tested materials,tomato mosaic virus (ToMV) was screened in field and laboratory,furthennore RAPD cluster analysis and gene amplification experiment were carried out,and specific primers of PCR amplification was carried outfinally. [ Result] Dilicioas cherry tomato,China tomato hybrid 9 ,early red jewel tomato,W262 -4,Oh -2 -2 - 11 ,yellow holy tomato and American tomato had stronger resistance to ToMV. According to the result of RAPD cluster analysis,the 20 tomato cultivars could be divided into three types, from which 9 varieties inclnding resistant and nonresistant varieties were selected amplify gene similar sequence. None band was found from those nonresistant varieties,while a band of about 300 bp was found from those resistant varieties,so the band was regarded as anti-ToMV gene similar sequence. [ Conclusion ] The reseach provides theoretical basis for breeding the tomato varieties which are resistant to ToMV.

  2. Multiplex Ligation-Dependent Probe Amplification Analysis of GATA4 Gene Copy Number Variations in Patients with Isolated Congenital Heart Disease

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    Valentina Guida


    Full Text Available GATA4 mutations are found in patients with different isolated congenital heart defects (CHDs, mostly cardiac septal defects and tetralogy of Fallot. In addition, GATA4 is supposed to be the responsible gene for the CHDs in the chromosomal 8p23 deletion syndrome, which is recognized as a malformation syndrome with clinical symptoms of facial anomalies, microcephaly, mental retardation, and congenital heart defects. Thus far, no study has been carried out to investigate the role of GATA4 copy number variations (CNVs in non-syndromic CHDs. To explore the possible occurrence of GATA4 gene CNVs in isolated CHDs, we analyzed by multiplex ligation-dependent probe amplification (MLPA a cohort of 161 non-syndromic patients with cardiac anomalies previously associated with GATA4 gene mutations. The patients were mutation-negative for GATA4, NKX2.5, and FOG2 genes after screening with denaturing high performance liquid chromatography. MLPA analysis revealed that normalized MLPA signals were all found within the normal range values for all exons in all patients, excluding a major contribution of GATA4 gene CNVs in CHD pathogenesis.

  3. Multiplex PCR amplification assay for the detection of blaSHV, blaTEM and blaCTX-M genes in Enterobacteriaceae. (United States)

    Monstein, H-J; Ostholm-Balkhed, A; Nilsson, M V; Nilsson, M; Dornbusch, K; Nilsson, L E


    Extended-spectrum beta-lactamases (ESBLs) are often mediated by (bla-)SHV, (bla)TEM and (bla)CTX-M genes in Enterobacteriaceae and other Gram-negative bacteria. Numerous molecular typing methods, including PCR-based assays, have been developed for their identification. To reduce the number of PCR amplifications needed we have developed a multiplex PCR assay which detects and discriminates between (bla-)SHV, (bla)TEM and (bla)CTX-M PCR amplicons of 747, 445 and 593 bp, respectively. This multiplex PCR assay allowed the identification of (bla-)SHV, (bla)TEM and (bla)CTX-M genes in a series of clinical isolates of Enterobacteriaceae with previously characterised ESBL phenotype. The presence of (bla)SHV, (bla)TEM and (bla)CTX-M genes was confirmed by partial DNA sequence analysis. Apparently, the universal well-established CTX-M primer pair used here to reveal plasmid-encoded (bla)CTX-M genes would also amplify the chromosomally located K-1 enzyme gene in all Klebsiella oxytoca strains included in the study.

  4. Experimental evolution of resistance to artemisinin combination therapy results in amplification of the mdr1 gene in a rodent malaria parasite.

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    Louise A Rodrigues

    Full Text Available BACKGROUND: Lacking suitable alternatives, the control of malaria increasingly depends upon Artemisinin Combination Treatments (ACT: resistance to these drugs would therefore be disastrous. For ACTs, the biology of resistance to the individual components has been investigated, but experimentally induced resistance to component drugs in combination has not been generated. METHODOLOGY/PRINCIPAL FINDINGS: We have used the rodent malaria parasite Plasmodium chabaudi to select in vivo resistance to the artesunate (ATN+mefloquine (MF version of ACT, through prolonged exposure of parasites to both drugs over many generations. The selection procedure was carried out over twenty-seven consecutive sub-inoculations under increasing ATN+MF doses, after which a genetically stable resistant parasite, AS-ATNMF1, was cloned. AS-ATNMF1 showed increased resistance to ATN+MF treatment and to artesunate or mefloquine administered separately. Investigation of candidate genes revealed an mdr1 duplication in the resistant parasites and increased levels of mdr1 transcripts and protein. There were no point mutations in the atpase6 or ubp1genes. CONCLUSION: Resistance to ACTs may evolve even when the two drugs within the combination are taken simultaneously and amplification of the mdr1 gene may contribute to this phenotype. However, we propose that other gene(s, as yet unidentified, are likely to be involved.

  5. Deletion/duplication mutation screening of TP53 gene in patients with transitional cell carcinoma of urinary bladder using multiplex ligation-dependent probe amplification. (United States)

    Bazrafshani, Mohammad Reza R; Nowshadi, Pouriaali A; Shirian, Sadegh; Daneshbod, Yahya; Nabipour, Fatemeh; Mokhtari, Maral; Hosseini, Fatemehsadat; Dehghan, Somayeh; Saeedzadeh, Abolfazl; Mosayebi, Ziba


    Bladder cancer is a molecular disease driven by the accumulation of genetic, epigenetic, and environmental factors. The aim of this study was to detect the deletions/duplication mutations in TP53 gene exons using multiplex ligation-dependent probe amplification (MLPA) method in the patients with transitional cell carcinoma (TCC). The achieved formalin-fixed paraffin-embedded tissues from 60 patients with TCC of bladder were screened for exonal deletions or duplications of every 12 TP53 gene exons using MLPA. The pathological sections were examined by three pathologists and categorized according to the WHO scoring guideline as 18 (30%) grade I, 22 (37%) grade II, 13 (22%) grade III, and 7 (11%) grade IV cases of TCC. None mutation changes of TP53 gene were detected in 24 (40%) of the patients. Furthermore, mutation changes including, 15 (25%) deletion, 17 (28%) duplication, and 4 (7%) both deletion and duplication cases were observed among 60 samples. From 12 exons of TP53 gene, exon 1 was more subjected to exonal deletion. Deletion of exon 1 of TP53 gene has occurred in 11 (35.4%) patients with TCC. In general, most mutations of TP53, either deletion or duplication, were found in exon 1, which was statistically significant. In addition, no relation between the TCC tumor grade and any type of mutation were observed in this research. MLPA is a simple and efficient method to analyze genomic deletions and duplications of all 12 exons of TP53 gene. The finding of this report that most of the mutations of TP53 occur in exon 1 is in contrast to that of the other reports suggesting that exons 5-8 are the most (frequently) mutated exons of TP53 gene. The mutations of exon 1 of TP53 gene may play an important role in the tumorogenesis of TCC.

  6. 肿瘤细胞回文序列介导的基因扩增机制研究进展%Research progress on the mechanism of gene amplification mediated by palindromes in tumor genome

    Institute of Scientific and Technical Information of China (English)

    刘佳玮; 徐晖; 张海员; 白静; 傅松滨; 周春水


    基因组不稳定是肿瘤细胞的特征性标志,基因扩增是一种特殊的基因组不稳定性的表现形式,其在激活原癌基因引发癌症的过程中起着重要的作用.基因扩增将导致细胞加速生长增殖或耐药生长等表型.人们已经广泛注意到其在癌症的诊断和治疗等方面的重要临床意义.但是,人们对基因扩增的分子机制的认识仍然十分有限,本文综述了回文序列介导的基因扩增的分子机制,提出在DNA断点处以无模板形式、新合成小回文序列可能是一部分肿瘤细胞中基因扩增的关键机制,为进一步探索基因扩增的分子机制提供理论参考.%Gene amplification is a common chromosomal aberration which is often the result of tumor genome instability,and plays a crucial role in the process of activating oncogenes and leading to cancers.As gene amplification often is evident in advanced stages of cancer,its clinical importance in diagnosis and treatment has been popularly recognized.However,the underlying mechanisms governing gene amplification are still not fully understood.Herein,we reviewed one of the well-defined gene amplification mechanisms:DNA double-strand breaks-triggered,palindromes or short DNA inverted repeatsmediated gene amplification model,which was established and validated in a variety of research organisms including T.thermophila,fission yeast,budding yeast and rodent cells.Based on the recent published reports,we put forward a new model for the palindromes mediated gene amplification mechanism,that is de novo synthesis of small palindromic sequences (usually several hundred base pairs in length or shorter) at the DNA breakpoints in a template-free manner is the key determinant for gene amplification in certain tumor genomes.Elucidating the potential mechanism and involved enzymes for the regulation and creation of novel palindromic sequences should shed more light onto the palindromes mediated gene amplification and the

  7. Discordant cellular response to pre-surgical letrozole in bilateral synchronous ER+ breast cancers with a KRAS mutation or FGFR1 gene amplification (United States)

    Balko, Justin M.; Mayer, Ingrid A.; Sanders, Melinda E.; Miller, Todd W.; Kuba, Maria G.; Meszoely, Ingrid M.; Wagle, Nikhil; Garraway, Levi A.; Arteaga, Carlos L.


    We describe herein a patient presenting with bilateral ER+ breast tumors who was enrolled in a clinical trial exploring molecular aberrations associated with hormone-refractory tumor cell proliferation. Short-term (2-week) hormonal therapy with the aromatase inhibitor letrozole substantially reduced proliferation as measured by Ki67 immunohistochemistry in one tumor, while the second was essentially unchanged. Extensive molecular and genetic workup of the two tumors yielded divergent lesions in the two tumors: an activating KRAS mutation in the responsive tumor, and an amplification of the FGFR1 locus in the treatment-refractory tumor. These findings provide an insight to possible mechanisms of resistance to antiestrogen therapy in ER+ breast cancers. First, they illustrate the necessity of clinically approved assays to identify FGFR1 gene amplification, which occur in ~5% of breast tumors and have been linked to antiestrogen resistance. It is quite possible that the addition of FGFR inhibitors to ER-targeted therapy will yield a superior antitumor effect and improved patient outcome. Second, they suggest that the role of activating mutations in RAS, although rare in breast cancer, may need to be explored in the context of ER+ breast tumors. PMID:22879364

  8. Target-induced reconfiguration of DNA probes for recycling amplification and signal-on electrochemical detection of hereditary tyrosinemia type I gene. (United States)

    Dou, Baoting; Yang, Cuiyun; Chai, Yaqin; Yuan, Ruo; Xiang, Yun


    By coupling target DNA-induced reconfiguration of the dsDNA probes with enzyme-assisted target recycling amplification, we describe the development of a signal-on electrochemical sensing approach for sensitive detection of hereditary tyrosinemia type I gene. The dsDNA probes are self-assembled on the sensing electrode, and the addition of the target DNA reconfigures and switches the dsDNA probes into active substrates for exonuclease III, which catalytically digests the probes and leads to cyclic reuse of the target DNA. The target DNA recycling and the removal of one of the ssDNA from the dsDNA probes by exonuclease III result in the formation of many hairpin structures on the sensor surface, which brings the electroactive methylene blue labels into proximity with the electrode and produces a significantly amplified current response for sensitive detection of the target gene down to 0.24 pM. This method is also selective to discriminate single-base mismatch and can be employed to detect the target gene in human serum samples. With the demonstration for the detection of the target gene, we expect the developed method to be a universal sensitive sensing platform for the detection of different nucleic acid sequences.

  9. Amplification of cellular oncogenes in solid tumors

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    Ozkan Bagci


    Full Text Available The term gene amplification refers to an increase in copy number of a gene. Upregulation of gene expression through amplification is a general mechanism to increase gene dosage. Oncogene amplifications have been shown in solid human cancers and they are often associated with progression of cancer. Defining oncogene amplification is useful since it is used as a prognostic marker in clinical oncology nowadays, especially v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (HER2 targeted agents are used in breast cancer patients with high level of HER2 overexpression as a therapeutic approach. However, patients without HER2 overexpression do not appear to benefit from these agents. We concluded that determination of oncogene amplification in solid tumors is an important factor in treatment of human cancers with many unknowns. We have referred to PubMed and some databases to prepare this article.

  10. Real-time PCR based on SYBR-Green I fluorescence: An alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions

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    Puisieux Alain


    Full Text Available Abstract Background Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive. Results We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC in peripheral blood mononuclear cells; the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy. Conclusion Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.

  11. Identification of planorbids from Venezuela by polymerase chain reaction amplification and restriction fragment length polymorphism of internal transcriber spacer of the RNA ribosomal gene

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    Caldeira Roberta L


    Full Text Available Snails of the genus Biomphalaria from Venezuela were subjected to morphological assessment as well as polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP analysis. Morphological identification was carried out by comparison of characters of the shell and the male and female reproductive apparatus. The PCR-RFLP involved amplification of the internal spacer region ITS1 and ITS2 of the RNA ribosomal gene and subsequent digestion of this fragment by the restriction enzymes DdeI, MnlI, HaeIII and MspI. The planorbids were compared with snails of the same species and others reported from Venezuela and present in Brazil, Cuba and Mexico. All the enzymes showed a specific profile for each species, that of DdeI being the clearest. The snails were identified as B. glabrata, B. prona and B. kuhniana.

  12. Gene amplification and immunohistochemical expression of ERBB2 and EGFR in cervical carcinogenesis. Correlation with cell-cycle markers and HPV presence. (United States)

    Conesa-Zamora, Pablo; Torres-Moreno, Daniel; Isaac, María A; Pérez-Guillermo, Miguel


    Although the members of the epidermal growth factor receptor family ERBB2 and EGFR are important therapeutic targets in the treatment of malignant neoplasias, little is known about their role in cervical carcinogenesis. Our objective was to evaluate the dysfunction of ERBB2 and EGFR at the gene copy number and protein expression level in neoplastic lesions of the uterine cervix with the aim of obtaining information about its role in cervical carcinogenesis and their possible use as therapeutic targets in these diseases. We studied gene amplification and protein expression of ERBB2 and EGFR and their relationship with Ki67, p16 and p53 and HPV presence in 22 normal/benign (N/B) cervices, 20 low-grade squamous intraepithelial lesions (LSILs), 70 high-grade SILs (HSILs) and 32 invasive squamous cervical carcinomas (ISCCs). No cases showed selective amplification of ERBB2 or EGFR but corresponding chromosome-specific probes displayed chromosome 17 and 7 polyploidy associated with the grade of the lesion (plesions (N/B plus LSIL 21.4% vs. HSIL plus ISCC 45.5%; p=0.007). No association was observed between EGFR expression and that of cell-cycle markers or HPV presence. Increased copy number of EGFR and ERBB2 is due to polyploidy of 7 and 17 chromosomes, this being a phenomenon associated with lesion severity and with an increase in the expression of cell-cycle markers. EGFR, but not ERBB2, is expressed in precursor lesions of squamous cervical neoplasia and is related to the neoplastic progression but not to proliferation marker expression and therefore ERBB2 and this calls into question the usefulness of ERBB2 as a therapeutic target.

  13. Correlation of HER2 overexpression with gene amplification and its relation to chromosome 17 aneuploidy: a 5-year experience with invasive ductal and lobular carcinomas. (United States)

    Nassar, Aziza; Khoor, Andras; Radhakrishnan, Reshmitha; Radhakrishnan, Anu; Cohen, Cynthia


    The HER2 oncogene shows expression or amplification, or both, in approximately 15% to 20% of breast cancers and has been associated with poor prognosis and a response to trastuzumab therapy. HER2 gene status determines the eligibility of breast cancer patients for trastuzumab therapy and a large fraction (41-56%) of these patients respond to targeted therapy. Several studies have related the increased expression of HER2 to an increased copy number of chromosome 17, rather than amplification of the HER2 gene. We compared the results of immunohistochemistry and fluorescence in situ hybridization in both invasive ductal and invasive lobular carcinomas, to determine the frequency of chromosome 17 aneuploidy associated with discordant results. In total, 390 invasive ductal carcinomas and 180 invasive lobular carcinomas diagnosed from January 2000 to December 2005 were included in the study only if results were available for immunohistochemistry (HercepTest; DAKO, Carpinteria, California) and fluorescence in situ hybridization (PathVysion HER2 DNA Probe Kit; Abbott Laboratories, Des Plaines, Illinois). Tumors classified as invasive ductal carcinomas were graded according to the Bloom-Richardson grading system. Correlation between the results of immunohistochemistry and fluorescence in situ hybridization was performed for all categories. Among invasive ductal carcinomas, 29% (115/390) showed chromosome 17 aneuploidy, mostly associated with grade 3/HER2 2+ (45%) or grade 2/HER2 3+ (55%) that were not amplified. Also, 34% (12/35) of invasive lobular carcinomas showed chromosome 17 aneuploidy; approximately one-third of these cases were HER2 2+ (33%) and HER2 3+ (37%) that were not amplified. Discordance between the results of immunohistochemistry and fluorescence in situ hybridization in both ductal and lobular carcinomas is largely associated with chromosome 17 aneuploidy.

  14. Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of the tdh and trh genes of Vibrio parahaemolyticus and related Vibrio species. (United States)

    Yamazaki, Wataru; Kumeda, Yuko; Misawa, Naoaki; Nakaguchi, Yoshitsugu; Nishibuchi, Mitsuaki


    Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are the major virulence determinants of Vibrio parahaemolyticus. TRH is further differentiated into TRH1 and TRH2 on the basis of genetic and phenotypic differences. We developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for sensitive and rapid detection of the tdh, trh1, and trh2 genes of V. parahaemolyticus. The LAMP assay was designed for both combined and individual detection of the tdh, trh1, and trh2 genes and combined detection of the trh1 and trh2 genes. Our results showed that it gave the same results as DNA probes and conventional PCR assays for 125 strains of V. parahaemolyticus, 3 strains of Grimontia hollisae, and 2 strains of Vibrio mimicus carrying the tdh, trh1, and trh2 genes in various combinations. No LAMP products were detected for any of the 20 bacterial strains lacking the tdh, trh1, and trh2 genes. The sensitivities of the LAMP assay for detection of tdh-, trh1-, and trh2-carrying V. parahaemolyticus strains in spiked shrimp samples were 0.8, 21.3, and 5.0 CFU per LAMP reaction tube, respectively. Starting with DNA extraction from a single colony and from spiked shrimp samples, the LAMP assay required only 27 to 60 min and less than 80 min, respectively. This is the first report of a rapid and specific LAMP assay for detection and differentiation of the tdh, trh1, and trh2 genes of V. parahaemolyticus and related Vibrio species.

  15. Development and Application of Loop-Mediated Isothermal Amplification Assays for Rapid Visual Detection of cry2Ab and cry3A Genes in Genetically-Modified Crops

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    Feiwu Li


    Full Text Available The cry2Ab and cry3A genes are two of the most important insect-resistant exogenous genes and had been widely used in genetically-modified crops. To develop more effective alternatives for the quick identification of genetically-modified organisms (GMOs containing these genes, a rapid and visual loop-mediated isothermal amplification (LAMP method to detect the cry2Ab and cry3A genes is described in this study. The LAMP assay can be finished within 60 min at an isothermal condition of 63 °C. The derived LAMP products can be obtained by a real-time turbidimeter via monitoring the white turbidity or directly observed by the naked eye through adding SYBR Green I dye. The specificity of the LAMP assay was determined by analyzing thirteen insect-resistant genetically-modified (GM crop events with different Bt genes. Furthermore, the sensitivity of the LAMP assay was evaluated by diluting the template genomic DNA. Results showed that the limit of detection of the established LAMP assays was approximately five copies of haploid genomic DNA, about five-fold greater than that of conventional PCR assays. All of the results indicated that this established rapid and visual LAMP assay was quick, accurate and cost effective, with high specificity and sensitivity. In addition, this method does not need specific expensive instruments or facilities, which can provide a simpler and quicker approach to detecting the cry2Ab and cry3A genes in GM crops, especially for on-site, large-scale test purposes in the field.

  16. An improved method for detection of Edwardsiella tarda by loop-mediated isothermal amplification by targeting the EsrB gene

    Institute of Scientific and Technical Information of China (English)

    XIE Guosi; ZHANG Qingli; HAN Nana; SHI Chengyin; WANG Xiuhua; LIU Qinghui; HUANG Jie


    Edwardsiella tarda is a major pathogen in aquatic environments that can cause heavy economic losses.An improved method for quick and accurate detection of E.tarda by loop-mediated isothermal amplification (LAMP) with two additional loop primers was developed by targeting the EsrB gene (EsrBLAMP).In this method,the Mg2+ concentration,reaction temperature,and reaction time were optimized to 8 mmol/L,61℃,and 40 min,respectively.The detection limit with the EsrB gene was as low as 10 copies,which is 100 times more sensitive than that of conventional polymerase chain reaction (PCR).The EsrB-LAMP assay was shown more sensitive and rapid than previously reported LAMP assays targeting the hemolysin gene (hemolysin-LAMP) for detection ofE.tarda.The EsrB-LAMP was also highly specific to E.tarda and had no cross-reaction with 13 other strains of bacteria.The assay can be carried out in a simple heating device and the EsrB-LAMP products can be visually detected by adding fluorescent dye to the reaction mixture.Taken together,the improved EsrB-LAMP diagnostic protocol has the potential for detection ofE.tarda from indoor and outdoor samples.

  17. Evaluation of multiplex ligation-dependent probe amplification analysis versus multiplex polymerase chain reaction assays in the detection of dystrophin gene rearrangements in an Iranian population subset

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    Nayereh Nouri


    Full Text Available Background: The Duchenne muscular dystrophy (DMD gene is located in the short arm of the X chromosome (Xp21. It spans 2.4 Mb of the human genomic DNA and is composed of 79 exons. Mutations in the Dystrophin gene result in DMD and Becker muscular dystrophy. In this study, the efficiency of multiplex ligation-dependent probe amplification (MLPA over multiplex polymerase chain reaction (PCR assays in an Iranian population was investigated. Materials and Methods: Multiplex PCR assays and MLPA analysis were carried out in 74 patients affected with DMD. Results: Multiplex PCR detected deletions in 51% of the patients with DMD. MLPA analysis could determine all the deletions detected by the multiplex PCR. Additionally, MLPA was able to identify one more deletion and duplication in patients without detectable mutations by multiplex PCR. Moreover, MLPA precisely determined the exact size of the deletions. Conclusion: Although MLPA analysis is more sensitive for detection of deletions and duplications in the dystrophin gene, multiplex PCR might be used for the initial analysis of the boys affected with DMD in the Iranian population as it was able to detect 95% of the rearrangements in patients with DMD.

  18. A PCR-based sex determination method for possible application in caprine gender selection by simultaneous amplification of the Sry and Aml-X genes. (United States)

    Phua, Alice Choon Yen; Abdullah, Ramli Bin; Mohamed, Zulqarnain


    Sex determination of livestock is performed to achieve the objectives of livestock breeding programmes. Techniques for sex determination have evolved from karyotyping to detecting Y-specific antigens and recently to the polymerase chain reaction (PCR), which appears to be the most sensitive, accurate, rapid and reliable method to date. In this study, a PCR-based sex determination method for potential application in goat breeding programmes was developed. Primers were designed to amplify a portion of the X amelogenin gene (Aml-X) on the X chromosome to give a 300 bp product and Sry gene on the Y chromosome to give a 116 bp product. PCR optimization was performed using DNA template extracted from a whole blood sample of Jermasia goats (German Fawn x Katjang) of both sexes. It was possible to identify the sex chromosomes by amplifying both male- and female-specific genes simultaneously in a duplex reaction with males yielding two bands and females yielding one band. The Aml-X primer set, which served as an internal control primer, did not interfere with amplification of the Y-specific sequence even when a low amount of DNA (1 ng) was used. The duplex reaction subjected to a blind test showed 100% (14/14) concordance, proving its accuracy and reliability. The primer sets used were found to be highly specific and were suitable for gender selection of goats.

  19. Rapid, Sensitive Detection of Bartonella quintana by Loop-Mediated Isothermal Amplification of the groEL Gene (United States)

    Hu, Shoukui; Niu, Lina; Luo, Lijuan; Song, Xiuping; Sun, Jimin; Liu, Qiyong


    Trench fever, caused by Bartonella quintana, is recognized as a re-emerging and neglected disease. Rapid and sensitive detection approaches are urgently required to monitor and help control B. quintana infections. Here, loop-mediated isothermal amplification (LAMP), which amplifies target DNA at a fixed temperature with high sensitivity, specificity and rapidity, was employed to detect B. quintana. Thirty-six strains, including 10 B. quintana, 13 other Bartonella spp., and 13 other common pathogens, were applied to verify and evaluate the LAMP assay. The specificity of the LAMP assay was 100%, and the limit of detection was 125 fg/reaction. The LAMP assay was compared with qPCR in the examination of 100 rhesus and 20 rhesus-feeder blood samples; the diagnostic accuracy was found to be 100% when LAMP was compared to qPCR, but the LAMP assay was significantly more sensitive (p < 0.05). Thus, LAMP methodology is a useful for diagnosis of trench fever in humans and primates, especially in low-resource settings, because of its rapid, sensitive detection that does not require sophisticated equipment. PMID:27916953

  20. Rapid, Sensitive Detection of Bartonella quintana by Loop-Mediated Isothermal Amplification of the groEL Gene

    Directory of Open Access Journals (Sweden)

    Shoukui Hu


    Full Text Available Trench fever, caused by Bartonella quintana, is recognized as a re-emerging and neglected disease. Rapid and sensitive detection approaches are urgently required to monitor and help control B. quintana infections. Here, loop-mediated isothermal amplification (LAMP, which amplifies target DNA at a fixed temperature with high sensitivity, specificity and rapidity, was employed to detect B. quintana. Thirty-six strains, including 10 B. quintana, 13 other Bartonella spp., and 13 other common pathogens, were applied to verify and evaluate the LAMP assay. The specificity of the LAMP assay was 100%, and the limit of detection was 125 fg/reaction. The LAMP assay was compared with qPCR in the examination of 100 rhesus and 20 rhesus-feeder blood samples; the diagnostic accuracy was found to be 100% when LAMP was compared to qPCR, but the LAMP assay was significantly more sensitive (p < 0.05. Thus, LAMP methodology is a useful for diagnosis of trench fever in humans and primates, especially in low-resource settings, because of its rapid, sensitive detection that does not require sophisticated equipment.

  1. Relation Among DNA Content, N-myc Gene Amplification and the Clinical Prognosis%小儿神经母细胞瘤DNA含量、N-myc基因扩增与临床预后的关系

    Institute of Scientific and Technical Information of China (English)

    刘获; 吴建新; 呼守生; 马汝柏; 林久治


    本文比较分析了18例神经母细胞瘤的DNA含量、N-myc基因扩增、临床分期、原发部位等因素与预后的关系.结果表明,有N-myc基因扩增的死亡率(7/10)明显高于无扩增的(1/8);DNA含量增高与肿瘤的恶性度成正比;临床分期与N-myc基因扩增情况相结合与病死率的关系极为密切.%In this study, 18 children with neuroblastoma were analysed on the relation among DNA content, N-myc gene amplification and the clinical staging. The results reveal; the mortality of patients with N-myc gene amplification (7/10) was higher than those without N-myc gene amplification (1/8); DNA content was related to malignancy of the tumor, combination of N-myc gene amplification and the clinical staging kept close link with the prognosis.

  2. Amplification of the aroA gene from Escherichia coli results in tolerance to the herbicide glyphosate.


    Rogers, S G; Brand, L A; Holder, S B; Sharps, E S; Brackin, M J


    The predominant cellular target of the herbicide glyphosate is thought to be the enzyme 5-enolpyruvylshikimate-3-phosphoric acid synthase (EPSP synthase). As a means of biologically testing this finding, we cloned a segment of DNA from Escherichia coli that encodes this enzyme. Clones carrying the gene for EPSP synthase were identified by genetic complementation. Cells that contain a multicopy plasmid carrying the EPSP synthase gene overproduce the enzyme 5- to 17-fold and exhibit at least an...

  3. A new morphologically distinct avian malaria parasite that fails detection by established polymerase chain reaction-based protocols for amplification of the cytochrome B gene. (United States)

    Zehtindjiev, Pavel; Križanauskienė, Asta; Bensch, Staffan; Palinauskas, Vaidas; Asghar, Muhammad; Dimitrov, Dimitar; Scebba, Sergio; Valkiūnas, Gediminas


    Plasmodium polymorphum n. sp. (Haemosporida, Plasmodiidae) was found in the skylark, Alauda arvensis (Passeriformes: Alaudidae), during autumnal migration in southern Italy. This organism is illustrated and described based on the morphology of its blood stages. The most distinctive feature of this malaria parasite is the clear preference of its blood stages (trophozoites, meronts, and gametocytes) for immature red blood cells, including erythroblasts. Based on preference of erythrocytic meronts for immature red blood cells, P. polymorphum is most similar to species of the subgenus Huffia . This parasite can be readily distinguished from all other bird malaria parasites, including Plasmodium ( Huffia ) spp., due to preferential development and maturation of its gametocytes in immature red blood cells, a unique character for avian Plasmodium spp. In addition, the margins of nuclei in blood stages of P. polymorphum are markedly smooth and distinct; this is also a distinct diagnostic feature of this parasite. Plasmodium polymorphum has been recorded only in the skylark; it is probably a rare parasite, whose host range and geographical distribution remain unclear. Microscopic examination detected a light infection of Plasmodium relictum (lineage GRW11, parasitemia of mitochondrial cytochrome b gene (cyt b ) of P. polymorphum from the microscopically positive sample by using published and newly designed primers for DNA amplification of avian Plasmodium spp. The light parasitemia of P. relictum was easily detectable using several polymerase chain reaction (PCR)-based assays, but P. polymorphum was undetectable in all applied assays. Quantitative PCR also showed the presence of light parasitemia (0.06%) of the lineage GRW11 in this sample. This supports the conclusion that the morphologically distinct parasite observed along with P. relictum and predominant in the sample is genetically dissimilar from the lineage GRW11 based on cyt b sequence. In samples with co

  4. 副溶血弧菌tdh基因LAMP检测技术的建立%Establishment of Loop-Mediated Isothermal Amplification for Detecting Vibrio Parahaemolyticus tdh Gene

    Institute of Scientific and Technical Information of China (English)

    程晓艳; 刘庆慧; 黄倢


    Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification technology which amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. In this paper, LAMP was applied for rapid detection of Vibrio parahaemolyticus. According to the sequence of the specific tdh gene of Vibrio parahaemolyticus, a set of primers was designed to amplify the specifial DNA sequences by LAMP. Moreover, the reaction conditions were optimized. The results showed the optimum LAMP assay for the rapid detection of Vibrio paiakaemolyticus was performed at 60.8 ℃ for 45 min. No crossreaction was found with other common bacteria which showed a good specificity. The LAMP assay had an detection limit of 35.5 fg/tube, which was 10 times lower than that of PCR assay. The LAMP assay could be finished within an hour requiring only a regular laboratory water bath for reaction. The detection of the strains isolated from scallop also proved the LAMP assay reliability.%环等温扩增技术(Loop-mediated isothermal amplification,LAMP)是一种在等温条件下高特异性、高效、快速地扩增靶基因的DNA扩增技术.根据副溶血弧菌特异性的毒力基因tdh保守序列,本文设计1套特异性引物对该基因进行环等温扩增,同时对反应条件进行优化,建立携带tdh基因的致病性副溶血弧菌的LAMP快速检测技术.结果表明,LAMP最适反应在60.8℃恒温、45 min内完成,与其它常见的细菌无交叉反应.LAMP方法的细菌DNA最低检出限为35.5 fg/反应管,灵敏度较PCR方法高10倍.对扇贝样品中分离的菌株的检测表明,LAMP方法对检测致病性副溶血弧菌具有良好的可靠性.

  5. Hyd5 gene-based detection of the major gushing-inducing Fusarium spp. in a loop-mediated isothermal amplification (LAMP) assay. (United States)

    Denschlag, Carla; Vogel, Rudi F; Niessen, Ludwig


    Fusarium graminearum and the closely related F. culmorum were found to be associated with over foaming of bottled beer (gushing) when contaminated brewing malt is used. The presence of highly surface active hydrophobins produced by these fungi upon growth on wheat or barley in the field or during malting may affect bubble formation and stability in gushing beers and other carbonated beverages. Aiming for a method for the rapid and user friendly analysis of unmalted and malted cereals during quality control in the brewing industry, a loop-mediated isothermal amplification (LAMP) assay for the detection of Fusarium spp. capable of producing the gushing inducing hydrophobin Hyd5p was set up. A set of primers was designed towards a 221 bp region within the hyd5 gene of F. culmorum. The LAMP product was verified by sequencing a 150 bp portion. Testing specificity with purified DNA from 99 different fungal species as well as barley and wheat showed that DNA synthesis only occurred during LAMP when DNA of the closely related species F. graminearum, F. culmorum, F. cerealis and F. lunulosporum were used as template. In-tube indirect detection of DNA amplification was applied using manganese-quenched calcein as fluorescence indicator for pyrophosphate produced during DNA synthesis. The assay had a detection limit of 0.74 pg of purified target DNA which corresponds 20 copy numbers per reaction within 30 minutes using a simple heating block. Analysis of Fusarium infected cereals revealed that the assay was able to detect F. graminearum at a level of 0.5% of infected grains in uninfected barley by analysis of surface washings without further sample preparation. Results show that the hyd5 based LAMP assay can be a rapid, useful and sensitive tool for quality control in the brewing and malting industry.

  6. Chromosomal localization of the murine RFC-1 gene encoding a folate transporter and its amplification in an antifolate resistant variant overproducing the transporter. (United States)

    Roy, K; Chiao, J H; Spengler, B A; Tolner, B; Yang, C H; Biedler, J L; Sirotnak, F M


    83 cells. These results document the amplification at the site of a putative HSR in an L1210 cell variant of the RFC-1 gene regulating expression of the one-carbon, reduced folate transporter.

  7. A novel gene amplification causes upregulation of the PatAB ABC transporter and fluoroquinolone resistance in Streptococcus pneumoniae. (United States)

    Baylay, Alison J; Ivens, Alasdair; Piddock, Laura J V


    Overexpression of the ABC transporter genes patA and patB confers efflux-mediated fluoroquinolone resistance in Streptococcus pneumoniae and is also linked to pneumococcal stress responses. Although upregulation of patAB has been observed in many laboratory mutants and clinical isolates, the regulatory mechanisms controlling expression of these genes are unknown. In this study, we aimed to identify the cause of high-level constitutive overexpression of patAB in M184, a multidrug-resistant mutant of S. pneumoniae R6. Using a whole-genome transformation and sequencing approach, we identified a novel duplication of a 9.2-kb region of the M184 genome which included the patAB genes. This duplication did not affect growth and was semistable with a low segregation rate. The expression levels of patAB in M184 were much higher than those that could be fully explained by doubling of the gene dosage alone, and inactivation of the first copy of patA had no effect on multidrug resistance. Using a green fluorescent protein reporter system, increased patAB expression was ascribed to transcriptional read-through from a tRNA gene upstream of the second copy of patAB. This is the first report of a large genomic duplication causing antibiotic resistance in S. pneumoniae and also of a genomic duplication causing antibiotic resistance by a promoter switching mechanism.

  8. Identification of Tunisian Leishmania spp. by PCR amplification of cysteine proteinase B (cpb) genes and phylogenetic analysis. (United States)

    Chaouch, Melek; Fathallah-Mili, Akila; Driss, Mehdi; Lahmadi, Ramzi; Ayari, Chiraz; Guizani, Ikram; Ben Said, Moncef; Benabderrazak, Souha


    Discrimination of the Old World Leishmania parasites is important for diagnosis and epidemiological studies of leishmaniasis. We have developed PCR assays that allow the discrimination between Leishmania major, Leishmania tropica and Leishmania infantum Tunisian species. The identification was performed by a simple PCR targeting cysteine protease B (cpb) gene copies. These PCR can be a routine molecular biology tools for discrimination of Leishmania spp. from different geographical origins and different clinical forms. Our assays can be an informative source for cpb gene studying concerning drug, diagnostics and vaccine research. The PCR products of the cpb gene and the N-acetylglucosamine-1-phosphate transferase (nagt) Leishmania gene were sequenced and aligned. Phylogenetic trees of Leishmania based cpb and nagt sequences are close in topology and present the classic distribution of Leishmania in the Old World. The phylogenetic analysis has enabled the characterization and identification of different strains, using both multicopy (cpb) and single copy (nagt) genes. Indeed, the cpb phylogenetic analysis allowed us to identify the Tunisian Leishmania killicki species, and a group which gathers the least evolved isolates of the Leishmania donovani complex, that was originated from East Africa. This clustering confirms the African origin for the visceralizing species of the L. donovani complex.

  9. Enumeration of total bacteria and bacteria with genes for proteolytic activity in pure cultures and in environmental samples by quantitative PCR mediated amplification. (United States)

    Bach, H-J; Tomanova, J; Schloter, M; Munch, J C


    Real-time quantitative PCR assays were developed for the absolute quantification of different groups of bacteria in pure cultures and in environmental samples. 16S rRNA genes were used as markers for eubacteria, and genes for extracellular peptidases were used as markers for potentially proteolytic bacteria. For the designed 16S rDNA TaqMan assay, specificity of the designed primer-probe combination for eubacteria, a high amplification efficiency over a wide range of starting copy numbers and a high reproducibility is demonstrated. Cell concentrations of Bacillus cereus, B. subtilis and Pseudomonas fluorescens in liquid culture were monitored by TaqMan-PCR using the 16S rDNA target sequence of Escherichia coli as external standard for quantification. Results agree with plate counts and microscopic counts of DAPI stained cells. The significance of 16S rRNA operon multiplicity to the quantification of bacteria is discussed.Furthermore, three sets of primer pair together with probe previously designed for targeting different classes of bacterial extracellular peptidases were tested for their suitability for TaqMan-PCR based quantification of proteolytic bacteria. Since high degeneracy of the probes did not allow accurate quantification, SybrGreen was used instead of molecular probes to visualize and quantify PCR products during PCR. The correlation between fluorescence and starting copy number was of the same high quality as for the 16S rDNA TaqMan assay for all the three peptidase gene classes. The detected amount of genes for neutral metallopeptidase of B. cereus, for subtilisin of B. subtilis and for alkaline metallopeptidase of P. fluorescens corresponded exactly to the numbers of bacteria investigated by the 16S rDNA targeting assay. The developed assays were applied for the quantification of bacteria in soil samples.

  10. Loop-Mediated Isothermal Amplification of the sefA Gene for Rapid Detection of Salmonella Enteritidis and Salmonella Gallinarum in Chickens. (United States)

    Gong, Jiansen; Zhuang, Linlin; Zhu, Chunhong; Shi, Shourong; Zhang, Di; Zhang, Linji; Yu, Yan; Dou, Xinhong; Xu, Bu; Wang, Chengming


    Salmonella spp. pose a threat to both human and animal health, with more than 2600 serovars having been reported to date. Salmonella serovars are usually identified by slide agglutination tests, which are labor intensive and time consuming. In an attempt to develop a more rapid screening method for the major poultry Salmonella serovars, we developed a loop-mediated isothermal amplification (LAMP) assay, which directly detected the sefA gene, a fimbrial operon gene existing in several specific serovars of Salmonella enterica including the major poultry serovars, namely Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) and Salmonella enterica serovar Gallinarum (Salmonella Gallinarum). With the 177 bacterial strains we tested, positive reactions were only observed with 85 strains of serovar Salmonella Enteritidis and Salmonella Gallinarum. The detection limit of the LAMP assay was 4 CFU/reaction with genomic DNAs of Salmonella Enteritidis (ATCC 13076) from pure culture and 400 CFU/ reaction with DNA extracted from spiked chicken feces. The LAMP assay was more sensitive than conventional culture, especially without enrichment, in detecting Salmonella Enteritidis (CMCC 50041) in the spiked fecal samples. The results show the sefA LAMP method is a rapid, sensitive, specific, and practical method for directly detection of Salmonella Enteritidis and Salmonella Gallinarum in chickens. The sefA LAMP assay can potentially serve as new on-site diagnostics in the poultry industry.

  11. Specific amplification of gene encoding N-terminal region of catalase-peroxidase protein (KatG-N) for diagnosis of disseminated MAC disease in HIV patients. (United States)

    Latawa, Romica; Singh, Krishna Kumar; Wanchu, Ajay; Sethi, Sunil; Sharma, Kusum; Sharma, Aman; Laal, Suman; Verma, Indu


    Disseminated Mycobacterium avium-intracellulare complex (MAC) infection is considered as severe complication of advanced HIV/AIDS disease. Currently available various laboratory investigations have not only limited ability to discriminate between MAC infection and tuberculosis but are also laborious and time consuming. The aim of this study was, therefore, to design a molecular-based strategy for specific detection of MAC and its differentiation from Mycobacterium tuberculosis (M. tb) isolated from the blood specimens of HIV patients. A simple PCR was developed based on the amplification of 120-bp katG-N gene corresponding to the first 40 amino acids of N-terminal catalase-peroxidase (KatG) protein of Mycobacterium avium that shows only ~13% sequence homology by clustal W alignment to N-terminal region of M. tb KatG protein. This assay allowed the accurate and rapid detection of MAC bacteremia, distinguishing it from M. tb in a single PCR reaction without any need for sequencing or hybridization protocol to be performed thereafter. This study produced enough evidence that a significant proportion of Indian HIV patients have disseminated MAC bacteremia, suggesting the utility of M. avium katG-N gene PCR for early detection of MAC disease in HIV patients. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Rapid, simple, and sensitive detection of the ompB gene of spotted fever group rickettsiae by loop-mediated isothermal amplification

    Directory of Open Access Journals (Sweden)

    Pan Lei


    Full Text Available Abstract Background Spotted fever caused spotted fever group rickettsiae (SFGR is prevalent throughout China. In this study, we describe a rapid, simple, and sensitive loop-mediated isothermal amplification (LAMP assay targeting the ompB gene of spotted fever group rickettsiae ideal for application in China. The LAMP assay has the potential to detect spotted fever group rickettsiae early in infection and could therefore serve as an alternative to existing methods. Methods A set of universal primers which are specific 7 common species of spotted fever group rickettsiae in China were designed using PrimerExplorer V4 software based on conserved sequences of ompB gene. The sensitivity, specificity and reproducibility of the LAMP were evaluated. The LAMP assay for detecting SFGR was compared with conventional PCR assays for sensitivity and specificity in early phase blood samples obtained from 11 infected human subjects. Results The sensitivity of the LAMP assay was five copies per reaction (25 μL total volume, and the assay did not detect false-positive amplification across 42 strains of 27 members of the order Rickettsiales and 17 common clinical pathogens. The LAMP assay was negative to typhus group rickettsiae including R. prowazekii and R. typhi for no available conserved sequences of ompB was obtained for designing primers. To evaluate the clinical applicability of the LAMP assay, a total of 11 clinical samples, 10 samples confirmed serologically (3 cases, ecologically (1 case, by real-time polymerase chain reaction (PCR; 2 cases, ecologically and by real-time PCR (1 case, and serologically and by real-time PCR (3 cases were analyzed by the ompB LAMP assay. Data were validated using a previously established nested PCR protocol and real-time PCR. A positive LAMP result was obtained for 8 of the 10 confirmed cases (sensitivity, 73%; specificity, 100%, while none of these samples were positive by nested PCR (sensitivity, 0%; specificity, 100

  13. Sex determination of cattle meat by polymerase chain reaction amplification of the amelogenin (AMELX/AMELY gene

    Directory of Open Access Journals (Sweden)

    P. Gokulakrishnan

    Full Text Available Aim: The objective of this study was to develop a simplified, efficient, and accurate protocol for sexing of cattle meat based on the amelogenin (AMELX/AMELY gene using PCR technique which is superior to earlier work in terms of band patterns. Materials and Methods: Based on the amelogenin gene located on the conservation region of X and Y chromosomes, a pair of primers was designed and the system of PCR was established to amplify a 241-bp fragment from the X chromosome in female cattle, and a 241-bp fragment from X chromosome and 178-bp fragment from the Y chromosome in male cattle, respectively. The accuracy and specificity of the primers was assessed using DNA template extracted from cattle meat samples of known sex. The protocol was subjected to a blind test showed 100% concordance, proving its accuracy and reliability. Results: PCR products of cattle meat samples after electrophoresis showed two bands (241, 178-bp for tissue from male while female tissue resulted in only one (241-bp band. Conclusions: Our findings show that the PCR assay based on the amelogenin gene is reliable for sex determination in cattle meat. [Vet World 2012; 5(9.000: 526-529

  14. Multiplex PCR-based simultaneous amplification of selectable marker and reporter genes for the screening of genetically modified crops. (United States)

    Randhawa, Gurinder Jit; Chhabra, Rashmi; Singh, Monika


    The development and commercialization of genetically modified (GM) crops with enhanced insect and herbicide resistance, abiotic stress tolerance, and improved nutritional quality has expanded dramatically. Notwithstanding the huge potential benefits of GM crops, the perceived environmental risks associated with these crops need to be addressed in proper perspective. One critical concern is the adventitious presence or unintentional mixing of GM seed in non-GM seed lots, which can seriously affect the global seed market. It would therefore be necessary though a challenging task to develop reliable, efficient, and economical assays for GM detection, identification, and quantification in non-GM seed lots. This can be systematically undertaken by preliminary screening for control elements and selectable or scorable (reporter) marker genes. In this study, simplex and multiplex polymerase chain reaction (PCR) assays individually as well as simultaneously amplifying the commonly used selectable marker genes, i.e., aadA, bar, hpt, nptII, pat encoding, respectively, for aminoglycoside-3'-adenyltransferase, Streptococcus viridochromogenes phosphinothricin-N-acetyltransferase, hygromycin phosphotransferase, neomycin phosphotransferase, Streptococcus hygroscopicus phosphinothricin-N-acetyltransferase, and a reporter gene uidA encoding beta-d-glucuronidase, were developed as a reliable tool for qualitative screening of GM crops. The efficiency of the assays was also standardized in the test samples prepared by artificial mixing of transgenic seed samples in different proportions. The developed multiplex PCR assays will be useful in verifying the GM status of a sample irrespective of the crop and GM trait.

  15. Isolation and characterisation of the human lung NK-2 receptor gene using rapid amplification of cDNA ends. (United States)

    Graham, A; Hopkins, B; Powell, S J; Danks, P; Briggs, I


    Functional cDNA clones for human NK-2 receptor were isolated from human lung RNA using a polymerase chain reaction (PCR) based method (RACE-PCR). In this method the cDNA was isolated as 5' end and 3'-end fragments; the entire cDNA was obtained by RNA-PCR. The sequence derived was 398 amino acids in length encoding an open-reading frame that was highly homologous to both the bovine and rat NK-2 receptor. The entire human cDNA sequence was cloned into a mammalian expression vector and mRNA was synthesised by in vitro transcription. Applications of tachykinins caused membrane current responses in Xenopus oocytes injected with the in vitro synthesised mRNA. The most potent of the three tachykinin peptides tested was neurokinin A. We have screened a human cosmid library and isolated a clone which contains the entire NK-2 receptor gene. The gene contains five exons and we have determined the complete sequence of the exons and the intron-exon junctions.

  16. Development and evaluation of a loop-mediated isothermal amplification (LAMP assay for rapid detection of Actinobacillus pleuropneumoniae based the dsbE-like gene

    Directory of Open Access Journals (Sweden)

    Hongwei Ji


    Full Text Available This paper reports on the development and validation of a loop-mediated isothermal amplification assay (LAMP for the rapid and specific detection of Actinobacillus pleuropneumoniae (A. pleuropneumoniae. A set of six primers were designed derived from the dsbE-like gene of A.pleuropneumoniae and validate the assay using 9 A. pleuropneumoniae reference/field strains, 132 clinical isolates and 9 other pathogens. The results indicated that positive reactions were confirmed for all A. pleuropneumoniae strains and specimens by LAMP at 63ºC for 60 min and no cross-reactivity were observed from other non-A.pleuropneumoniae including Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Bordetella bronchiseptica, Streptococcus suis, Salmonella enterica, Staphylococcus, porcine reproductive and respiratory syndrome virus (PRRSV, and Pseudorabies virus. The detection limit of the conventional PCR was 10² CFU per PCR test tube, while that of the LAMP was 5 copies per tube. Therefore, the sensitivity of LAMP was higher than that of PCR. Moreover, the LAMP assay provided a rapid yet simple test of A. pleuropneumoniae suitable for laboratory diagnosis and pen-side detection due to ease of operation and the requirement of only a regular water bath or heat block for the reaction.

  17. Molecular characterization of Cryptosporidium xiaoi in goat kids in Bangladesh by nested PCR amplification of 18S rRNA gene

    Institute of Scientific and Technical Information of China (English)

    AMAM; Zonaed; Siddiki; Sohana; Akter; Mina; Zinat; Farzana; Bibi; Ayesa; Rasel; Das; Mohammad; Alamgir; Hossain


    Objective:To investigate the prevalence of Cryptosporidium goat kids in selected areas of Bangladesh and to elucidate the potential zoonotic hazards.Methods:In the present study,we have used Ziehl-Neelsen staining and nested PCR approach to identify and characterize the Cryptosporidium sp.from diarrhoeic feces of goat kids.A total of 100 diarrhoeic feces samples were collected from Chittagong region in Southern Bangladesh.For nested PCR analysis,specific primers for amplification of 581 base pair fragments of 18 S rRNA gene were used.Results:A total of 15%and 3%samples were found positive in microscopic study and in nested PCR analysis respectively.Phylogenetic analysis of sequence data showed similarity with that of Cryptosporidium xiaoi recorded from sheep and goat.Conclusions:To our knowledge,this is the first report of Cryptosporidium xiaoi responsible for diarrhoea in goat kids in Bangladesh.Further study can highlight their zoonotic significance along with genetic diversity in other host species inside the country.

  18. Rapid and sensitive detection of Dasheen mosaic virus infecting elephant foot yam by reverse transcription loop mediated isothermal amplification of coat protein gene. (United States)

    Kamala, S; Makeshkumar, T


    Dasheen mosaic virus (DsMV), the pathogen causing mosaic disease of elephant foot yam (Amorphophallus paeoniifoilius) is disseminated mainly through vegetative propagation of the tubers. For the rapid and sensitive detection of the virus, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay based on the coat protein gene has been developed. A final concentration of 5.4 mM magnesium sulphate and 0.7 M betaine in the reaction mixture was found to be optimum for getting characteristic ladder like bands of the amplified product after gel electrophoresis. The reaction was set at 65°C for 50 min followed by reaction termination at 86°C for 5 min in a water bath. The sensitivity of the assay was found to be 100 times higher than that of RT-PCR. The virus was indexed successfully from tubers of elephant foot yam. In tube detection of the DsMV was carried out using fluorescence detection reagents. The assay was validated with field samples from various regions of Kerala state, India. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. G-quadruplex structures and CpG methylation cause drop-out of the maternal allele in polymerase chain reaction amplification of the imprinted MEST gene promoter.

    Directory of Open Access Journals (Sweden)

    Aaron J Stevens

    Full Text Available We observed apparent non-Mendelian behaviour of alleles when genotyping a region in a CpG island at the 5' end of the maternally imprinted human MEST isoform. This region contains three single nucleotide polymorphisms (SNPs in total linkage disequilibrium, such that only two haplotypes occur in the human population. Only one haplotype was detectable in each subject, never both, despite the use of multiple primers and several genotyping methods. We observed that this region contains motifs capable of forming several G-quadruplex structures. Circular dichroism spectroscopy and native polyacrylamide gel electrophoresis confirmed that at least three G-quadruplexes form in vitro in the presence of potassium ions, and one of these structures has a Tm of greater than 99°C in polymerase chain reaction (PCR buffer. We demonstrate that it is the methylated maternal allele that is always lost during PCR amplification, and that formation of G-quadruplexes and presence of methylated cytosines both contributed to this phenomenon. This observed parent-of-origin specific allelic drop-out has important implications for analysis of imprinted genes in research and diagnostic settings.

  20. G-Quadruplex Structures and CpG Methylation Cause Drop-Out of the Maternal Allele in Polymerase Chain Reaction Amplification of the Imprinted MEST Gene Promoter (United States)

    Cree, Simone L.; Gibb, Andrew; Miller, Allison L.; Doudney, Kit; Aitchison, Alan; Eccles, Michael R.; Joyce, Peter R.; Filichev, Vyacheslav V.; Kennedy, Martin A.


    We observed apparent non-Mendelian behaviour of alleles when genotyping a region in a CpG island at the 5′ end of the maternally imprinted human MEST isoform. This region contains three single nucleotide polymorphisms (SNPs) in total linkage disequilibrium, such that only two haplotypes occur in the human population. Only one haplotype was detectable in each subject, never both, despite the use of multiple primers and several genotyping methods. We observed that this region contains motifs capable of forming several G-quadruplex structures. Circular dichroism spectroscopy and native polyacrylamide gel electrophoresis confirmed that at least three G-quadruplexes form in vitro in the presence of potassium ions, and one of these structures has a Tm of greater than 99°C in polymerase chain reaction (PCR) buffer. We demonstrate that it is the methylated maternal allele that is always lost during PCR amplification, and that formation of G-quadruplexes and presence of methylated cytosines both contributed to this phenomenon. This observed parent-of-origin specific allelic drop-out has important implications for analysis of imprinted genes in research and diagnostic settings. PMID:25437198

  1. A Loop-Mediated Isothermal Amplification Assay Targeting 16S rRNA Gene for Rapid Detection of Anaplasma phagocytophilum Infection in Sheep and Goats. (United States)

    Wang, Jinhong; Zhang, Yan; Wang, Xiaoxing; Cui, Yanyan; Yan, Yaqun; Wang, Rongjun; Jian, Fuchun; Zhang, Longxian; Ning, Changshen


    Anaplasma phagocytophilum is a zoonotic pathogen and the causative agent of human granulocytic anaplasmosis in humans and tick-borne fever in various kinds of animals. In the present study, a loop-mediated isothermal amplification (LAMP) assay for rapid detection of A. phagocytophilum was developed using primers specific to 16S rRNA gene of this organism. The LAMP assay was performed at 65 C for 60 min and terminated at 80 C for 10 min. The optimal reaction conditions under which no cross-reaction was observed with other closely related tick-borne parasites ( Anaplasma bovis , Anaplasma ovis , Theileria luwenshuni, Babesia motasi, and Schistosoma japonicum ) were established. The assay exhibited much higher sensitivity compared with conventional polymerase chain reaction (PCR) (1 copy vs. 1,000 copies). To evaluate the applicability of the LAMP assay, 94 field samples of sheep blood were analyzed for A. phagocytophilum infection by using LAMP, nested PCR, and conventional PCR assays at the same time. A positive LAMP result was obtained from 53 (56.4%) of the 94 samples, whereas only 12 (12.8%) and 3 (3.2%) tested positive by nested and conventional PCR, respectively. In conclusion, this LAMP assay is a specific, sensitive, and rapid method for the detection of A. phagocytophilum in sheep/goats.

  2. 应用双重原位杂交检测胃癌HER2基因扩增%Amplification of HER2 gene in gastric carcinoma detected by dual in-situ hybridization

    Institute of Scientific and Technical Information of China (English)

    冼丽芳; 黄香婷; 文剑明; 叶玉清


    Objective To explore the status of HER2 gene amplification and its product HER2 protein expression in gastric carcinoma, so as to aid in patient selection for anti-HER2 targeted chemotherapy.Methods Eighty-five cases of gastric carcinoma biopsy tissues were collected.The status of HER2 gene amplification was detected by dual in situ hybridization (dual-ISH).And HER2 protein was detected by immunohistochemistry.Results HER2 gene amplification was detected in 10/85 ( 11.8% )cases of gastric carcinoma,and no amplification was detected in 75/85 (88.2% ) cases.In the 10 cases with HER2 amplification,HER2 immunoreaction scorings of 3 +,2 + and 0/1 + were present in 7,2 and 1 cases,respectively.In the 75 cases without HER2 amplification,HER2 immunoreaction scorings of 3 +,2 + and 0/1 + were present in 0,18 (24.0% ) and 57 (76.0% ) cases,respectively.Histologically,most gastric carcinoma with amplification of HER2 gene was moderately differentiated tubular adenocarcinoma.Conclusions HER2 gene dual-ISH technique is a reliable and objective method for detecting HER2 gene amplification in gastric carcinoma biopsy. Clinically, only few gastric carcinomas show HER2 gene amplification and are suitable candidates for anti-HER2 targeted chemotherapy.%目的 探讨胃癌HER2基因扩增状态及其基因产物蛋白表达,以协助临床筛选抗HER2靶向治疗患者.方法 收集胃癌活检标本共85例,进行HER2基因扩增和蛋白表达检测.HER2基因扩增状态采用双重原位杂交检测(HER2 dual-ISH detection),而HER2基因蛋白采用免疫组织化学[全程在全自动染色机(Ventana)]进行检测.结果 85例胃癌病例中,有HER2基因扩增10例,扩增率为11.8%,无扩增75例,占88.2%.在有HER2基因扩增的10例病例中,7例HER2蛋白免疫组织化学为3+,2例为2+,1例为-/1+;75例无基因扩增的病例,HER2蛋白免疫组织化学3+为0例、2+为18例(24.0%),-/1+为57例(76.0%).HER2基因扩增与HER2

  3. Development and evaluation of a loop-mediated isothermal amplification assay for detection of Ehrlichia canis DNA in naturally infected dogs using the p30 gene. (United States)

    Pinhanelli, V C; Costa, P N M; Silva, G; Aguiar, D M; Silva, C M L; Fachin, A L; Marins, M


    Canine monocytic ehrlichiosis (CME) is a common tick-borne disease caused by the rickettsial bacterium Ehrlichia canis (Rickettsiales: Anaplasmataceae). In view of the different stages and variable clinical signs of CME, which can overlap with those of other infections, a conclusive diagnosis can more readily be obtained by combining clinical and hematological evaluations with molecular diagnostic methods. In this study, a loop-mediated isothermal amplification (LAMP) assay targeting the p30 gene of E. canis was developed. The assay was developed using DNA extracted from E. canis-infected cultures of the macrophage cell line DH82 and samples from dogs testing positive for E. canis DNA by PCR. The LAMP assay was compared to a p30-based PCR assay, using DNA extracted from EDTA-anticoagulated blood samples of 137 dogs from an endemic region in Brazil. The LAMP assay was sensitive enough to detect a single copy of the target gene, and identified 74 (54.0%) E. canis DNA-positive samples, while the p30 PCR assay detected 50 positive samples (36.5%) among the field samples. Agreement between the two assays was observed in 42 positive and 55 negative samples. However, 32 positive samples that were not detected by the PCR assay were identified by the LAMP assay, while eight samples identified as E. canis-positive by PCR showed negative results in LAMP. The developed E. canis LAMP assay showed the potential to maximize the use of nucleic acid tests in a veterinary clinical laboratory, and to improve the diagnosis of CME.

  4. PCR amplification and high throughput sequencing of immunoglobulin heavy chain genes from formalin-fixed paraffin-embedded human biopsies. (United States)

    Tabibian-Keissar, Hilla; Schibby, Ginette; Michaeli, Miri; Rakovsky-Shapira, Aviya; Azogui-Rosenthal, Noemie; Dunn-Walters, Deborah K; Rosenblatt, Kinneret; Mehr, Ramit; Barshack, Iris


    The use of high throughput sequencing (HTS) technologies in biomedicine is expanding in a variety of fields in recent years. The 454 system is an HTS platform that is ideally suited to characterize B cell receptor (BCR) repertoires by sequencing of immunoglobulin (Ig) genes, as it is able to sequence stretches of several hundred nucleotides. Most studies that used this platform for antibody repertoire analyses have started from fresh or frozen tissues or peripheral blood samples, and rely on starting with optimal quality DNA. In this paper we demonstrate that BCR repertoire analysis can be done using DNA from formalin-fixed paraffin-embedded (FFPE) human tissue samples. The heterogeneity of BCR repertoires we obtained confirms the plausibility of HTS of DNA from FFPE specimens. The establishment of experimental protocols and computational tools that enable sequence data analysis from the low quality DNA of FFPE tissues is important for enabling research, as it would enable the use of the rich source of preserved samples in clinical biobanks and biopsy archives.

  5. Efficient PCR-Based Amplification of Diverse Alcohol Dehydrogenase Genes from Metagenomes for Improving Biocatalysis: Screening of Gene-Specific Amplicons from Metagenomes (United States)

    Kariya, Satomi; Kurokawa, Junji


    Screening of gene-specific amplicons from metagenomes (S-GAM) has tremendous biotechnological potential. We used this approach to isolate alcohol dehydrogenase (adh) genes from metagenomes based on the Leifsonia species adh gene (lsadh), the enzyme product of which can produce various chiral alcohols. A primer combination was synthesized by reference to homologs of lsadh, and PCR was used to amplify nearly full-length adh genes from metagenomic DNAs. All adh preparations were fused with lsadh at the terminal region and used to construct Escherichia coli plasmid libraries. Of the approximately 2,000 colonies obtained, 1,200 clones were identified as adh positive (∼60%). Finally, 40 adh genes, Hladh-001 to Hladh-040 (for homologous Leifsonia adh), were identified from 223 clones with high efficiency, which were randomly sequenced from the 1,200 clones. The Hladh genes obtained via this approach encoded a wide variety of amino acid sequences (8 to 99%). After screening, the enzymes obtained (HLADH-012 and HLADH-021) were confirmed to be superior to LSADH in some respects for the production of anti-Prelog chiral alcohols. PMID:25085492

  6. A broad spectrum, one-step reverse-transcription PCR amplification of the neuraminidase gene from multiple subtypes of influenza A virus

    Directory of Open Access Journals (Sweden)

    Chen Wenbin


    Full Text Available Abstract Background The emergence of high pathogenicity strains of Influenza A virus in a variety of human and animal hosts, with wide geographic distribution, has highlighted the importance of rapid identification and subtyping of the virus for outbreak management and treatment. Type A virus can be classified into subtypes according to the viral envelope glycoproteins, hemagglutinin and neuraminidase. Here we review the existing specificity and amplification of published primers to subtype neuraminidase genes and describe a new broad spectrum primer pair that can detect all 9 neuraminidase subtypes. Results Bioinformatic analysis of 3,337 full-length influenza A neuraminidase segments in the NCBI database revealed semi-conserved regions not previously targeted by primers. Two degenerate primers with M13 tags, NA8F-M13 and NA10R-M13 were designed from these regions and used to generate a 253 bp cDNA product. One-step RT-PCR testing was successful in 31/32 (97% cases using a touchdown protocol with RNA from over 32 different cultured influenza A virus strains representing the 9 neuraminidase subtypes. Frozen blinded clinical nasopharyngeal aspirates were also assayed and were mostly of subtype N2. The region amplified was direct sequenced and then used in database searches to confirm the identity of the template RNA. The RT-PCR fragment generated includes one of the mutation sites related to oseltamivir resistance, H274Y. Conclusion Our one-step RT-PCR assay followed by sequencing is a rapid, accurate, and specific method for detection and subtyping of different neuraminidase subtypes from a range of host species and from different geographical locations.

  7. Sponge-associated actinobacterial diversity: validation of the methods of actinobacterial DNA extraction and optimization of 16S rRNA gene amplification. (United States)

    Yang, Qi; Franco, Christopher M M; Zhang, Wei


    Experiments were designed to validate the two common DNA extraction protocols (CTAB-based method and DNeasy Blood & Tissue Kit) used to effectively recover actinobacterial DNA from sponge samples in order to study the sponge-associated actinobacterial diversity. This was done by artificially spiking sponge samples with actinobacteria (spores, mycelia and a combination of the two). Our results demonstrated that both DNA extraction methods were effective in obtaining DNA from the sponge samples as well as the sponge samples spiked with different amounts of actinobacteria. However, it was noted that in the presence of the sponge, the bacterial 16S rRNA gene could not be amplified unless the combined DNA template was diluted. To test the hypothesis that the extracted sponge DNA contained inhibitors, dilutions of the DNA extracts were tested for six sponge species representing five orders. The results suggested that the inhibitors were co-extracted with the sponge DNA, and a high dilution of this DNA was required for the successful PCR amplification for most of the samples. The optimized PCR conditions, including primer selection, PCR reaction system and program optimization, further improved the PCR performance. However, no single PCR condition was found to be suitable for the diverse sponge samples using various primer sets. These results highlight for the first time that the DNA extraction methods used are effective in obtaining actinobacterial DNA and that the presence of inhibitors in the sponge DNA requires high dilution coupled with fine tuning of the PCR conditions to achieve success in the study of sponge-associated actinobacterial diversity.

  8. PCR amplification of a multi-copy mitochondrial gene (cox3) improves detection of Cytauxzoon felis infection as compared to a ribosomal gene (18S). (United States)

    Schreeg, Megan E; Marr, Henry S; Griffith, Emily H; Tarigo, Jaime L; Bird, David M; Reichard, Mason V; Cohn, Leah A; Levy, Michael G; Birkenheuer, Adam J


    Cytauxzoon felis is a tick-transmitted protozoan parasite that infects felids. Clinical disease caused by acute C. felis infection rapidly progresses in domestic cats, leading to high morbidity and mortality. Accurately diagnosing cytauxzoonosis as soon as possible during acute infection would allow for earlier initiation of antiprotozoal therapy which could lead to higher survival rates. Molecular detection of parasite rRNA genes (18S) by PCR has previously been shown to be a sensitive method of diagnosing C. felis infections. Based on evidence from related apicomplexan species, we hypothesized that C. felis mitochondrial genes would exist at higher copy numbers than 18S and would be a more sensitive diagnostic target. In this study we have designed a PCR assay targeting the C. felis mitochondrial gene cytochrome c oxidase subunit III (cox3). Herein we demonstrate that (1) the cox3 PCR can detect as low as 1 copy of DNA target and can detect C. felis in samples with known mitochondrial sequence heterogeneity, (2) cox3 copy number is increased relative to 18S in blood and tissue samples from acutely infected cats, and (3) the cox3 PCR is more sensitive than 18S PCR for detection of C. felis during early infections.

  9. Loop-mediated isothermal amplification assay targeting the blaCTX-M9 gene for detection of extended spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae. (United States)

    Thirapanmethee, Krit; Pothisamutyothin, Kanokporn; Nathisuwan, Surakit; Chomnawang, Mullika T; Wiwat, Chanpen


    Extended-spectrum β-lactamases (ESBLs) produced by Enterobacteriaceae are one of the resistance mechanisms to most β-lactam antibiotics. ESBLs are currently a major problem in both hospitals and community settings worldwide. Rapid and reliable means of detecting ESBL-producing bacteria is necessary for identification, prevention and treatment. Loop-mediated isothermal amplification (LAMP) is a technique that rapidly amplifies DNA with high specificity and sensitivity under isothermal conditions. This study was aimed to develop a convenient, accurate and inexpensive method for detecting ESBL-producing bacteria by a LAMP technique. ESBLs-producing Escherichia coli and Klebsiella pneumoniae were isolated from a tertiary hospital in Bangkok, Thailand and reconfirmed by double-disk synergy test. A set of four specific oligonucleotide primers of LAMP for detection of bla(CTX-M9) gene was designed based on bla(CTX-M9) from E. coli (GenBank Accession No. AJ416345). The LAMP reaction was amplified under isothermal temperature at 63°C for 60 min. Ladder-like patterns of band sizes from 226 bp of the bla(CTX-M9) DNA target was observed. The LAMP product was further analyzed by restriction digestion with MboI and TaqI endonucleases. The fragments generated were approximately 168, 177 and 250 bp in size for MboI digestion and 165, 193, 229, 281 and 314 bp for TaqI digestion, which is in agreement with the predicted sizes. The sensitivity of the LAMP technique to bla(CTX-M9) was greater than that of the PCR method by at least 10,000-fold. These results showed that the LAMP primers specifically amplified only the bla(CTX-M9) gene. Moreover, the presence of LAMP amplicon was simply determined by adding SYBR Green I in the reaction. In conclusion, this technique for detection of ESBLs is convenient, reliable and easy to perform routinely in hospitals or laboratory units in developing countries.

  10. A novel method for the purification of DNA by capturing nucleic acid and magnesium complexes on non-woven fabric filters under alkaline conditions for the gene diagnosis of tuberculosis by loop-mediated isothermal amplification (LAMP). (United States)

    Fukasawa, Tadashi; Oda, Naozumi; Wada, Yasunao; Tamaru, Aki; Fukushima, Yukari; Nakajima, Chie; Suzuki, Yasuhiko


    A novel method for purifying DNA from clinical samples based on the complex formation of DNA and magnesium ion (Mg(2+)) was developed for the detection of Mycobacterium tuberculosis. The formation of a DNA-Mg(2+) complex under alkaline conditions was observed by analyzing electrophoretic mobility reduction of DNA on agarose gel. The DNA-Mg(2+) complex increases the efficacy of DNA recovery from the sample solution on polyethylene terephthalate non-woven fabric (PNWF) filters. Among the various divalent metal cations, only Mg(2+) was associated with this effect. The applicability of DNA recovered on the PNWF filter was examined for the gene amplification method; loop-mediated isothermal amplification (LAMP). DNA on the PNWF filter could be used for the amplification of specific DNA fragments without elution from the filter. Using this method, DNA was directly purified from M. tuberculosis spiked sputum and examined by LAMP assay, showing a high sensitivity in comparison to the commercially available DNA extraction kit. These results indicated that the method developed in this study is useful for rapid gene diagnosis of tuberculosis.

  11. PCR Amplification System of DNA Barcoding Genes ITS, ITS2 and rbcL from Xanthium%DNA条形码基因ITS·ITS2及rbcL在苍耳属可采用相同的PCR条件

    Institute of Scientific and Technical Information of China (English)

    胡伟毅; 汪连军; 盛志超


    [目的]为植物 DNA条形码标准基因筛选研究提供参考,减少植物 DNA条形码研究中的工作量。[方法]对7种苍耳属植物 ITS、ITS2及 rbcL基因采用相同的扩增条件(95℃4 min;[35 cycles:94℃30 s;52℃45 s;72℃45 s];72℃10 min;4℃保存)。[结果]3种DNA条形码基因同时成功扩增。[结论]这说明植物 DNA条形码基因中 ITS、ITS2及 rbcL的PCR条件存在合并可能性。%[Objective] This study aimed to provide reference and reduce the workload for screening standard DNA barcoding genes of plants. [Method] Three DNA bar-coding genes ITS, ITS2 and rbcL were amplified from seven Xanthium species un-der the same PCR condition: PCR amplification was started with initial denaturation at 95 ℃ for 4 min, fol owed by 35 cycles of denaturation at 94 ℃ for 30 s, anneal-ing at 52 ℃ for 45 s, and extension at 72 ℃ for 45 s; the amplification was com-pleted by holding the reaction mixture at 72 ℃ for 10 min to al ow complete exten-sion of PCR, and the PCR products were stored at 4 ℃. [Result] Three DNA bar-coding genes ITS, ITS2 and rbcL were al amplified successful y. [Conclusion] This study indicates that PCR amplification conditions for DNA barcoding genes ITS, ITS2 and rbcL in plants may be consistent.

  12. Preparing a re-sequencing DNA library of 2 cancer candidate genes using the ligation-by-amplification protocol by two PCR reactions

    Institute of Scientific and Technical Information of China (English)


    To meet the needs of large-scale genomic/genetic studies, the next-generation massively parallelized sequencing technologies provide high throughput, low cost and low labor-intensive sequencing service, with subsequent bioinformatic software and laboratory methods developed to expand their applications in various types of research. PCR-based genomic/genetic studies, which have significant usage in association studies like cancer research, haven’t benefited much from those next-generation sequencing technologies, because the shortgun re-sequencing strategy used by such sequencing machines as the Illumina/Solexa Genome Analyzer may not be applied to direct re-sequencing of short-length target regions like those in PCR-based genomic/genetic studies. Although several methods have been proposed to solve this problem, including microarray-based genomic selections and selector-based technologies, they require advanced equipment and procedures which limit their applications in many laboratories. By contrast, we overcame such potential drawbacks by utilizing a ligation by amplification (LBA) protocol, a method using a pair of Universal Adapters to randomly ligate target regions in a two-step-PCR procedure, whose Long LBA products were easily fragmented and sequenced on the next-generation sequencing machine. In this concept-proven study, we chose the consensus coding sequences of two human cancer genes: BRCA1 and BRCA2 as target regions, specifically designed LBA primer pairs to amplify and randomly ligate them. 70 target sequences were successfully amplified and ligated into Long LBA products, which were then fragmented to construct DNA libraries for sequencing on both a conventional Sanger sequencer ABI 3730xl DNA Analyzer and the next-generation ’synthesis by sequencing technology’ Illumina/Solexa Genome Analyzer. Bioinformatic analysis demonstrated the utility and efficiency (including the coverage and depth of each target sequence and the SNPs detection

  13. Preparing a re-sequencing DNA library of 2 cancer candidate genes using the ligation-by-amplification protocol by two PCR reactions

    Institute of Scientific and Technical Information of China (English)

    SU YeYang; LIN Lin; TIAN Geng; CHEN Chen; LIU Tao; XU Xingya; QI XinPeng; ZHANG XiuQing; YANG HuanMing


    To meet the needs of large-scale genomic/genetic studies, the next-generation massively parallelized sequencing technologies provide high throughput, low cost and low labor-intensive sequencing ser-vice, with subsequent bioinformatic software and laboratory methods developed to expand their ap-plications in various types of research. PCR-based genomic/genetic studies, which have significant usage in association studies like cancer research, haven't benefited much from those next-generation sequencing technologies, because the shortgun re-sequencing strategy used by such sequencing machines as the Illumina/Solexa Genome Analyzer may not be applied to direct re-sequencing of short-length target regions like those in PCR-based genomic/genetic studies. Although several meth-ods have been proposed to solve this problem, including microarray-based genomic selections and selector-based technologies, they require advanced equipment and procedures which limit their ap-plications in many laboratories. By contrast, we overcame such potential drawbacks by utilizing a liga-tion by amplification (LBA) protocol, a method using a pair of Universal Adapters to randomly ligate target regions in a two-step-PCR procedure, whose Long LBA products were easily fragmented and sequenced on the next-generation sequencing machine. In this concept-proven study, we chose the consensus coding sequences of two human cancer genes: BRCA1 and BRCA2 as target regions, spe-cifically designed LBA primer pairs to amplify and randomly ligate them. 70 target sequences were successfully amplified and ligated into Long LBA products, which were then fragmented to construct DNA libraries for sequencing on both a conventional Sanger sequencer ABI 3730xl DNA Analyzer and the next-generation 'synthesis by sequencing technology' IlluminalSolexa Genome Analyzer. Bioin-formatic analysis demonstrated the utility and efficiency (including the coverage and depth of each target sequence and the SNPs detection

  14. Diagnóstico de Mycoplasma genitalium por amplificación de los genes MgPa y ARN ribosomal 16S Diagnosis of Mycoplasma genitalium by MgPa and rRNA 16S gene amplification

    Directory of Open Access Journals (Sweden)

    Carmen Fernández-Molina


    Full Text Available OBJETIVO: El microorganismo Mycoplasma genitalium se ha relacionado con la uretritis no gonocócica (UNG. La técnica de PCR se ha convertido en el principal método de detección de este patógeno. En consecuencia, debe aplicarse un método de diagnóstico mediante la amplificación de fragmentos de ADN por la técnica PCR. MATERIAL Y MÉTODOS: Se seleccionaron los cebadores MGF-MGR y MgPaF-MgPaR, complementarios de los genes de ARNr 16S y MgPa de M. genitalium, respectivamente. Se efectuaron ensayos de especificidad y sensibilidad y se estudiaron muestras clínicas. RESULTADOS: La PCR con cada grupo de cebadores utilizado fue específica sólo para M. genitalium y la sensibilidad fue mayor con el grupo de cebadores MGF-MGR. En el estudio de 34 muestras clínicas, 18.5% fue positivo a M. genitalium y se encontró un mayor número de muestras positivas al utilizar los cebadores MgPaF-MgPaR. CONCLUSIONES: Debe aplicarse en la práctica clínica el diagnóstico de M. genitalium mediante la amplificación del ADN por PCR en los pacientes con UNG.OBJECTIVE: Mycoplasma genitalium has been associated with nongonococcal urethritis (NGU. Diagnosis by PCR has become the primary detection method for this organism. Thus, diagnosis by DNA amplification using the PCR technique should be utilized. MATERIAL AND METHODS: GMF/GMR and MgpF/MgpR primer pairs, complementary to the M. genitalium 16S rRNA and MgPa genes, respectively, were selected. Specificity and sensibility assays were conducted and clinical samples were studied. RESULTS: The PCR with each primer pair was specific only for M. genitalium, and the sensibility was higher with the GMF/GMR primers. In the study of 34 clinical samples, 18,5% were positive for M. genitalium, with more positive samples when the MgpF/MgpR primers were used. CONCLUSIONS: DNA amplification by PCR should be applied in clinical practice to the diagnosis of M. genitalium in patients with NGU should using.

  15. Can Genomic Amplification of Human Telomerase Gene and C-MYC in Liquid-Based Cytological Specimens Be Used as a Method for Opportunistic Cervical Cancer Screening? (United States)

    Gao, Kun; Eurasian, Menglan; Zhang, Jieqing; Wei, Yuluan; Zheng, Qian; Ye, Hongtao; Li, Li


    To evaluate the effectiveness of five methods including the ThinPrep cytological test (TCT), liquid-based cytology, the human papillomavirus (HPV) test, detection of the TERC and C-MYC genes and visual inspection with acetic acid/Lugol's iodine (VIA/VILI) for opportunistic cervical cancer screening, and to explore whether genomic amplification of the human telomerase gene and C-MYC in liquid-based cytological specimens can be used as a method for opportunistic cervical cancer screening. Data were collected prospectively from 1,010 consecutive patients who visited the gynecology clinic and agreed to participate in opportunistic cervical cancer screening at our institution from November 2010 to July 2011. The five methods mentioned above were used for the screening in all cases. The histopathological diagnosis served as the gold standard for the evaluation. A comparison between the five screening methods for the diagnosis of high-grade cervical intraepithelial neoplasia (CIN II and III) was performed for their sensitivity, specificity, false-positive rate, false-negative rate, accuracy rate, positive likelihood ratio and negative likelihood ratio. A comprehensive comparison of the different combination programs for screening was performed according to the analysis of the receiver operating characteristic (ROC) curve area. The accuracy of the five screening methods for the diagnosis of high-grade CIN (CIN II and III) was compared in the different age groups. A joint model for the diagnosis using different combinations of the five methods was developed according to the analysis by the SAS 8.0 software. The model was used to evaluate the accuracy of the different combination programs for the diagnosis of high-grade CIN, and the results were confirmed by the histopathological examination. The sensitivity and specificity of the single screen method (TCT, HPV test, detection of the TERC and C-MYC genes, and VIA/VILI method) for CIN II was 80.9, 70.2, 72.3, 76.6, and 72

  16. Lack of specificity for PCR assays targeting human Bacteroides 16S rRNA gene: cross-amplification with fish feces (United States)

    Methods focused on members of the genus Bacteroides have been increasingly utilized in microbial source tracking studies for identifying and quantifying sources of non-point fecal contamination. We present results using real-time PCR to show significant cross-amplification of a human-specific Bacter...

  17. Altered DNA Binding and Amplification of Human Breast Cancer Suppressor Gene BRCA1 Induced by a Novel Antitumor Compound, [Ru(η6-p-phenylethacrynateCl2(pta

    Directory of Open Access Journals (Sweden)

    Adisorn Ratanaphan


    Full Text Available The ruthenium-based complex [Ru(η6-p-phenylethacrynateCl2(pta] (pta = 1,3,5-triaza-7-phosphatricyclo-[]decane, termed ethaRAPTA, is an interesting antitumor compound. The elucidation of the molecular mechanism of drug activity is central to the drug development program. To this end, we have characterized the ethaRAPTA interaction with DNA, including probing the sequence specific modified DNA structural stability and DNA amplification using the breast cancer suppressor gene 1 (BRCA1 of human breast and colon adenocarcinoma cell lines as models. The preference of ethaRAPTA base binding is in the order A > G > T > C. Once modified, the ethaRAPTA-induced BRCA1 structure has higher thermal stability than the modified equivalents of its related compound, RAPTA-C. EthaRAPTA exhibits a higher efficiency than RAPTA-C in inhibiting BRCA1 amplification. With respect to both compounds, the inhibition of BRCA1 amplification is more effective in an isolated system than in cell lines. These data provide evidence that will help to understand the process of elucidating the pathways involved in the response induced by ethaRAPTA.

  18. Identification of Staphylococcus aureus, S. intermedius and S. hyicus by PCR amplification of coa and nuc genes Identificação de Staphylococcus aureus, S. intermedius e S. hyicus através de seqüências dos genes coa e nuc

    Directory of Open Access Journals (Sweden)

    Wladimir Padilha da Silva


    Full Text Available Sixty-five strains of coagulase positive staphylococci (Staphylococcus aureus, S. intermedius and S. hyicus were identified at species level by PCR amplification of the coa gene, specific for S. aureus, and of the nuc gene, specific for S. intermedius and for S. hyicus.Sessenta e cinco cepas de estafilococos coagulase positiva foram identificadas em nível de espécie, através da amplificação, por PCR, de seqüências do gene coa, específicas para S. aureus, e do gene nuc, específicas para S. intermedius e para S. hyicus.

  19. The Seneca Amplification Construction

    Directory of Open Access Journals (Sweden)

    Wallace Chafe


    Full Text Available The polysynthetic morphology of the Northern Iroquoian languages presents a challenge to studies of clause combining. The discussion here focuses on a Seneca construction that may appear within a single clause but may also straddle clause boundaries. It amplifies the information provided by a referent, here called the trigger, that is introduced by the pronominal prefix within a verb or occasionally in some other way. The particle neh signals that further information about that referent will follow. This construction is found at four levels of syntactic complexity. At the first level the trigger and its amplification occur within the same prosodic phrase and the amplification is a noun. At the second level the amplification occurs in a separate prosodic phrase but remains a noun. At the third level the amplification exhibits verb morphology but has been lexicalized with a nominal function. At the fourth level the amplification functions as a full clause and neh serves as a marker of clause combining. Several varieties of amplification are discussed, as are cases in which the speaker judges that no amplification is needed. It is suggested that the typologically similar Caddo language illustrates a situation in which this construction could never arise, simply because Caddo verbs lack the pronominal element that triggers the construction in Seneca.

  20. Gene amplification: developments, and applications for medical diagnostics. January 1985-February 1989 (Citations from the Biobusiness data base). Report for January 1985-February 1989

    Energy Technology Data Exchange (ETDEWEB)


    This bibliography contains citations concerning genetic amplification, a revolutionary new DNA probe technology. Companies on the cutting edge of this technology are discussed, and mention is made of their licensing agreements. Applications in clinical diagnosis for such diseases as acquired immune deficiency syndrome and leukemia, and applications for prenatal testing, and forensic testing are examined. (Contains 55 citations fully indexed and including a title list.)

  1. Amplification of NOON States

    CERN Document Server

    Agarwal, G S; Rai, Amit


    We examine the behavior of a Non Gaussian state like NOON state under phase insensitive amplification. We derive analytical result for the density matrix of the NOON state for arbitrary gain of the amplifier. We consider cases of both symmetric and antisymmetric amplification of the two modes of the NOON state. We quantitatively evaluate the loss of entanglement by the amplifier in terms of the logarithmic negativity parameter. We find that NOON states are more robust than their Gaussian counterparts.

  2. Amplification of NOON States



    We examine the behavior of a Non Gaussian state like NOON state under phase insensitive amplification. We derive analytical result for the density matrix of the NOON state for arbitrary gain of the amplifier. We consider cases of both symmetric and antisymmetric amplification of the two modes of the NOON state. We quantitatively evaluate the loss of entanglement by the amplifier in terms of the logarithmic negativity parameter. We find that NOON states are more robust than their Gaussian coun...

  3. 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 (United States)


    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 C-MYC test showed lower sensitivity (80.0% vs. 84.0%) and higher specificity (77.7% vs. 64.3%). Using a cut-off value of 5% or more aberrant cells, the TERC test showed the highest combination of sensitivity and specificity. The CIN2+ group showed more high-level TERC gene copy number (GCN) cells than did the normal/CIN1 group (p C-MYC, no significant difference between the two histological categories was detected (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

  4. 苔藓植物matK基因PCR反应体系的建立和优化%Establishment and Optimization of matK gene-PCR Amplification System for Bryophytes

    Institute of Scientific and Technical Information of China (English)

    郑红建; 张安世


    [Objective] To establish and optimize the matK gene-PCR reaction system for Bryophytes. [ Method ] Using genomic DNA of Bryo-phytes extracted via an improved CTAB method, single factor analysis was performed to investigate the impacts of DNA template concentration, primer concentration, 2 x Taq MasterMix concentration on matK gene-PCR amplification and to optimize this system for Bryophytes. [Result] The matK gene-PCR amplification(10.0 μl) suitable for Bryophytes was determined to be composed of 0.5 μl DNA, 0.2 μl primer and 5.6 μl 2 × Taq MasterMix. [ Conclusion] The study laid basis for analyzing the molecular systematics of Bryophytes.%[目的]建立和优化适合于苔藓植物matK基因的PCR反应体系.[方法]以鳞叶藓为材料,利用改良CTAB法提取了基因组DNA,利用单因素试验分析了DNA模板、引物和2×Taq MasterMix的浓度对苔藓植物matK基因PCR反应的影响,对适合苔藓植物的matK基因PCR反应条件进行了优化.[结果]适合苔藓植物的matK基因PCR反应体系为10.0 μl,其中含DNA模板0.5μl,正反引物各0.2μl,2×Taq MasterMix 5.6 μl.[结论]为苔藓植物的分子系统学等研究奠定了基础.

  5. Efficacy Improvement of PCR Amplification of CAG Trinucleotide Repeats in the Coding Sequence of Spinocerebellar Ataxia Type II Gene%提高SCA2编码区CAG三核苷酸重复的PCR扩增效率

    Institute of Scientific and Technical Information of China (English)

    汤熙翔; 夏家辉


    To improve the efficacy of PCR amplification of CAG trinucleotide repeats in the coding sequence of spinocerebellar ataxia type II gene(69.2% G+C), hot-start PCR, base-replacement PCR, and the addition of enhancers(1%~12.5% DMSO , 1%~25% glycerol ,1%~12.5% formamide) were performed and compared with normal PCR . The results showed that hot-start PCR, base-replacement PCR and the addition of enhancers(1%~10% DMSO , 5%~20% glycerol , 1%~10% formamide) improved the amplification efficacy of the GC rich region. Gene diagnosis in 70 SCA pedgrees and 60 spontaneous SCA patients were also conducted.%以遗传性脊髓小脑共济失调II型基因(spinocerebellar ataxia type II gene SCA2)编码区内的CAG三核苷酸重复为研究对象(G+C含量为69.2%),比较了热启动PCR、碱基替代PCR、添加增效剂(1%~12.5%二甲亚砜、1%~25%甘油、1%~12.5%甲酰胺)与常规PCR的扩增效率,发现热启动PCR、碱基替代PCR及添加增效剂(1%~10%二甲亚砜、5%~20%甘油、1%~10%甲酰胺)能提高该GC富集区的扩增效率,并对70个SCA家系及60个散发SCA患者进行了SCA2的基因诊断。

  6. Comparison of loop-mediated isothermal amplification (LAMP) and nested-PCR assay targeting the RE and B1 gene for detection of Toxoplasma gondii in blood samples of children with leukaemia. (United States)

    Fallahi, Shirzad; Seyyed Tabaei, Seyyed Javad; Pournia, Yadollah; Zebardast, Nozhat; Kazemi, Bahram


    Toxoplasmosis diagnosis constitutes an important measure for disease prevention and control. In this paper, a newly described DNA amplification technique, loop-mediated isothermal amplification (LAMP), and nested-PCR targeting the repeated element (RE) and B1 gene, were compared to each other for the detection of Toxoplasma gondii DNA in blood samples of children with leukaemia. One hundred ten blood samples from these patients were analyzed by LAMP and nested-PCR. Out of 50 seropositive samples (IgM+, IgG+), positive results were obtained with 92% and 86% on RE, B1-LAMP and 82% and 68% on RE, B1-nested PCR analyses, respectively. Of the 50 seronegative samples, three, two and one samples were detected positive by RE-LAMP, B1-LAMP and RE-nested PCR assays, respectively, while none were detected positive by B1-nested PCR. None of the 10 IgM-, IgG+ samples was detected positive after testing LAMP and nested-PCR assays in duplicate. This is the first report of a study in which the LAMP method was applied with high sensitivity and efficacy for the diagnosis of T. gonii in blood samples of children with leukaemia.

  7. Amplification and flagellin typing of pseudomonas aeruginosa by molecular method

    Directory of Open Access Journals (Sweden)

    gholamreza goudarzi


    Conclusion: This method could be utilized to determine flagellated (Motile and non-flagellated strains of P. aeruginosa, genotyping, amplification of full coding sequence of fliC gene in order to clone and express recombinant flagellin protein.

  8. Comparative analysis of the EGFR, HER2, c-MYC, and MET variations in colorectal cancer determined by three different measures: gene copy number gain, amplification status and the 2013 ASCO/CAP guideline criterion for HER2 testing of breast cancer. (United States)

    Kwak, Yoonjin; Yun, Sumi; Nam, Soo Kyung; Seo, An Na; Lee, Kyu Sang; Shin, Eun; Oh, Heung-Kwon; Kim, Duck Woo; Kang, Sung Bum; Kim, Woo Ho; Lee, Hye Seung


    The purpose of this study was to explore gene copy number (GCN) variation of EGFR, HER2, c-MYC, and MET in patients with primary colorectal cancer (CRC). Dual-colour silver-enhanced in situ hybridization was performed in tissue samples of 334 primary CRC patients. The amplification status (GCN ratio ≥2) and GCN gain (average GCN ≥4) data for the EGFR, HER2, c-MYC and MET genes were obtained. GCN variation was also assessed by the criterion of the 2013 ASCO/CAP guidelines for HER2 testing. Amplification of EGFR, HER2, c-MYC and MET was detected in 8 (2.4%), 20 (6.0%), 29 (8.7%), and 14 (4.2%) patients, respectively. Of 66 patients with at least one amplified gene, five exhibited co-amplification of genes studied (HER2-MET co-amplification: two patients; HER2-c-MYC co-amplification: two patients; EGFR-c-MYC co-amplification: one patient). There were 109 patients with GCN gains of one or more genes (EGFR: 11/334, HER2: 29/334, c-MYC; 60/334, MET: 48/334) and 32.1% (35/109) had multiple GCN gains. When each GCN was assessed by the criterion of the ASCO/CAP 2013 guideline for HER2 testing, 116 people showed positive or equivocal results for one or more genes. The cumulative amplification status had no association with patients' outcome. However, the cumulative results of the GCN gain and GCN status determined according to the ASCO/CAP guideline had a significant prognostic correlation in the univariate analysis (P values of 0.006 and 0.022, respectively). In the multivariate analysis, GCN gain and GCN status were independent prognostic factors (P values of 0.010 and 0.017, respectively). In this study, we evaluated GCN variation of four genes in a large sample of Korean CRC patients. The amplification status was not related to patient outcome. However, the GCN gain and GCN status according to the ASCO/CAP 2013 guideline were independent prognostic factors.

  9. Biomaterials in light amplification (United States)

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


    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

  10. Development of Visual Loop-Mediate Isothermal Amplification Assay for CpTI gene%抗虫转CpTI基因成分现场可视化检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    王永; 兰青阔; 朱珠; 赵新; 陈锐; 刘娜; 李欧静; 郭永泽; 程奕


    It is important to detect Cowpea Trypsin Inhibitor (CpTI) gene in food or feed materials in order to prevent the illegal diffusion. A rapid visual loop-mediated isothermal amplification (LAMP) method assay for CpTI gene detection was developed. A set of primers were designed according to the nucleotide sequence of the target CpTI gene, seven genetically modified organisms (GMO) were detected by LAMP for method specificity, and meanwhile the mode of artificially contaminated food was constructed to evaluate the sensitivity of LAMP assay and qualitative PCR method. The results showed that the CpTI gene had specific amplification, but the non- CpTI GMO submitted negative reactions. Sensitivity of LAMP assay for Kefeng No.6 genetically modified rice was 0.005%. In conclusion, the LAMP assay developed in the present study is a specific, sensitive, simple and convenient method for the rapid screening of CpTI gene in contaminated foods.%豇豆胰蛋白酶抑制剂(CpTI)基因是目前仅次于Bt基因的广谱性抗虫基因,常用于转基因水稻和转基因棉花的基因工程研究中。根据CpTI基因特异性碱基序列设计引物,应用可视化环介导等温扩增技术,对转基因水稻科丰6号进行扩增,同时建立转基因成分人工污染的食品模型,比较了 LAMP 法与定性PCR方法检测的敏感性。结果表明:该方法仅对CpTI基因产生特异性扩增,灵敏度达0.005%;所建立的CpTI基因现场可视化筛查方法具有较高的特异性与灵敏性,操作简单、快速,可用于含有CpTI基因转基因成分污染的快速检测。

  11. Real-time fluorescence PCR for HER2 gene amplification%实时荧光PCR法检测}HER2基因扩增方法的建立

    Institute of Scientific and Technical Information of China (English)

    邓艳华; 吴荃; 陈华云; 李明


    Objective To evaluate the double standard curve of Real-time fluorescence PCR to detect human epidermalgrowth factor reeeptor-2(HER2) gene amplification for breast cancer diagnosis and treatment. Methods Genomie DNA was extracted in 500 eases fresh tissue samples of breast cancer for real-time fluorescence PCR and quantified by double standard curve. Data was analyzed by calculating the concentration ratio of the target gene to the referenced gene detected. Fluorescent in situ hybridization (FISH) was chosen as the control method for HER2 detection. Results 72 cases of positive samples and 419 negative samples were detected by this method. The sensitivity was 85.9%, the specificity was 98.79% and the accuracy is 96.74%. Compared with the FISH test, the results of both methods have good consistency. Conclusion The method of double standard curve of Real-time fluoreacence PCR to detect HER2 gene amplification has a wide application prospect for breast cancer diagnosis and treatment.%目的 探讨应用双标准曲线的实时荧光PCR法检测原癌基因人类表皮生长因子受体2(HER2)基因扩增在临床乳腺癌诊治中的可行性.方法 收集500例乳腺癌术后新鲜组织标本,抽提组织DNA进行实时荧光PCR检测,采用双标准曲线法定量,通过计算目的 基因浓度和内标基因浓度的比值来判断HER2基因的扩增情况.选择灵敏度和特异度均较高的荧光原位杂交方法作为对照方法.结果 检测阳性标本72例,阴性样本419例.该方法检测的灵敏度为85.9%,特异度为98.79%,准确度为96.74.与荧光原位杂交法(FISH)检测结果相比,二者具有较好的一致性.结论 双标准曲线的实时荧光PCR法用于检测HER2基因扩增相对准确可靠,有较好的临床应用前景.

  12. Analysis of 525 samples to determine the usefulness of PCR amplification and sequencing of the 16S rRNA gene for diagnosis of bone and joint infections. (United States)

    Fenollar, Florence; Roux, Véronique; Stein, Andréas; Drancourt, Michel; Raoult, Didier


    The 16S rRNA gene PCR in the diagnosis of bone and joint infections has not been systematically tested. Five hundred twenty-five bone and joint samples collected from 525 patients were cultured and submitted to 16S rRNA gene PCR detection of bacteria in parallel. The amplicons with mixed sequences were also cloned. When discordant results were observed, culture and PCR were performed once again. Bacteria were detected in 139 of 525 samples. Culture and 16S rRNA gene PCR yielded identical documentation in 475 samples. Discrepancies were linked to 13 false-positive culture results, 5 false-positive PCR results, 9 false-negative PCR results, 16 false-negative culture results, and 7 mixed infections. Cloning and sequencing of 16S rRNA gene amplicons in 6 of 8 patients with mixed infections identified 2 to 8 bacteria per sample. Rarely described human pathogens such as Alcaligenes faecalis, Comamonas terrigena, and 21 anaerobes were characterized. We also detected, by 16S rRNA gene PCR, four previously identified bacteria never reported in human infection, Alkanindiges illinoisensis, dehydroabietic acid-degrading bacterium DhA-73, unidentified Hailaer soda lake bacterium, and uncultured bacterium clone HuCa4. Seven organisms representing new potential species were also detected. PCR followed by cloning and sequencing may help to identify new pathogens involved in mixed bone infection.

  13. Development of a cost-efficient novel method for rapid, concurrent genotyping of five common single nucleotide polymorphisms of the brain derived neurotrophic factor (BDNF) gene by tetra-primer amplification refractory mutation system. (United States)

    Wang, Cathy K; Xu, Michael S; Ross, Colin J; Lo, Ryan; Procyshyn, Ric M; Vila-Rodriguez, Fidel; White, Randall F; Honer, William G; Barr, Alasdair M


    Brain derived neurotrophic factor (BDNF) is a molecular trophic factor that plays a key role in neuronal survival and plasticity. Single nucleotide polymorphisms (SNPs) of the BDNF gene have been associated with specific phenotypic traits in a large number of neuropsychiatric disorders and the response to psychotherapeutic medications in patient populations. Nevertheless, due to study differences and occasionally contrasting findings, substantial further research is required to understand in better detail the association between specific BDNF SNPs and these psychiatric disorders. While considerable progress has been made recently in developing advanced genotyping platforms of SNPs, many high-throughput probe- or array-based detection methods currently available are limited by high costs, slow processing times or access to advanced instrumentation. The polymerase chain reaction (PCR)-based, tetra-primer amplification refractory mutation system (T-ARMS) method is a potential alternative technique for detecting SNP genotypes efficiently, quickly, easily, and cheaply. As a tool in psychopathology research, T-ARMS was shown to be capable of detecting five common SNPs in the BDNF gene (rs6265, rs988748, rs11030104, 11757G/C and rs7103411), which are all SNPs with previously demonstrated clinical relevance to schizophrenia and depression. The present technique therefore represents a suitable protocol for many research laboratories to study the genetic correlates of BDNF in psychiatric disorders. Copyright Copyright © 2015 John Wiley & Sons, Ltd.

  14. Comparison between NuGEN's WT-Ovation Pico and one-direct amplification systems. (United States)

    Morse, Alison M; Carballo, Valentina; Baldwin, Donald A; Taylor, Christopher G; McIntyre, Lauren M


    Differential gene expression between groups of homogenous cell types is a biological question whose time has come. RNA can be extracted from small numbers of cells, such as those isolated by laser-capture microdissection, but the small amounts obtained often require amplification to enable whole genome transcriptome profiling by technologies such as microarray analysis and RNA-seq. Recently, advances in amplification procedures make amplification directly from whole cell lysates possible. The aim of this study was to compare two amplification systems for variations in observed RNA abundance attributable to the amplification procedure for use with small quantities of cells isolated by laser-capture microdissection. Arabidopsis root cells undergoing giant cell formation as a result of nematode infestation and uninfested control root cells were laser-captured and used to evaluate two amplification systems. One, NuGEN's WT-Ovation Pico (Pico) amplification system, uses total RNA as starting material, and the other, NuGEN's WT-One-Direct (One-Direct) amplification system, uses lysate containing the captured cells. The reproducibility of whole genome transcript profiling and correlations of both systems were investigated after microarray analysis. The One-Direct system was less reproducible and more variable than the Pico system. The Pico amplification kit resulted in the detection of thousands of differentially expressed genes between giant cells and control cells. This is in marked contrast to the relatively few genes detected after amplification with the One-Direct amplification kit.

  15. High copy number variation of cancer-related microRNA genes and frequent amplification of DICER1 and DROSHA in lung cancer (United States)

    Czubak, Karol; Lewandowska, Marzena Anna; Klonowska, Katarzyna; Roszkowski, Krzysztof; Kowalewski, Janusz; Figlerowicz, Marek; Kozlowski, Piotr


    A growing body of evidence indicates that miRNAs may be a class of genetic elements that can either drive or suppress oncogenesis. In this study we analyzed the somatic copy number variation of 14 miRNA genes frequently found to be either over- or underexpressed in lung cancer, as well as two miRNA biogenesis genes, DICER1 and DROSHA, in non-small-cell lung cancer (NSCLC). Our analysis showed that most analyzed miRNA genes undergo substantial copy number alteration in lung cancer. The most frequently amplified miRNA genes include the following: miR-30d, miR-21, miR-17 and miR-155. We also showed that both DICER1 and DROSHA are frequently amplified in NSCLC. The copy number variation of DICER1 and DROSHA correlates well with their expression and survival of NSCLC and other cancer patients. The increased expression of DROSHA and DICER1 decreases and increases the survival, respectively. In conclusion, our results show that copy number variation may be an important mechanism of upregulation/downregulation of miRNAs in cancer and suggest an oncogenic role for DROSHA. PMID:26156018

  16. Identification of a novel, invasive, not-yet-cultivated Treponema sp in the large intestine of pigs by PCR amplification of the 16S rRNA gene

    DEFF Research Database (Denmark)

    Mølbak, Lars; Schou, Kirstine Klitgaard; Jensen, Tim Kåre;


    Laser capture microdissection in combination with fluorescent in situ hybridization was used to identify an unknown species of spirochetes from the pig colonic mucosa. The 16S rRNA gene was PCR amplified, and the closest related type strain was Treponema bryantii(T) (90.1%). The spirochete, here...

  17. Spurious Amplification of a Plasmodium vivax Small-Subunit RNA Gene by Use of Primers Currently Used To Detect P. knowlesi▿ (United States)

    Imwong, Mallika; Tanomsing, Naowarat; Pukrittayakamee, Sasithon; Day, Nicholas P. J.; White, Nicholas J.; Snounou, Georges


    The PCR primers commonly used to detect Plasmodium knowlesi infections in humans were found to cross-react stochastically with P. vivax genomic DNA. A nested primer set that targets one of the P. knowlesi small-subunit rRNA genes was validated for specificity and for sensitivity of detection of <10 parasite genomes. PMID:19812279

  18. Amplification of Surface Antigen P43 Gene and Its Application in Detection of Toxoplasma Gondii in Allogeneic Hematopoietic Stem Cell Transplantation

    Institute of Scientific and Technical Information of China (English)

    ZHOUYongan; YUXinbing; 等


    Objective:To establish a rapid,specific and sensitive diagnostic technique for the human Toxoplasma gondii infection in the recipi-ents with allogeneic hematopoietic stem cell transplantation and discuss its clinical significance.Methods:30 patients undergoing allogeneic hematopoietic stem cell transplantation were detected by using ELISA and PCR.Results:Among 30 recipients undergiong allogeneic hematopoietic stem cell transplantation,3 were positive for Toxoplasma gondiii antigen and 5 for surface antigen p43 gene with the positive rate being 13.3% and 16.67% respectively.20 healthy people(negative for anti-Tox antibody)were also tested by using ELISA and PCR.Conclusion:PCR is an accurate,relatively rapid,sensitive and specific method for detecting P43 gene of Toxoplasma gondii.Be-canuse PCR can be applied to a variety of different clinical samples,it can be considered as a valuable additional tool for identification of Toxoplasma gondii infections.

  19. Evaluation of PCR primer selectivity and phylogenetic specificity by using amplification of 16S rRNA genes from betaproteobacterial ammonia-oxidizing bacteria in environmental samples. (United States)

    Junier, Pilar; Kim, Ok-Sun; Hadas, Ora; Imhoff, Johannes F; Witzel, Karl-Paul


    The effect of primer specificity for studying the diversity of ammonia-oxidizing betaproteobacteria (betaAOB) was evaluated. betaAOB represent a group of phylogenetically related organisms for which the 16S rRNA gene approach is especially suitable. We used experimental comparisons of primer performance with water samples, together with an in silico analysis of published sequences and a literature review of clone libraries made with four specific PCR primers for the betaAOB 16S rRNA gene. With four aquatic samples, the primers NitA/NitB produced the highest frequency of ammonia-oxidizing-bacterium-like sequences compared to clone libraries with products amplified with the primer combinations betaAMOf/betaAMOr, betaAMOf/Nso1255g, and NitA/Nso1225g. Both the experimental examination of ammonia-oxidizing-bacterium-specific 16S rRNA gene primers and the literature search showed that neither specificity nor sensitivity of primer combinations can be evaluated reliably only by sequence comparison. Apparently, the combination of sequence comparison and experimental data is the best approach to detect possible biases of PCR primers. Although this study focused on betaAOB, the results presented here more generally exemplify the importance of primer selection and potential primer bias when analyzing microbial communities in environmental samples.

  20. Hardness amplification in nondeterministic logspace


    Gupta, Sushmita


    A hard problem is one which cannot be easily computed by efficient algorithms. Hardness amplification is a procedure which takes as input a problem of mild hardness and returns a problem of higher hardness. This is closely related to the task of decoding certain error-correcting codes. We show amplification from mild average case hardness to higher average case hardness for nondeterministic logspace and worst-to-average amplification for nondeterministic linspace. Finally we explore possible ...

  1. The ERI-6/7 helicase acts at the first stage of an siRNA amplification pathway that targets recent gene duplications.

    Directory of Open Access Journals (Sweden)

    Sylvia E J Fischer


    Full Text Available Endogenous small interfering RNAs (siRNAs are a class of naturally occuring regulatory RNAs found in fungi, plants, and animals. Some endogenous siRNAs are required to silence transposons or function in chromosome segregation; however, the specific roles of most endogenous siRNAs are unclear. The helicase gene eri-6/7 was identified in the nematode Caenorhabditis elegans by the enhanced response to exogenous double-stranded RNAs (dsRNAs of the null mutant. eri-6/7 encodes a helicase homologous to small RNA factors Armitage in Drosophila, SDE3 in Arabidopsis, and Mov10 in humans. Here we show that eri-6/7 mutations cause the loss of 26-nucleotide (nt endogenous siRNAs derived from genes and pseudogenes in oocytes and embryos, as well as deficiencies in somatic 22-nucleotide secondary siRNAs corresponding to the same loci. About 80 genes are eri-6/7 targets that generate the embryonic endogenous siRNAs that silence the corresponding mRNAs. These 80 genes share extensive nucleotide sequence homology and are poorly conserved, suggesting a role for these endogenous siRNAs in silencing of and thereby directing the fate of recently acquired, duplicated genes. Unlike most endogenous siRNAs in C. elegans, eri-6/7-dependent siRNAs require Dicer. We identify that the eri-6/7-dependent siRNAs have a passenger strand that is ∼19 nt and is inset by ∼3-4 nts from both ends of the 26 nt guide siRNA, suggesting non-canonical Dicer processing. Mutations in the Argonaute ERGO-1, which associates with eri-6/7-dependent 26 nt siRNAs, cause passenger strand stabilization, indicating that ERGO-1 is required to separate the siRNA duplex, presumably through endonucleolytic cleavage of the passenger strand. Thus, like several other siRNA-associated Argonautes with a conserved RNaseH motif, ERGO-1 appears to be required for siRNA maturation.

  2. 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: [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)


    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.

  3. Targeting MET Amplification as a New Oncogenic Driver

    Directory of Open Access Journals (Sweden)

    Hisato Kawakami


    Full Text Available 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.

  4. Plasma HER2 amplification in cell-free DNA during neoadjuvant chemotherapy in breast cancer

    DEFF Research Database (Denmark)

    Bechmann, Troels; Andersen, Rikke Fredslund; Pallisgaard, Niels


    Measurement of human epidermal growth factor receptor 2 (HER2) gene amplification in cell-free DNA (cfDNA) is an evolving technique in breast cancer, enabling liquid biopsies and treatment monitoring. The present study investigated the dynamics of plasma HER2 gene copy number and amplification in...... in cfDNA during neoadjuvant chemotherapy....

  5. Primers to block the amplification of symbiotic apostome ciliate 18S rRNA gene in a PCR-based copepod diet study (United States)

    Yi, Xiaoyan; Zhang, Huan; Liu, Guangxing


    Pelagic copepods play an important role in the marine food web. However, a full understanding of the ecological status of this zooplankton group depends on the careful study of their natural diets. In previous PCR-based copepod diet studies, we found many apostome ciliates that live symbiotically under the exoskeleton of the copepods, and their sequences were often over-represented in the 18S rRNA gene (18S rDNA) libraries. As a first step to address this issue, we designed three apostome ciliate 18S rDNA blocking primers, and tested their blocking efficiency against apostome ciliate 18s rDNA under various PCR conditions. Using a semi-quantitative PCR method, we optimized the conditions to efficiently amplify the 18S rDNA of the prey while simultaneously excluding the symbiotic apostome ciliates. This technique will facilitate PCR-based diet studies of copepods and other zooplankton in their natural environments.

  6. Amplification, cloning and expression of Brucella melitensis bp26 gene (OMP28 isolated from Markazi province (Iran and purification of Bp26 Protein

    Directory of Open Access Journals (Sweden)

    Hosseini, S.D.


    Full Text Available Brucellosis is a debilitative disease that imposes costs on both economy and society. It is shown that although the vaccine can prevent abortion, it does not provide complete protection against infection. In Iran, Brucella melitensis is a common causative agent for brucellosis and BP26 protein of this bacterium having a good antigenesity and an important vaccine candidate. In this study B. melitensis bp26 gene was cloned first in to PTZ57R/T vector and accessed on the PET28a vector and sequenced. Recombinant vector transformed and expressed in to E. coli BL21 (DE3 and then recombinant protein was purified with Ni-NTA column of chromatography against His tag. Obtained rOmp28 could be used as a research experimental tool to find its potential as a detection kit and vaccine candidate.

  7. A New Method for Sex Determination Based on Detection of SRY, STS and Amelogenin Gene Regions with Simultaneous Amplification of Their Homologous Sequences by a Multiplex PCR

    Directory of Open Access Journals (Sweden)



    Full Text Available We have developed a new method for sex determination based on simultaneous detection of the SRY (sex-determining region Y, STS (steroid sulfatase and amelogenin (AMELX and AMELY gene regions and their homologous sequences. The sex of 246 blood samples was correctly determined by this method. An AMELY-deleted male sample, which would have been erroneously considered female based solely on analysis of the amelogenin locus, was successfully identified as male by the present method. The detection limit of this method was 63 pg of genomic DNA, and the male DNA component could be detected from mixed samples having a male:female ratio as low as 1:10. This method was useful for degraded DNA and possessed the human specificity. Practical application to 35 autopsy cases is described.

  8. A new method for sex determination based on detection of SRY, STS and amelogenin gene regions with simultaneous amplification of their homologous sequences by a multiplex PCR. (United States)

    Morikawa, Toshio; Yamamoto, Yuji; Miyaishi, Satoru


    We have developed a new method for sex determination based on simultaneous detection of the SRY (sex-determining region Y), STS (steroid sulfatase) and amelogenin (AMELX and AMELY) gene regions and their homologous sequences. The sex of 246 blood samples was correctly determined by this method. An AMELY-deleted male sample, which would have been erroneously considered female based solely on analysis of the amelogenin locus, was successfully identified as male by the present method. The detection limit of this method was 63 pg of genomic DNA, and the male DNA component could be detected from mixed samples having a male:female ratio as low as 1:10. This method was useful for degraded DNA and possessed the human specificity. Practical application to 35 autopsy cases is described.

  9. Nucleic acid amplification: Alternative methods of polymerase chain reaction. (United States)

    Fakruddin, Md; Mannan, Khanjada Shahnewaj Bin; Chowdhury, Abhijit; Mazumdar, Reaz Mohammad; Hossain, Md Nur; Islam, Sumaiya; Chowdhury, Md Alimuddin


    Nucleic acid amplification is a valuable molecular tool not only in basic research but also in application oriented fields, such as clinical medicine development, infectious diseases diagnosis, gene cloning and industrial quality control. A comperehensive review of the literature on the principles, applications, challenges and prospects of different alternative methods of polymerase chain reaction (PCR) was performed. PCR was the first nucleic acid amplification method. With the advancement of research, a no of alternative nucleic acid amplification methods has been developed such as loop mediated isothermal amplification, nucleic acid sequence based amplification, strand displacement amplification, multiple displacement amplification. Most of the alternative methods are isothermal obviating the need for thermal cyclers. Though principles of most of the alternate methods are relatively complex than that of PCR, they offer better applicability and sensitivity in cases where PCR has limitations. Most of the alternate methods still have to prove themselves through extensive validation studies and are not available in commercial form; they pose the potentiality to be used as replacements of PCR. Continuous research is going on in different parts of the world to make these methods viable technically and economically.

  10. Isothermal Amplification of Nucleic Acids. (United States)

    Zhao, Yongxi; Chen, Feng; Li, Qian; Wang, Lihua; Fan, Chunhai


    Isothermal amplification of nucleic acids is a simple process that rapidly and efficiently accumulates nucleic acid sequences at constant temperature. Since the early 1990s, various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). These isothermal amplification methods have been used for biosensing targets such as DNA, RNA, cells, proteins, small molecules, and ions. The applications of these techniques for in situ or intracellular bioimaging and sequencing have been amply demonstrated. Amplicons produced by isothermal amplification methods have also been utilized to construct versatile nucleic acid nanomaterials for promising applications in biomedicine, bioimaging, and biosensing. The integration of isothermal amplification into microsystems or portable devices improves nucleic acid-based on-site assays and confers high sensitivity. Single-cell and single-molecule analyses have also been implemented based on integrated microfluidic systems. In this review, we provide a comprehensive overview of the isothermal amplification of nucleic acids encompassing work published in the past two decades. First, different isothermal amplification techniques are classified into three types based on reaction kinetics. Then, we summarize the applications of isothermal amplification in bioanalysis, diagnostics, nanotechnology, materials science, and device integration. Finally, several challenges and perspectives in the field are discussed.

  11. Efficient audio power amplification - challenges

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, Michael A.E.


    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)

  12. Comparison of Gene Amplification Effect under Three Methods of PCR Based on Degenerate Primers%基于兼并引物的 PCR 扩增特定基因的效果比较

    Institute of Scientific and Technical Information of China (English)

    李志强; 何德; 李翠新


    Three PCR methods(conventional PCR, touchdown PCR, nested PCR) were used to amplify the DNA topoisomeraseⅡfragments of Pleurotus ostreatus and P.eryngii var ferulae with degenerate prim-ers,which were designed with the gene sequences of DNA topoisomerase Ⅱfrom NCBI.Their PCR sensi-tivity and specificity were compared .The result showed that the sensitivity and specificity of nested PCR were highest , and its sensitivity was 100 fold higher than conventional PCR , which indicated that the nested PCR was more suitable for PCR amplification under degenerate primers .This study will have some guidance significance to obtain the specific gene through degenerate primers .%根据NCBI上的DNA拓扑异构酶Ⅱ基因序列设计兼并引物,采用常规PCR、降落PCR、巢式PCR三种方法扩增糙皮侧耳和阿魏侧耳的拓扑异构酶Ⅱ部分基因序列,比较三种方法扩增效果的特异性、灵敏度。结果表明,巢式PCR的灵敏度和特异性都优于常规PCR和降落PCR,并且巢式PCR的灵敏度是常规PCR灵敏度的100倍以上,更加适合兼并引物扩增。这对利用兼并引物获取特异基因具有指导意义。

  13. 乳液PCR扩增复杂基因序列的方法建立及普通PCR的比较%The Construction of the Emulsion PCR Amplification Complex Gene Sequence and Its Comparison with Conventional PCR

    Institute of Scientific and Technical Information of China (English)

    章爱娣; 徐敏轩; 韦婷; 周东蕊


    该研究分析普通PCR的不足,通过较多实验研究初步建立乳液PCR方法,改善PCR实验技术.采用人工合成的乳酸杆菌质粒为模板,不同成分配比条件下的乳液PCR和普通PCR对质粒序列进行扩增,对扩增产物进行琼脂糖凝胶电泳,电泳结束后对凝胶进行Gelred染色并用GS-800灰度扫描仪成像.进一步分析乳液PCR和普通PCR扩增产物的特异性以及不同成分配比条件下乳液PCR的扩增质量.通过比较两种PCR方法的扩增结果可得,在相同条件下,乳液PCR扩增特异性高于普通PCR扩增,并确定当水相中加入100g/LBSA,水油体积比在1:2.5时,破乳水饱和乙醚体积比为1:1时,水油相在1700rpm条件下连续混合5min时扩增效果最好.%In the present study, the shortage of Amplification Complex Gene Sequence by Conventional PCR was analyzed, and then a protocol was constructed to minimize these problems. With the plasmid of Lactobacillus as a template, different components of the emulsion PCR and the conventional PCR plasmid template were effectively amplified to promote the amplified product with PCR to carry on agarose electrophoresis. With that accomplished, the Gelred dyeing was done to the gelatin and the image formation was obtained by a GS-800 gradation scanner. Then a further analysis was made to get the specialities of the emulsion PCR and the conventional PCR amplified products and the producing quality by emulsion PCR. In terms of different components, it was found out by contrasting the two methods that the speciality of the emulsion PCR amplification was higher than that of the conventional PCR with the same conditions. Moreover, when 100 g/1 BSA was added to aqueous phase with the volume ratio of water-in-oil(w/o) at 1:2.5, the volume ratio of breaking water-saturated diethyl ether at 1:1 and water and oil mixture was thoroughly mixed in 5 min at 1700 rpm, the best amplification effect was obtained.

  14. Engineering targeted chromosomal amplifications in human breast epithelial cells. (United States)

    Springer, Simeon; Yi, Kyung H; Park, Jeenah; Rajpurohit, Anandita; Price, Amanda J; Lauring, Josh


    Chromosomal amplifications are among the most common genetic alterations found in human cancers. However, experimental systems to study the processes that lead to specific, recurrent amplification events in human cancers are lacking. Moreover, some common amplifications, such as that at 8p11-12 in breast cancer, harbor multiple driver oncogenes, which are poorly modeled by conventional overexpression approaches. We sought to develop an experimental system to model recurrent chromosomal amplification events in human cell lines. Our strategy is to use homologous-recombination-mediated gene targeting to deliver a dominantly selectable, amplifiable marker to a specified chromosomal location. We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using the E. coli inosine monophosphate dehydrogenase (IMPDH) enzyme as a marker. We applied selective pressure using IMPDH inhibitors. Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR and FISH. Genome-wide array comparative genomic hybridization confirmed that amplifications had occurred on the short arm of chromosome 8, without changes on 8q or other chromosomes. Patterns of amplification were variable and similar to those seen in primary human breast cancers, including "sawtooth" patterns, distal copy number loss, and large continuous regions of copy number gain. This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a model to analyze oncogene cooperativity in amplifications harboring multiple candidate driver genes.

  15. Transcriptional Amplification in Tumor Cells with Elevated c-Myc (United States)

    Lin, Charles Y.; Lovén, Jakob; Rahl, Peter B.; Paranal, Ronald M.; Burge, Christopher B.; Bradner, James E.; Lee, Tong Ihn; Young, Richard A.


    Summary Elevated expression of the c-Myc transcription factor occurs frequently in human cancers and is associated with tumor aggression and poor clinical outcome. The effect of high levels of c-Myc on global gene regulation is poorly understood, but is widely thought to involve newly activated or repressed “Myc target genes”. We report here that in tumor cells expressing high levels of c-Myc, the transcription factor accumulates in the promoter regions of active genes and causes transcriptional amplification, producing increased levels of transcripts within the cell's gene expression program. Thus, rather than binding and regulating a new set of genes, c-Myc amplifies the output of the existing gene expression program. These results provide an explanation for the diverse effects of oncogenic c-Myc on gene expression in different tumor cells and suggest that transcriptional amplification reduces rate-limiting constraints for tumor cell growth and proliferation. PMID:23021215

  16. Amplification and Sequence Analysis of CD14 Gene in Nanyang Yellow Cattle%南阳黄牛CD14基因扩增及序列分析

    Institute of Scientific and Technical Information of China (English)

    马蓓蓓; 龙塔; 薛云; 周峰; 赵战勤


    The CD14 gene in Nanyang yellow cattle was segmently amplificated by polymerase chain reaction (PCR) method. Primers were designesd according to CD14 gene sequence of Holstein in GenBank. The complete sequence of 2 969 bp, containing complete coding region of CD14,5 ' and 3 ' non-coding region, were obtained. Sequence analysis showed that CD14 gene in Nanyang Yellow cattle contains ORF of 966 bp in length encoded 321 amino acids,comprised of A( 18.4% ) ,T( 18.4% ) ,C(32. 9% ) ,G(30. 2% ). 2 bases of coding region were changed compared to CD14 gene of Holstein, but none of amino acid changed. 7 bases of non-coding region at the end of 5 ' and 3' were changed compared to CD14 gene of Holstein. The homology rates of nucleotide sequences of CD14 gene among Nanyang yellow cattle and Holstein cow, buffalo, sheep, goats,pigs, monkeys, gorillas, human, mouse were decreased in tum, and were 99. 8% , 98. 2% , 96. 8% , 92. 8% ,83. 5% ,19. 8% , 79. 7% , 79. 5% , 71. 9% respectively. The results of categorize from phylogenetic tree were similar to the traditional classification results. There is no transmembrane domains by means of protein structure prediction of CD14 gene sequence in Nanyang yellow cattle.%根据GenBank发表的荷斯坦奶牛CD14基因的序列设计引物,通过PCR方法对南阳黄牛的CD14基因进行分段扩增并测序,拼接后得到包含CD14完整编码区以及5'端和3'端非编码区的2 969 bp的全序列.序列分析结果表明:南阳黄牛CD14基因开放阅读框全长966 bp,共编码321个氨基酸,碱基组成分别为A(18.4%)、T(18.4%)、C(32.9%)、G(30.2%),编码区与荷斯坦奶牛CD14基因相比发生了2个碱基突变,没有引起氨基酸的改变.在5'端和3'端存在较长的非编码区,与荷斯坦奶牛CD14基因相比发生了7个碱基突变.南阳黄牛的CD14基因与荷斯坦奶牛、水牛、绵羊、山羊、猪、猕猴、大猩猩、人、小鼠的同源性依次降低,分别为99.8%、98.2%、96.8

  17. Large Brillouin Amplification in Silicon

    CERN Document Server

    Kittlaus, Eric A; Rakich, Peter T


    Strong Brillouin coupling has only recently been realized in silicon using a new class of optomechanical waveguides that yield both optical and phononic confinement. Despite these major advances, appreciable Brillouin amplification has yet to be observed in silicon. Using a new membrane-suspended silicon waveguide we report large Brillouin amplification for the first time, reaching levels greater than 5 dB for modest pump powers, and demonstrate a record low (5 mW) threshold for net amplification. This work represents a crucial advance necessary to realize high-performance Brillouin lasers and amplifiers in silicon.

  18. Identification of deletions and duplications in the Duchenne muscular dystrophy gene and female carrier status in western India using combined methods of multiplex polymerase chain reaction and multiplex ligation-dependent probe amplification

    Directory of Open Access Journals (Sweden)

    Rashna S Dastur


    Full Text Available Background: The technique of multiplex ligation-dependent probe amplification (MLPA assay is an advanced technique to identify deletions and duplications of all the 79 exons of DMD gene in patients with Duchenne/Becker muscular dystrophy (DMD/BMD and female carriers. Aim: To use MLPA assay to detect deletions which remained unidentified on multiplex polymerase chain reaction (mPCR analysis, scanning 32 exons of the "hot spot" region. Besides knowing the deletions and/or duplications, MLPA was also used to determine the carrier status of the females at risk. Materials and Methods: Twenty male patients showing no deletions on mPCR and 10 suspected carrier females were studied by MLPA assay using P-034 and P-035, probe sets (MRC Holland covering all the 79 exons followed by capillary electrophoresis on sequencing system. Results: On MLPA analysis, nine patients showed deletions of exons other than 32 exons screened by mPCR represented by absence of peak. Value of peak areas were double or more in four patients indicating duplications of exons. Carrier status was confirmed in 50% of females at risk. Conclusion: Combining the two techniques, mPCR followed by MLPA assay, has enabled more accurate detection and extent of deletions and duplications which otherwise would have remained unidentified, thereby increasing the mutation pick up rate. These findings have also allowed prediction of expected phenotype. Determining carrier status has a considerable significance in estimating the risk in future pregnancies and prenatal testing options to limit the birth of affected individuals.

  19. Relationship between human telomerase gene amplification and infection of human papillomavirus in cervical disease between Uygur and Han patients in Xinjiang%新疆维吾尔族、汉族妇女宫颈病变人类染色体端粒酶基因扩增与人乳头瘤病毒感染相关性研究

    Institute of Scientific and Technical Information of China (English)

    韩英; 艾星子·艾里; 段玲; 陈志芳; 苏晶; 丁岩


    Objective To investigate the difference of human telomerase gene amplification in cervical exfoliated cells between Uygur and Hah patients with cervical diseases, and their relationship with high-risk subtypes of human papillomavirus infection.Methods Fifty Uygur and 52 Han patients with cervical diseases were detected with fluorescence in situ hybridization to compare the amplification of human telomerase gene, and human papillomavirus infection was detected.Results Human telomerase gene amplification rates were 62.00% and 51.92% in Uygur and Han patients with cervical disease.With the progression of disease, the human telomerase gene amplification rate increased significantly.There were significant differences in average human telomerase gene multiples amplification between multiple infection and single infection(P<0.05), and no significant differences among the infections of each human papillomavirus subtype in Uygur and Han patients.However,there was significant difference in the total of human telomerase gene multiple amplification between Uygur and Han patients ( P< 0.05 ).Conclusion Human telomerase gene amplification is correlated with high-risk human papillomavirus infection, and it might be a predictor of progression of cervical cancer.Multiple high-risk subtype of human papillomavirus infection may be one of causes of the high incidence of cervical cancer in Uygur patients.%目的:探讨新疆地区维吾尔族(维族)、汉族妇女宫颈病变脱落细胞中人类染色体端粒酶基因(human telomerase gene,hTERC)的表达差异及其与人乳头瘤病毒(human papillomavirus,HPV)高危亚型感染的相关性.方法:采用荧光原位杂交技术,比较50例维族和52例汉族宫颈病变患者hTERC基因扩增情况,同时检测HPV感染情况.结果:维族宫颈病变患者hTERC基因扩增率为62.00%,汉族宫颈病变hTERC基因扩增率为51.92%.随病变进展.维、汉族官颈病变患者hTERC基因扩增均明显增加.维、汉

  20. 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


    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)

  1. 食品中弓形菌16S rRNA特异性扩增检测方法的建立%Development of a specific amplification method of Arcobacter 16S rRNA gene in foods

    Institute of Scientific and Technical Information of China (English)


    针对弓形菌16SrRNA基因合成1对引物,通过对聚合酶链式反应(PCR)扩增条件的优化,建立了检测弓形菌的PCR方法。3株弓形菌标准菌株PCR产物测序结果与NCBI上公布的弓形菌16S rRNA基因序列进行比对,比对结果表明3株弓形菌测序结果与NCBI上公布的弓形菌16S rRNA基因序列同源性均在99%以上。3株弓形菌标准菌株均特异性地扩增出了长度为1202bp的片段,其他19株不同种类的菌株均无扩增产物出现。55份食品样品用Johnson-Murano肉汤增菌后用此法进行检测,其中6份样品为弓形菌阳性,阳性率为10.9%。上述实验结果表明,方法特异性强、操作简便,节省了检测时间,可用于食品中弓形菌的快速检测。%One pair of primers were used to amplify 16S rRNA sequence of Arcobacter,and a PCR method for detection of Arcobacter was developed with optimization of PCR amplification conditions.PCR products of 3 different Arcobacter type strains were sequenced and compared with 16S rRNA genes of Arcobacter published on NCBI.It's proved that the homology between the PCR products and 16S rRNA genes of Arcobacter published were all above 99%.3 different type strains of Arcobacter produced a 1202 bp amplified band,while all of the other 19 different bacteria didn't produced any band.55 food samples were detected whether Arcobacter present or not by the PCR method following enrichment in Johnson-Murano broth,and 6 samples were detected positive for this microorganism,namely with the positive ratio of 10.9%.The result suggested that the method developed was specific,easy to operate and timesaving,and could be used for rapid detection of Arcobacter in foods.

  2. Quantification of HER2 autoantibodies in the amplification phenomenon of HER2 in breast cancer

    DEFF Research Database (Denmark)

    Lauterlein, Jens-Jacob L; Petersen, Eva R B; Olsen, Dorte Aa


    Gene amplification of HER2 (human epidermal growth factor receptor 2) is a well-known phenomenon in various cancers. However, little is known about the mechanism of the gene amplification phenomenon itself. Autoantibodies to cellular receptors have been described in several cancer types. We hypot...... hypothesised that autoantibodies against HER2 might have a stimulatory capacity and could be the cause of the HER2 gene amplification phenomenon. To investigate this, we developed a test for the detection of autoantibodies against HER2 in serum (S-HER2Ab)....

  3. Transcriptome dynamics of transgene amplification in Chinese hamster ovary cells. (United States)

    Vishwanathan, Nandita; Le, Huong; Jacob, Nitya M; Tsao, Yung-Shyeng; Ng, Sze-Wai; Loo, Bernard; Liu, Zhong; Kantardjieff, Anne; Hu, Wei-Shou


    Dihydrofolate reductase (DHFR) system is used to amplify the product gene to multiple copies in Chinese Hamster Ovary (CHO) cells for generating cell lines which produce the recombinant protein at high levels. The physiological changes accompanying the transformation of the non-protein secreting host cells to a high producing cell line is not well characterized. We performed transcriptome analysis on CHO cells undergoing the selection and amplification processes. A host CHO cell line was transfected with a vector containing genes encoding the mouse DHFR (mDHFR) and a recombinant human IgG (hIgG). Clones were isolated following selection and subcloned following amplification. Control cells were transfected with a control plasmid which did not have the hIgG genes. Although methotrexate (MTX) amplification increased the transcript level of the mDHFR gene significantly, its effect on both hIgG heavy and light chain genes was more modest. The subclones appeared to retain the transcriptome signatures of their parental clones, however, their productivity varied among those derived from the same clone. The transcript levels of hIgG transgenes of all subclones fall in a narrower range than the product titer, alluding to the role of many functional attributes, other than transgene transcript, on productivity. We cross examined functional class enrichment during selection and amplification as well as between high and low producers and discerned common features among them. We hypothesize that the role of amplification is not merely increasing transcript levels, but also enriching survivors which have developed the cellular machinery for secreting proteins, leading to an increased frequency of isolating high-producing clones. We put forward the possibility of assembling a hyper-productivity gene set through comparative transcriptome analysis of a wide range of samples.

  4. Double trouble: medical implications of genetic duplication and amplification in bacteria. (United States)

    Craven, Sarah H; Neidle, Ellen L


    Gene amplification allows organisms to adapt to changing environmental conditions. This type of increased gene dosage confers selectable benefits, typically by augmenting protein production. Gene amplification is a reversible process that does not require permanent genetic change. Although transient, altered gene dosage has significant medical impact. Recent examples of amplification in bacteria, described here, affect human disease by modifying antibiotic resistance, the virulence of pathogens, vaccine efficacy and antibiotic biosynthesis. Amplification is usually a two-step process whereby genetic duplication (step one) promotes further increases in copy number (step two). Both steps have important evolutionary significance for the emergence of innovative gene functions. Recent genome sequence analyses illustrate how genome plasticity can affect the evolution and immunogenic properties of bacterial pathogens.

  5. Isothermal strand displacement amplification (iSDA): a rapid and sensitive method of nucleic acid amplification for point-of-care diagnosis. (United States)

    Toley, Bhushan J; Covelli, Isabela; Belousov, Yevgeniy; Ramachandran, Sujatha; Kline, Enos; Scarr, Noah; Vermeulen, Nic; Mahoney, Walt; Lutz, Barry R; Yager, Paul


    We present a method of rapid isothermal amplification of DNA without initial heat denaturation of the template, and methods and probes for (a) real-time fluorescence detection and (b) lateral flow detection of amplicons. Isothermal strand displacement amplification (iSDA) can achieve >10(9)-fold amplification of the target sequence in isothermal DNA amplification methods. iSDA initiates at sites where DNA base pairs spontaneously open or transiently convert into Hoogsteen pairs, i.e. "breathe", and proceeds to exponential amplification by repeated nicking, extension, and displacement of single strands. We demonstrate successful iSDA amplification and lateral flow detection of 10 copies of a Staphylococcus aureus gene, NO.-inducible l-lactate dehydrogenase (ldh1) (Richardson, Libby, and Fang, Science, 2008, 319, 1672-1676), in a clean sample and 50 copies in the presence of high concentrations of genomic DNA and mucins in isothermal amplification reactions. Finally, we demonstrate the multiplexing capability of iSDA by the simultaneous amplification of the target gene and an engineered internal control sequence. The speed, sensitivity, and specificity of iSDA make it a powerful method for point-of-care molecular diagnosis.

  6. Sexing Bovine Embryos Using PCR Amplification of Bovine SRY Sequence

    Institute of Scientific and Technical Information of China (English)

    曾溢滔; 张美兰; 陈美珏; 周霞娣; 黄英; 任兆瑞; 黄淑帧; 胡明信; 吴学清; 高建明; 张斌; 徐慧如


    This study analyses the bovine SRY DNA sequence by direct sequencing procedure, followed by the designation of the PCR primers specific for bovine SRY. Using PCR amplification of bovine SRY gene, the embryo sex was determined. The results of the embryo sex identification were confirmed after the embryo transfer and pregnancies.

  7. Direct Extraction and Amplification of DNA from Soil. (United States)

    Trevors, Jack T.; Leung, K.


    Presents an exercise that describes the direct extraction and purification of DNA from a small soil sample. Also discusses the subsequent amplification of a 343-bp Tn7 transposate A gene fragment (tnsA) from a strain of Pseudomonas aureofaciens 3732RNL11. Contains 21 references. (DDR)

  8. Direct Extraction and Amplification of DNA from Soil. (United States)

    Trevors, Jack T.; Leung, K.


    Presents an exercise that describes the direct extraction and purification of DNA from a small soil sample. Also discusses the subsequent amplification of a 343-bp Tn7 transposate A gene fragment (tnsA) from a strain of Pseudomonas aureofaciens 3732RNL11. Contains 21 references. (DDR)

  9. Uncertainties in Site Amplification Estimation (United States)

    Cramer, C. H.; Bonilla, F.; Hartzell, S.


    Typically geophysical profiles (layer thickness, velocity, density, Q) and dynamic soil properties (modulus and damping versus strain curves) are used with appropriate input ground motions in a soil response computer code to estimate site amplification. Uncertainties in observations can be used to generate a distribution of possible site amplifications. The biggest sources of uncertainty in site amplifications estimates are the uncertainties in (1) input ground motions, (2) shear-wave velocities (Vs), (3) dynamic soil properties, (4) soil response code used, and (5) dynamic pore pressure effects. A study of site amplification was conducted for the 1 km thick Mississippi embayment sediments beneath Memphis, Tennessee (see USGS OFR 04-1294 on the web). In this study, the first three sources of uncertainty resulted in a combined coefficient of variation of 10 to 60 percent. The choice of soil response computer program can lead to uncertainties in median estimates of +/- 50 percent. Dynamic pore pressure effects due to the passing of seismic waves in saturated soft sediments are normally not considered in site-amplification studies and can contribute further large uncertainties in site amplification estimates. The effects may range from dilatancy and high-frequency amplification (such as observed at some sites during the 1993 Kushiro-Oki, Japan and 2001 Nisqually, Washington earthquakes) or general soil failure and deamplification of ground motions (such as observed at Treasure Island during the 1989 Loma Prieta, California earthquake). Examples of two case studies using geotechnical data for downhole arrays in Kushiro, Japan and the Wildlife Refuge, California using one dynamic code, NOAH, will be presented as examples of modeling uncertainties associated with these effects. Additionally, an example of inversion for estimates of in-situ dilatancy-related geotechnical modeling parameters will be presented for the Kushiro, Japan site.

  10. Multigene amplification and massively parallel sequencing for cancer mutation discovery (United States)

    Dahl, Fredrik; Stenberg, Johan; Fredriksson, Simon; Welch, Katrina; Zhang, Michael; Nilsson, Mats; Bicknell, David; Bodmer, Walter F.; Davis, Ronald W.; Ji, Hanlee


    We have developed a procedure for massively parallel resequencing of multiple human genes by combining a highly multiplexed and target-specific amplification process with a high-throughput parallel sequencing technology. The amplification process is based on oligonucleotide constructs, called selectors, that guide the circularization of specific DNA target regions. Subsequently, the circularized target sequences are amplified in multiplex and analyzed by using a highly parallel sequencing-by-synthesis technology. As a proof-of-concept study, we demonstrate parallel resequencing of 10 cancer genes covering 177 exons with average sequence coverage per sample of 93%. Seven cancer cell lines and one normal genomic DNA sample were studied with multiple mutations and polymorphisms identified among the 10 genes. Mutations and polymorphisms in the TP53 gene were confirmed by traditional sequencing. PMID:17517648

  11. Comparison of multiplex ligation dependent probe amplification to immunohistochemistry for assessing HER-2/neu amplification in invasive breast cancer. (United States)

    Purnomosari, D; Aryandono, T; Setiaji, K; Nugraha, S B; Pals, G; van Diest, P J


    The HER-2/neu transmembrane tyrosine kinase receptor is both a prognostic marker and a therapeutic target for breast cancer. Accurate determination of HER-2/neu status is a prerequisite for selecting breast tumors for HER-2/neu immunotherapy or for taxan based chemotherapy. Unfortunately, there is no consensus concerning how this determination should be reached. We compared assessment of HER-2/neu status using Multiplex ligation-dependent probe amplification (MLPA) and immunohistochemistry (IHC). The patient group comprised 60 Indonesian breast cancers patients. IHC was performed on paraffin sections using the CB11 antibody from Novocastra. Results were scored according to the Hercept test. For MLPA, DNA was extracted from frozen samples, PCR amplified with a probe set containing three hemi-primer sets for the HER-2 locus and another nine control probes spread over chromosome 17 and other chromosomes, and analyzed on a gene scanner. A ratio above two for at least two HER-2 locus probes compared to the control probes was regarded as amplification. IHC for HER-2/neu was negative in 36 cases, and 24 cases (40%) showed expression. Seven, eight and nine of the latter cases were 1+, 2+ and 3+ positive, respectively. Forty-seven cases showed no amplification by MLPA, and 13 cases (22%) were amplified. Comparison of IHC and MPLA showed that none of the 36 IHC-negative or seven IHC 1+ cases was amplified. Five of the eight (63%) 2+ cases were amplified, and eight of nine (89%) of the IHC 3+ tumors showed gene amplification by MLPA assay. For HER-2/neu, there is a good correlation between gene amplification detected by MLPA and overexpression by IHC in invasive breast cancer. It appears that MLPA can detect the HER-2 amplified cases in the IHC 2+ class. Because MLPA is quick and inexpensive, it is an attractive method for detecting HER-2/neu amplification in daily laboratory practice.

  12. 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;


    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 ...... to a second round of treatment if left untreated during a sufficient period of time. [Mol Cancer Ther 2009;8(2):424-32]....

  13. Isothermal Amplification of Insect DNA (United States)

    The loop-mediated isothermal amplification of DNA (LAMP) method can amplify a target DNA sequence at a constant temperature in about one hour. LAMP has broad application in agriculture and medicine because of the need for rapid and inexpensive diagnoses. LAMP eliminates the need for temperature cycl...

  14. Rapid amplification of cDNA ends (RACE). (United States)

    Yeku, Oladapo; Frohman, Michael A


    Rapid Amplification of cDNA ends (RACE) provides an inexpensive and powerful tool to quickly obtain full-length cDNA when the sequence is only partially known. Starting with an mRNA mixture, gene-specific primers generated from the known regions of the gene and non-specific anchors, full-length sequences can be identified in as little as 3 days. RACE can also be used to identify alternative transcripts of a gene when the partial or complete sequence of only one transcript is known. In the following sections, we outline details for rapid amplification of 5(') and 3(') cDNA ends using the "new RACE" technique.

  15. Retrieval and Amplification of DNA from Unstained Histopathological Sections

    Institute of Scientific and Technical Information of China (English)

    DonnaC.MONTAGUE; BeverlyD.LYN-COOK; 等


    Testing of compounds for carcinogenic potential in vivo involves various experimental designs.A few of these techniques are directed to demonstrate the genotoxicity and mutagenicity of the compound by histopathology.These changes shown by histochemical means include monoclonal antibody directed cellular markers.Development of the polymerase chain reaction technique(PCR)for amplification of DNA has facilitated the investigation of molecular events related to the formation of malignant neoplasms.We describe here a method for screening tissues for mutations of the H-ras gene using monoclonal antibodies directed toward normal and mutant p21 proteins.Formalin-fixed,paraffinembedded tissue sections are used to subsequently confirm the gene mutation by PCR amplification of the H-ras gene.The results indicated a successful application of this technique to demonstrate the presence of p21 oncoprotein in the tissues tested.

  16. 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.

  17. Miniaturized isothermal nucleic acid amplification, a review. (United States)

    Asiello, Peter J; Baeumner, Antje J


    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.

  18. 肠道病毒71型 VP1基因的扩增、克隆、表达及蛋白分析%Amplification,cloning,expression and protein analysis of enterovirus 71 VP1 gene

    Institute of Scientific and Technical Information of China (English)

    刘云锋; 周珍文; 关锐梨; 关启鸿; 骆鸣勇; 周帅


    Objective To conduct the amplification,cloning,bioinformatics analysis,prokaryotic expression and purification of enterovirus 71 VP1 gene segment and to initially confirm the biological activity of the recombinant expression product.Methods A pair of specific primers was designed according to GenBank EV71 sequence,viral RNA as a template was extracted from the throat swab specimens in the EV71 patients.EV71 VP1 gene was amplified by RT-PCR.After enzyme digestion,the expression vector pET28a was inserted.The prokaryotic expression vector of pET28a-EV71 VP1 was constructed.Then the E.coli DH5a transforma-tion was performed.IPTG was adopted for induction expression.The expression results were analyzed by using SDS-PAGE and Western blot.The bioinformatics analysis of the sequenced results was performed by the software.Expressed protein was purified and the plates were coated,ELISA was used to test the VP1 specific IgG antibody in serum samples of EV71 positive and COX A16-positive patients.Results The BLAST alignment showed that the homology of the objective gene EV71 VP1 was 99% com-pared with other strains(JQ766207.1)in GenBank.EV71 VP1 protein was about 32×103 ,which mainly existed in the form of in-clusion body.The bioinformatics analysis showed that EV71 VP1 protein was a hydrophilic protein,without transmembrane region and N-terminal signal peptide sequence,the tertiary structure existed.The ELISA results showed that the specific IgG OD value in EV71-positive patients was(2.425±0.521),OD value in COX A16 positive patients was(1.205 ±0.314),the normal control OD value was(0.353±0.128).The sensitivity and specificity of EV71 VP1 protein detection were 84% and 88% respectively.Conclu-sion The pET28a-EV71 VP1 expression vector is successfully constructed;the preliminary analysis on the serum of the infected patients by ELISA shows that the obtained objective protein has higher sensitivity and specificity,which is initially confirmed to have biological activity

  19. Spheromak Impedance and Current Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Fowler, T K; Hua, D D; Stallard, B W


    It is shown that high current amplification can be achieved only by injecting helicity on the timescale for reconnection, {tau}{sub REC}, which determines the effective impedance of the spheromak. An approximate equation for current amplification is: dI{sub TOR}{sup 2}/dt {approx} I{sup 2}/{tau}{sub REC} - I{sub TOR}{sup 2}/{tau}{sub closed} where I is the gun current, I{sub TOR} is the spheromak toroidal current and {tau}{sub CLOSED} is the ohmic decay time of the spheromak. Achieving high current amplification, I{sub TOR} >> I, requires {tau}{sub REC} <<{tau}{sub 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 {tau}{sub 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 {proportional_to} I, or I{sub TOR} {approx} I. Program implications are discussed.

  20. A robust method for the amplification of RNA in the sense orientation

    Directory of Open Access Journals (Sweden)

    Quackenbush John


    Full Text Available Abstract Background Small quantities of RNA (1–4 μg total RNA available from biological samples frequently require a single round of amplification prior to analysis, but current amplification strategies have limitations that may restrict their usefulness in downstream genomic applications. The Eberwine amplification method has been extensively validated but is limited by its ability to produce only antisense RNA. Alternatives lack extensive validation and are often confounded by problems with bias or yield attributable to their greater biological and technical complexity. Results To overcome these limitations, we have developed a straightforward and robust protocol for amplification of RNA in the sense orientation. This protocol is based upon Eberwine's method but incorporates elements of more recent amplification techniques while avoiding their complexities. Our technique yields greater than 100-fold amplification, generates long transcript, and produces mRNA that is well suited for use with microarray applications. Microarrays performed with RNA amplified using this protocol demonstrate minimal amplification bias and high reproducibility. Conclusion The protocol we describe here is readily adaptable for the production of sense or antisense, labeled or unlabeled RNA from intact or partially-degraded prokaryotic or eukaryotic total RNA. The method outperforms several commercial RNA amplification kits and can be used in conjunction with a variety of microarray platforms, such as cDNA arrays, oligonucleotide arrays, and Affymetrix GeneChip™ arrays.

  1. Progress in HER2 testing in breast cancer: multiplex ligation-dependent probe amplification and automated immunohistochemistry

    NARCIS (Netherlands)

    Moelans, C.B.


    One of the most frequent genetic changes in sporadic breast cancer is amplification of the HER2 gene, usually resulting in protein overexpression on the cell membrane and growth activation of the cells. Breast cancer patients that have this HER2 amplification have a worse prognosis but can be treate

  2. Functional MYCN signature predicts outcome of neuroblastoma irrespective of MYCN amplification (United States)

    Valentijn, Linda J.; Koster, Jan; Haneveld, Franciska; Aissa, Rachida Ait; van Sluis, Peter; Broekmans, Marloes E. C.; Molenaar, Jan J.; van Nes, Johan; Versteeg, Rogier


    Neuroblastoma is a pediatric tumor of the sympathetic nervous system. MYCN (V-myc myelocytomatosis viral-related oncogene, neuroblastoma derived [avian]) is amplified in 20% of neuroblastomas, and these tumors carry a poor prognosis. However, tumors without MYCN amplification also may have a poor outcome. Here, we identified downstream targets of MYCN by shRNA-mediated silencing MYCN in neuroblastoma cells. From these targets, 157 genes showed an expression profile correlating with MYCN mRNA levels in NB88, a series of 88 neuroblastoma tumors, and therefore represent in vivo relevant MYCN pathway genes. This 157-gene signature identified very poor prognosis tumors in NB88 and independent neuroblastoma cohorts and was more powerful than MYCN amplification or MYCN expression alone. Remarkably, this signature also identified poor outcome of a group of tumors without MYCN amplification. Most of these tumors have low MYCN mRNA levels but high nuclear MYCN protein levels, suggesting stabilization of MYCN at the protein level. One tumor has an MYC amplification and high MYC expression. Chip-on-chip analyses showed that most genes in this signature are directly regulated by MYCN. MYCN induces genes functioning in cell cycle and DNA repair while repressing neuronal differentiation genes. The functional MYCN-157 signature recognizes classical neuroblastoma with MYCN amplification, as well as a newly identified group marked by MYCN protein stabilization. PMID:23091029

  3. CCNE1 amplification is associated with aggressive potential in endometrioid endometrial carcinomas


    Nakayama, Kentaro; RAHMAN, MOHAMMED TANJIMUR; Rahman, Munmun; NAKAMURA, Kohei; Ishikawa, Masako; Katagiri, Hiroshi; Sato, Emi; Ishibashi, Tomoka; IIDA, KOUJI; Ishikawa, Noriyuki; Kyo, Satoru


    The clinicopathological significance of amplification was investigated of the gene encoding cyclin E (CCNE1) and we assessed whether CCNE1 was a potential target in endometrioid endometrial carcinomas. CCNE1 amplification and CCNE1 or F-box and WD repeat domain-containing 7 (FBXW7) expression in endometrial endometrioid carcinoma was assessed by immunohistochemistry and fluorescence in situ hybridization. CCNE1 knockdown by small interfering RNA (siRNA) was used to assess the CCNE1 function. ...

  4. Dynamics and Control of DNA Sequence Amplification

    CERN Document Server

    Marimuthu, Karthikeyan


    DNA amplification is the process of replication of a specified DNA sequence \\emph{in vitro} through time-dependent manipulation of its external environment. A theoretical framework for determination of the optimal dynamic operating conditions of DNA amplification reactions, for any specified amplification objective, is presented based on first-principles biophysical modeling and control theory. Amplification of DNA is formulated as a problem in control theory with optimal solutions that can differ considerably from strategies typically used in practice. Using the Polymerase Chain Reaction (PCR) as an example, sequence-dependent biophysical models for DNA amplification are cast as control systems, wherein the dynamics of the reaction are controlled by a manipulated input variable. Using these control systems, we demonstrate that there exists an optimal temperature cycling strategy for geometric amplification of any DNA sequence and formulate optimal control problems that can be used to derive the optimal tempe...

  5. Comparative evaluation of PCR amplification of RLEP, 16S rRNA, rpoT and Sod A gene targets for detection of M. leprae DNA from clinical and environmental samples

    Directory of Open Access Journals (Sweden)

    Ravindra P Turankar


    Conclusion: Amongst all the gene targets used in this study, PCR positivity using RLEP gene target was the highest in all the clinical and environmental samples. Further, the RLEP gene target was able to detect 53% of blood samples as positive in BI-negative leprosy cases indicating its future standardization and use for diagnostic purposes.

  6. Dynamics and control of DNA sequence amplification

    Energy Technology Data Exchange (ETDEWEB)

    Marimuthu, Karthikeyan [Department of Chemical Engineering and Center for Advanced Process Decision-Making, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 (United States); Chakrabarti, Raj, E-mail:, E-mail: [Department of Chemical Engineering and Center for Advanced Process Decision-Making, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 (United States); Division of Fundamental Research, PMC Advanced Technology, Mount Laurel, New Jersey 08054 (United States)


    DNA amplification is the process of replication of a specified DNA sequence in vitro through time-dependent manipulation of its external environment. A theoretical framework for determination of the optimal dynamic operating conditions of DNA amplification reactions, for any specified amplification objective, is presented based on first-principles biophysical modeling and control theory. Amplification of DNA is formulated as a problem in control theory with optimal solutions that can differ considerably from strategies typically used in practice. Using the Polymerase Chain Reaction as an example, sequence-dependent biophysical models for DNA amplification are cast as control systems, wherein the dynamics of the reaction are controlled by a manipulated input variable. Using these control systems, we demonstrate that there exists an optimal temperature cycling strategy for geometric amplification of any DNA sequence and formulate optimal control problems that can be used to derive the optimal temperature profile. Strategies for the optimal synthesis of the DNA amplification control trajectory are proposed. Analogous methods can be used to formulate control problems for more advanced amplification objectives corresponding to the design of new types of DNA amplification reactions.

  7. Multiplex ligation-dependent probe amplification (MLPA) screening for exon copy number variation in the calcium sensing receptor gene: no large rearrangements identified in patients with calcium metabolic disorders

    DEFF Research Database (Denmark)

    Nissen, Peter H; Christensen, Signe E; Wallace, Andrew;


    samples were previously found negative for CASR mutations. Multiplex ligation-dependent probe amplification was used to screen the patients for exon copy number variations. Results. All exons were amplified with mean normalised ratios between 0.98 and 1.06. We did not identify any exon copy number......Summary Background. Mutation screening of the CASR by DNA sequencing is commonly used in the diagnosis of disorders of calcium metabolism, such as familial hypocalciuric hypercalcaemia (FHH). Exon copy number variation is not detected by currently used molecular genetic screening methods, and might...... be a genetic cause of inherited forms of hyper- or hypocalcaemia caused by the CASR. Objective. We wanted to further evaluate possible genetic causes for disorders of calcium metabolism, by investigating the prevalence of exon copy number variations, such as large deletions or duplications of the CASR...

  8. Chromogenic in situ hybridization to detect HER-2/neu gene amplification in histological and ThinPrep-processed breast cancer fine-needle aspirates: a sensitive and practical method in the trastuzumab era. (United States)

    Vocaturo, Amina; Novelli, Flavia; Benevolo, Maria; Piperno, Giulia; Marandino, Ferdinando; Cianciulli, Anna Maria; Merola, Roberta; Donnorso, Raffaele Perrone; Sperduti, Isabella; Buglioni, Simonetta; Mottolese, Marcella


    The increasing evidence of trastuzumab efficacy in breast cancer (BC) patients means that an accurate and reproducible evaluation of HER-2 statusis of paramount importance in histological and in cytological samples. Currently, the two main methods used to analyze HER-2 amplification or overexpression are fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Although the two methods are strongly correlated for histological tissue, the evaluation of tumor morphology through FISH may be difficult and fluorescence fades quickly. These limitations can be overcome by chromogenic in situ hybridization (CISH), which can visualize the amplification product along with morphological features. In view of this, in the present study, we analyzed the usefulness of CISH on formalin-fixed, paraffin-embedded (FFPE) BC specimens and investigated whether CISH can be a valid technique in the determination of HER-2 status for fine-needle aspirates (FNAs) processed by liquid-based cytology. The results we obtained in a retrospective series of 111 FFPE BC specimens demonstrated good concordance between CISH and IHC and between CISH and FISH. The former concordance was comparable with that observed between FISH and IHC. When CISH was applied to a prospective series of 53 FNAs, from surgically removed BC, our data showed evidence of a higher concordance of results between liquid-based cytology and the companion FFPE tissues using CISH rather than HercepTesttrade mark. Therefore, CISH analysis, which is avaluable and reproducible alternative to FISH for selecting breast cancer patients for trastuzumab therapy, can lower false-positive immunocytochemistry findings in ThinPrep-processed FNAs.

  9. Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh. (United States)

    Bej, A K; Patterson, D P; Brasher, C W; Vickery, M C; Jones, D D; Kaysner, C A


    Vibrio parahaemolyticus is an important human pathogen which can cause gastroenteritis when consumed in raw or partially-cooked seafood. A multiplex PCR amplification-based detection of total and virulent strains of V. parahaemolyticus was developed by targeting thermolabile hemolysin encoded by tl, thermostable direct hemolysin encoded by tdh, and thermostable direct hemolysin-related trh genes. Following optimization using oligonucleotide primers targeting tl, tdh and trh genes, the multiplex PCR was applied to V. parahaemolyticus from 27 clinical, 43 seafood, 15 environmental, 7 strains obtained from various laboratories and 19 from oyster plants. All 111 V. parahaemolyticus isolates showed PCR amplification of the tl gene; however, only 60 isolates showed amplification of tdh, and 43 isolates showed amplification of the trh gene. Also, 18 strains showed amplification of the tdh gene, but these strains did not show amplification of the trh gene. However, one strain exhibited amplification for the trh but not the tdh gene, suggesting both genes need to be targeted in a PCR amplification reaction to detect all hemolysin-producing strains of this pathogen. The multiplex PCR approach was successfully used to detect various strains of V parahaemolyticus in seeded oyster tissue homogenate. Sensitivity of detection for all three target gene segments was at least between 10(1)-10(2) cfu per 10 g of alkaline peptone water enriched seeded oyster tissue homogenate. This high level of sensitivity of detection of this pathogen within 8 h of pre-enrichment is well within the action level (10(4) cfu per 1 g of shell stock) suggested by the National Seafood Sanitation Program guideline. Compared to conventional microbiological culture methods, this multiplex PCR approach is rapid and reliable for accomplishing a comprehensive detection of V. parahaemolyticus in shellfish.

  10. Ancient DNA: genomic amplification of Roman and medieval bovine bones

    Directory of Open Access Journals (Sweden)

    A. Valentini


    Full Text Available Cattle remains (bones and teeth of both roman and medieval age were collected in the archaeological site of Ferento (Viterbo, Italy with the aim of extracting and characterising nucleic acids. Procedures to minimize contamination with modern DNA and to help ancient DNA (aDNA preservation of the archaeological remains were adopted. Different techniques to extract aDNA (like Phenol/chloroform extraction from bovine bones were tested to identify the method that applies to the peculiar characteristics of the study site. Currently, aDNA investigation is mainly based on mtDNA, due to the ease of amplification of the small and high-copied genome and to its usefulness in evolutionary studies. Preliminary amplification of both mitochondrial and nuclear aDNA fragments from samples of Roman and medieval animals were performed and partial specific sequences of mitochondrial D-loop as well as of nuclear genes were obtained. The innovative amplification of nuclear aDNA could enable the analysis of genes involved in specific animal traits, giving insights of ancient economic and cultural uses, as well as providing information on the origin of modern livestock population.

  11. Rolling circle amplification of complete nematode mitochondrial genomes. (United States)

    Tang, Sha; Hyman, Bradley C


    To enable investigation of nematode mitochondrial DNA evolution, methodology has been developed to amplify intact nematode mitochondrial genomes in preparative yields using a rolling circle replication strategy. Successful reactions were generated from whole cell template DNA prepared by alkaline lysis of the rhabditid nematode Caenorhabditis elegans and a mermithid nematode, Thaumamermis cosgrovei. These taxa, representing the two major nematode classes Chromodorea and Enoplea, maintain mitochondrial genomes of 13.8 kb and 20.0 kb, respectively. Efficient amplifications were conducted on template DNA isolated from individual or pooled nematodes that were alive or stored at -80 degrees C. Unexpectedly, these experiments revealed that multiple T. cosgrovei mitochondrial DNA haplotypes are maintained in our local population. Rolling circle amplification products can be used as templates for standard PCR reactions with specific primers that target mitochondrial genes or for direct DNA sequencing.

  12. Meta-analysis of EGFR mutation,EGFR gene amplification and KRAS mutation in prima-ry and metastatic NSCLC%NSCLC原发肿瘤和转移瘤中EGFR基因突变、基因扩增及KRAS基因突变的Meta分析

    Institute of Scientific and Technical Information of China (English)

    陈宽冰; 王一北; 石文君; 宣莹


    Objective:To determine EGFR mutation,EGFR gene amplification and KRAS mutation in primary and metastatic NSCLC in order to explore the implications in clinical practice. Methods:We searched the Chinese and English database to identify all the articles about the predictive biomarkers of EGFR-TKIs treatment. Meta-analysis was completed by Review Manager 5. 2 software. Results:11 literatrues were included in Meta-analysis. The RR in the group of EGFR mutation,EGFR gene amplification and KRAS mutation were 0. 86(0. 65-1. 15),0. 73(0. 44-1. 20)and 1. 39(0. 95-2. 03)respectively. Conclusion:Combined detection of KRAS and EGFR mutations in pri-mary NSCLC was considered to be the optimal approach to provide promising predictive biomarkers for targeted thera-py. EGFR amplification from metastatic tumors may be helpful for further identification of patients targeted therapy.%目的:检测NSCLC原发肿瘤和转移瘤中EGFR基因突变、基因扩增及KRAS基因突变情况,为临床靶向治疗提供更多的循证医学证据。方法:计算机文献检索中英文数据库,收集国内外公开发表的有关EG-FR-TKI靶向治疗预测性生物标记的文献研究,采用Cochrane协作网提供Review Manager 5.2软件进行Meta分析。结果:筛选文献,共11篇纳入研究。Meta分析结果显示,EGFR基因突变组、基因扩增组及KRAS基因突变组RR值分别为0.86(0.65-1.15)、0.73(0.44-1.20)及1.39(0.95-2.03)。结论:联合检测EGFR基因和KRAS基因在原发肿瘤中的突变情况,可作为EGFR-TKIs一线治疗较好的预测性生物标记。检测转移瘤中EGFR基因的扩增情况能够帮助进一步筛查哪些患者对靶向治疗更加敏感。

  13. Multiscale image contrast amplification (MUSICA) (United States)

    Vuylsteke, Pieter; Schoeters, Emile P.


    This article presents a novel approach to the problem of detail contrast enhancement, based on multiresolution representation of the original image. The image is decomposed into a weighted sum of smooth, localized, 2D basis functions at multiple scales. Each transform coefficient represents the amount of local detail at some specific scale and at a specific position in the image. Detail contrast is enhanced by non-linear amplification of the transform coefficients. An inverse transform is then applied to the modified coefficients. This yields a uniformly contrast- enhanced image without artefacts. The MUSICA-algorithm is being applied routinely to computed radiography images of chest, skull, spine, shoulder, pelvis, extremities, and abdomen examinations, with excellent acceptance. It is useful for a wide range of applications in the medical, graphical, and industrial area.

  14. Risk Perception and Social Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Smith, R.E. [Environment Agency (United Kingdom)


    This paper seeks to consider social amplification as it applies to risk perception. Perceptions of the magnitude of a risk are conditioned by issues such as the degree of uncertainty in probability and consequences, the nature of the consequences and the relative weightings placed on probability and consequences. Risk perceptions are also influenced by factors such as confidence in the operator of an industrial process, trust in the regulator and the perceived fairness of regulatory decision-making. Different people may hold different views about these issues and there may also be difficulties in communication. The paper identifies and discusses self-reinforcing mechanisms, which will be labelled 'lock-in' here. They appear to apply in many situations where social amplification is observed. Historically, the term 'lock-in' has been applied mainly in the technological context but, in this paper, four types of lock-in are identified, namely scientific/technological, economic, social and institutional lock-in. One type of lock-in tends to lead to the next and all are buttressed by people's general acceptance of the familiar, fear of the unknown and resistance to change. The regulator seeks to make decisions which achieve the common good rather than supporting or perpetuating any set of vested interests. In this regard the locked-in positions of stakeholders, whether organisations, interest groups, or individual members of the public, are obstacles and challenges. Existing methods of consultation are unsatisfactory in terms of achieving a proper and productive level of dialogue with stakeholders.

  15. HER2 Amplification Has no Prognostic Value in Sporadic and Hereditary Ovarian Tumours

    Directory of Open Access Journals (Sweden)

    Brożek Izabela


    Full Text Available Abstract Whereas HER2 amplification is a well-known phenomenon in breast tumours, its frequency and clinical importance in ovarian cancer have not been established. The aim of the study was to compare the frequency of HER2 amplification in hereditary (BRCA-positive and sporadic (BRCA-negative ovarian tumours and to estimate the association of this gene alteration on clinical outcome in ovarian cancer patients. We analysed HER2 amplification in 53 ovarian tumours: 20 from mutation carriers (18 in BRCA1 and 2 in BRCA2 gene and 33 from non-carriers. Fluorescence in situ hybridization for HER2 was performed on 'touch' slides from frozen tumour samples or formalin-fixed, paraffin-embedded tissue. Our results indicate that high amplification (HER2: centromere ratio>5 is an infrequent phenomenon in ovarian tumours (6/53 cases. It occurs in both hereditary (4/20 and sporadic (2/33 tumours and no difference in the frequency of HER2 amplification exists between these groups. There is no significant difference in the clinical outcome of patients with HER2 amplified and non-amplified tumours (p = 0.3. Our results suggest a different biological role of HER2 amplification in ovarian and breast cancer.

  16. Optimization and evaluation of T7 based RNA linear amplification protocols for cDNA microarray analysis

    Directory of Open Access Journals (Sweden)

    Børresen-Dale Anne-Lise


    Full Text Available Abstract Background T7 based linear amplification of RNA is used to obtain sufficient antisense RNA for microarray expression profiling. We optimized and systematically evaluated the fidelity and reproducibility of different amplification protocols using total RNA obtained from primary human breast carcinomas and high-density cDNA microarrays. Results Using an optimized protocol, the average correlation coefficient of gene expression of 11,123 cDNA clones between amplified and unamplified samples is 0.82 (0.85 when a virtual array was created using repeatedly amplified samples to minimize experimental variation. Less than 4% of genes show changes in expression level by 2-fold or greater after amplification compared to unamplified samples. Most changes due to amplification are not systematic both within one tumor sample and between different tumors. Amplification appears to dampen the variation of gene expression for some genes when compared to unamplified poly(A+ RNA. The reproducibility between repeatedly amplified samples is 0.97 when performed on the same day, but drops to 0.90 when performed weeks apart. The fidelity and reproducibility of amplification is not affected by decreasing the amount of input total RNA in the 0.3–3 micrograms range. Adding template-switching primer, DNA ligase, or column purification of double-stranded cDNA does not improve the fidelity of amplification. The correlation coefficient between amplified and unamplified samples is higher when total RNA is used as template for both experimental and reference RNA amplification. Conclusion T7 based linear amplification reproducibly generates amplified RNA that closely approximates original sample for gene expression profiling using cDNA microarrays.

  17. Pulse Compression And Raman Amplification In Optical Fibres (United States)

    Byron, Kevin C.


    Experimental and theoretical investigations on Raman amplification in fibres have been carried out and simultaneous amplification and pulse compression observed. With a fibre design optimised for amplification high gain may be obtained at practical pump power levels.

  18. Restricted 12p Amplification and RAS Mutation in Human Germ Cell Tumors of the Adult Testis (United States)

    Roelofs, Helene; Mostert, Marijke C.; Pompe, Kirsten; Zafarana, Gaetano; van Oorschot, Monique; van Gurp, Ruud J. H. L. M.; Gillis, Ad J. M.; Stoop, Hans; Beverloo, Berna; Oosterhuis, J. Wolter; Bokemeyer, Carsten; Looijenga, Leendert H. J.


    Human testicular germ-cell tumors of young adults (TGCTs), both seminomas and nonseminomas, are characterized by 12p overrepresentation, mostly as isochromosomes, of which the biological and clinical significance is still unclear. A limited number of TGCTs has been identified with an additional high-level amplification of a restricted region of 12p including the K-RAS proto-oncogene. Here we show that the incidence of these restricted 12p amplifications is ∼8% in primary TGCTs. Within a single cell formation of i(12p) and restricted 12p amplification is mutually exclusive. The borders of the amplicons cluster in short regions, and the amplicon was never found in the adjacent carcinoma in situ cells. Seminomas with the restricted 12p amplification virtually lacked apoptosis and the tumor cells showed prolonged in vitro survival like seminoma cells with a mutated RAS gene. However, no differences in proliferation index between these different groups of seminomas were found. Although patients with a seminoma containing a homogeneous restricted 12p amplification presented at a significantly younger age than those lacking it, the presence of a restricted 12p amplification/RAS mutation did not predict the stage of the disease at clinical presentation and the treatment response of primary seminomas. In 55 primary and metastatic tumors from 44 different patients who failed cisplatinum-based chemotherapy, the restricted 12p amplification and RAS mutations had the same incidence as in the consecutive series of responding patients. These data support the model that gain of 12p in TGCTs is related to invasive growth. It allows tumor cells, in particular those showing characteristics of early germ cells (ie, the seminoma cells), to survive outside their specific microenvironment. Overexpression of certain genes on 12p probably inhibits apoptosis in these tumor cells. However, the copy numbers of the restricted amplification of 12p and K-RAS mutations do not predict response

  19. Linking Arctic amplification and local feedbacks (United States)

    Balcerak, Ernie


    Climate simulations show that as the Earth warms, the Arctic warms more than the average global warming. However, models differ on how much more the Arctic warms, and although scientists have proposed a variety of mechanisms to explain the Arctic warming amplification, there is no consensus on the main reasons for it. To shed light on this issue, Hwang et al. investigated the relationship between Arctic amplification and poleward energy transport and local Arctic feedbacks, such as changes in cloud cover or ice loss, across a group of models. The researchers noted that differences in atmospheric energy transport did not explain the ranges of polar amplification; rather, models with more amplification showed less energy transport into high latitudes. The authors found that decreasing energy transport is due to a coupled relationship between Arctic amplification and energy transport: Arctic amplification reduces the equator-to-pole temperature gradient, which strongly decreases energy transport. They suggest that this coupled relationship should be taken into account in studies of Arctic amplification. (Geophysical Research Letters, doi:10.1029/2011GL048546, 2011)

  20. MITF基因扩增预测重组人血管内皮抑素联合达卡巴嗪一线治疗晚期黑色素瘤疗效的临床研究%Predictive value of MITF gene amplification in metastatic melanoma patients receiving recombined human endostatin and dacarbazine as first-line treatment

    Institute of Scientific and Technical Information of China (English)

    马家芳; 孔燕; 崔传亮; 梁龙; 张虔男; 唐碧霞; 代杰; 郭军


    目的 探讨小眼畸形相关转录因子(MITF)基因扩增对重组人血管内皮抑素(恩度)联合达卡巴嗪一线治疗晚期黑色素瘤的疗效预测作用.方法 收集60例接受恩度联合达卡巴嗪一线治疗晚期黑色素瘤患者的石蜡包埋组织样本,采用实时定量PCR检测MITF基因扩增,观察患者的疾病控制率和远期疗效.结果 56例晚期黑色素瘤样本成功检测,MITF基因扩增率为51.8% (29/56),其中肢端、非肢端皮肤、黏膜、原发不明黑色素瘤中的MITF基因扩增率分别为31.2%、63.2%、80.0%和36.4%(P=0.050).MITF基因扩增组与无扩增组患者接受恩度联合达卡巴嗪治疗的疾病控制率分别为58.6%(17/29)和81.5% (22/27),差异无统计学意义(P=0.063);两组的中位无进展生存期分别为6.4个月(95%CI:0.5~12.2个月)和8.4个月(95%CI:6.8~10.3个月),差异亦无统计学意义(P=0.169).结论 中国晚期黑色素瘤患者MITF基因扩增率较高,检测MITF基因扩增可能有助于预测恩度联合达卡巴嗪治疗晚期黑色素瘤的疗效,总生存结果有待进一步随访.%Objective To investigate the predictive effect of microphthalmia-associated transcription factor(MITF) gene amplification on clinical outcome of recombined human endostatin(endostar) plus dacarbazine as first-line treatment for patients with metastatic melanoma.Methods MITF gene amplification in tumor tissues obtained from 60 metastatic melanoma patients were determined by quantitative real-time PCR.All the patients were followed for disease control rate and long-term efficacy.Results Of 60 tumor tissues,56 were evaluable for MITF gene detection.MITF gene amplification was detected in 29 of 56 patients(51.8%),31.2% (5/16) of acral,63.2% (12/19) of non-aeral,80.0% (8/10) of mucosal and 36.4% (4/11) of unknown primary melanoma (P =0.050).The disease control rate of patients with or without MITF gene amplification were 58.6% and 81.5% (P =0

  1. EGFR Amplification and Glioblastoma Stem-Like Cells

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    Katrin Liffers


    Full Text Available Glioblastoma (GBM, the most common malignant brain tumor in adults, contains a subpopulation of cells with a stem-like phenotype (GS-cells. GS-cells can be maintained in vitro using serum-free medium supplemented with epidermal growth factor, basic fibroblast growth factor-2, and heparin. However, this method does not conserve amplification of the Epidermal Growth Factor Receptor (EGFR gene, which is present in over 50% of all newly diagnosed GBM cases. GS-cells with retained EGFR amplification could overcome the limitations of current in vitro model systems and contribute significantly to preclinical research on EGFR-targeted therapy. This review recapitulates recent methodological approaches to expand stem-like cells from GBM with different EGFR status in order to maintain EGFR-dependent intratumoral heterogeneity in vitro. Further, it will summarize the current knowledge about the impact of EGFR amplification and overexpression on the stem-like phenotype of GBM-derived GS-cells and different approaches to target the EGFR-dependent GS-cell compartment of GBM.

  2. Diagnostic Devices for Isothermal Nucleic Acid Amplification

    Directory of Open Access Journals (Sweden)

    Chia-Chen Chang


    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.

  3. Advanced backward Raman amplification seeding (United States)

    Malkin, Vladimir; Fisch, Nathaniel


    Next generations of ultrapowerful laser pulses, reaching exawatt and zetawatt powers within reasonably compact facilities, might be based on the backward Raman amplification (BRA) in plasmas. Amplified pulse intensities hundreds times higher than the pump intensity are already observed experimentally. More advanced BRA stages should produce even higher intensities. The largest nonfocused intensity, limited primarily by instabilities associated with the relativistic electron nonlinearity of the amplified laser pulse, is, roughly speaking, 0.1 of the fully relativistic value. It corresponds to the amplified pulse final (and shortest) duration be about the electron plasma wave period. The needed seed pulse should be at least that short then to stay ahead of the amplified pulse, rather than be shadowed by it (which would much reduce the seeding efficiency). However, at earlier BRA stages, when the amplified pulse is longer, the optimal duration of the seed pulse is also longer. This work proposes the use of self-contracting seed pulses for further optimizing the advanced BRA.

  4. Quantum Amplitude Amplification and Estimation

    CERN Document Server

    Brassard, G; Mosca, M; Tapp, A; Brassard, Gilles; Hoyer, Peter; Mosca, Michele; Tapp, Alain


    Consider a Boolean function $\\chi: X \\to \\{0,1\\}$ that partitions set $X$ between its good and bad elements, where $x$ is good if $\\chi(x)=1$ and bad otherwise. Consider also a quantum algorithm $\\mathcal A$ such that $A \\ket{0} = \\sum_{x\\in X} \\alpha_x \\ket{x}$ is a quantum superposition of the elements of $X$, and let $a$ denote the probability that a good element is produced if $A \\ket{0}$ is measured. If we repeat the process of running $A$, measuring the output, and using $\\chi$ to check the validity of the result, we shall expect to repeat $1/a$ times on the average before a solution is found. *Amplitude amplification* is a process that allows to find a good $x$ after an expected number of applications of $A$ and its inverse which is proportional to $1/\\sqrt{a}$, assuming algorithm $A$ makes no measurements. This is a generalization of Grover's searching algorithm in which $A$ was restricted to producing an equal superposition of all members of $X$ and we had a promise that a single $x$ existed such tha...

  5. Genetic Heterogeneity of HER2 Amplification and Telomere Shortening in Papillary Thyroid Carcinoma

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    Paola Caria


    Full Text Available Extensive research is dedicated to understanding if sporadic and familial papillary thyroid carcinoma are distinct biological entities. We have previously demonstrated that familial papillary thyroid cancer (fPTC cells exhibit short relative telomere length (RTL in both blood and tissues and that these features may be associated with chromosome instability. Here, we investigated the frequency of HER2 (Human Epidermal Growth Factor Receptor 2 amplification, and other recently reported genetic alterations in sporadic PTC (sPTC and fPTC, and assessed correlations with RTL and BRAF mutational status. We analyzed HER2 gene amplification and the integrity of ALK, ETV6, RET, and BRAF genes by fluorescence in situ hybridization in isolated nuclei and paraffin-embedded formalin-fixed sections of 13 fPTC and 18 sPTC patients. We analyzed BRAFV600E mutation and RTL by qRT-PCR. Significant HER2 amplification (p = 0.0076, which was restricted to scattered groups of cells, was found in fPTC samples. HER2 amplification in fPTCs was invariably associated with BRAFV600E mutation. RTL was shorter in fPTCs than sPTCs (p < 0.001. No rearrangements of other tested genes were observed. These findings suggest that the association of HER2 amplification with BRAFV600E mutation and telomere shortening may represent a marker of tumor aggressiveness, and, in refractory thyroid cancer, may warrant exploration as a site for targeted therapy.

  6. FGFR-1 amplification in metastatic lymph-nodal and haematogenous lobular breast carcinoma

    Directory of Open Access Journals (Sweden)

    Brunello Eleonora


    Full Text Available Abstract Background Lobular breast carcinoma usually shows poor responsiveness to chemotherapies and often lacks targeted therapies. Since FGFR1 expression has been shown to play pivotal roles in primary breast cancer tumorigenesis, we sought to analyze the status of FGFR1 gene in a metastatic setting of lobular breast carcinoma, since promising FGFR1 inhibitors has been recently developed. Methods Fifteen tissue metastases from lobular breast carcinomas with matched primary infiltrative lobular breast carcinoma were recruited. Eleven cases showed loco-regional lymph-nodal and four haematogenous metastases. FGFR-1 gene (8p12 amplification was evaluated by chromogenic in situ hybridization (CISH analysis. Her-2/neu and topoisomerase-IIα gene status was assessed. E-cadherin and Hercept Test were also performed. We distinguished amplification (>6 or cluster of signals versus gains (3–6 signals of the locus specific FGFR-1 gene. Results Three (20% primary lobular breast carcinomas showed >6 or cluster of FGFR1 signals (amplification, six cases (40% had a mean of three (range 3–6 chromogenic signals (gains whereas in 6 (40% was not observed any abnormality. Three of 15 metastasis (20% were amplified, 2/15 (13,4% did not. The ten remaining cases (66,6% showed three chromogenic signals. The three cases with FGFR-1 amplification matched with those primary breast carcinomas showing FGFR-1 amplification. The six cases showing FGFR-1 gains in the primary tumour again showed FGFR-1 gains in the metastases. Four cases showed gains of FGFR-1 gene signals in the metastases and not in the primary tumours. Her-2/neu gene amplification was not observed in all cases but one (6% case. Topoisomerase-IIα was not amplified in all cases. Conclusions 1 a subset of metastatic lobular breast carcinoma harbors FGFR-1 gene amplification or gains of chromogenic signals; 2 a minor heterogeneity has been observed after matching primary and metastatic carcinomas; 3 in the

  7. Can Anomalous Amplification be Attained Without Postselection?

    CERN Document Server

    Martínez-Rincón, Julián; Viza, Gerardo I; Howell, John C


    We present a parameter estimation technique based on performing joint measurements of a weak interaction away from the weak-value-amplification approximation. Two detectors are used to collect full statistics of the correlations between two weakly entangled degrees of freedom. Without the need of postselection, the protocol resembles the anomalous amplification of an imaginary-weak-value-like response. The amplification is induced in the difference signal of both detectors allowing robustness to different sources of technical noise, and offering in addition the advantages of balanced signals for precision metrology. All of the Fisher information about the parameter of interest is collected, and a phase controls the amplification response. We experimentally demonstrate the proposed technique by measuring polarization rotations in a linearly polarized laser pulse. The effective sensitivity and precision of a split detector is increased when compared to a conventional continuous-wave balanced detection technique...

  8. Rapid detection and identification of Candida albicans and Torulopsis (Candida) glabrata in clinical specimens by species-specific nested PCR amplification of a cytochrome P-450 lanosterol-alpha-demethylase (L1A1) gene fragment. (United States)

    Burgener-Kairuz, P; Zuber, J P; Jaunin, P; Buchman, T G; Bille, J; Rossier, M


    PCR of a Candida albicans cytochrome P-450 lanosterol-alpha-demethylase (P450-L1A1) gene segment is a rapid and sensitive method of detection in clinical specimens. This enzyme is a target for azole antifungal action. In order to directly detect and identify the clinically most important species of Candida, we cloned and sequenced 1.3-kbp fragments of the cytochrome P450-L1A1 genes from Torulopsis (Candida) glabrata and from Candida krusei. These segments were compared with the published sequences from C. albicans and Candida tropicalis. Amplimers for gene sequences highly conserved throughout the fungal kingdom were first used; positive PCR results were obtained for C. albicans, T. glabrata, C. krusei, Candida parapsilosis, C. tropicalis, Cryptococcus neoformans, and Trichosporon beigelii DNA extracts. Primers were then selected for a highly variable region of the gene, allowing the species-specific detection from purified DNA of C. albicans, T. glabrata, C. krusei, and C. tropicalis. The assay sensitivity as tested for C. albicans in seeded clinical specimens such as blood, peritoneal fluid, or urine was 10 to 20 cells per 0.1 ml. Compared with results obtained by culture, the sensitivity, specificity, and efficiency of the species-specific nested PCR tested with 80 clinical specimens were 71, 95, and 83% for C. albicans and 100, 97, and 98% for T. glabrata, respectively.

  9. Determining the repertoire of immunodominant proteins via whole-genome amplification of intracellular pathogens.

    Directory of Open Access Journals (Sweden)

    Michael J Dark

    Full Text Available Culturing many obligate intracellular bacteria is difficult or impossible. However, these organisms have numerous adaptations allowing for infection persistence and immune system evasion, making them some of the most interesting to study. Recent advancements in genome sequencing, pyrosequencing and Phi29 amplification, have allowed for examination of whole-genome sequences of intracellular bacteria without culture. We have applied both techniques to the model obligate intracellular pathogen Anaplasma marginale and the human pathogen Anaplasma phagocytophilum, in order to examine the ability of phi29 amplification to determine the sequence of genes allowing for immune system evasion and long-term persistence in the host. When compared to traditional pyrosequencing, phi29-mediated genome amplification had similar genome coverage, with no additional gaps in coverage. Additionally, all msp2 functional pseudogenes from two strains of A. marginale were detected and extracted from the phi29-amplified genomes, highlighting its utility in determining the full complement of genes involved in immune evasion.

  10. Efficient newly designed primers for the amplification and sequencing of bird mitochondrial genomes. (United States)

    Amer, Sayed A M; Ahmed, Mohamed Mohamed; Shobrak, Mohammed


    In the present study, 27 mitochondrial genomes of diverse avian supra-orders were collected from the Genbank database and their genes were aligned separately. From the alignments, the conserved sequences were selected to design novel conserved primers for amplification and sequencing of the different mitochondrial genes. The reproducibility of these primers was tested in the amplification and sequencing of diverse avian supra-order mitochondrial genomes and was confirmed. This method helped in designing a new set of primers to accelerate both the amplification and the sequencing of bird mitogenomes. It also aids in building mitogenome markers in studying the genetic framework of endemic birds as a preliminary strategy for conservation management of them.

  11. Fibroblast growth factor receptor 1 amplification in non-small cell lung cancer by quantitative real-time PCR.

    Directory of Open Access Journals (Sweden)

    Shirish M Gadgeel

    Full Text Available INTRODUCTION: Amplification of the fibroblast growth factor receptor 1 (FGFR1 gene has been described in tumors of non-small-cell lung cancer (NSCLC patients. Prior reports showed conflicting rates of amplification frequency and clinical relevance. MATERIALS AND METHODS: We developed a reliable real-time quantitative PCR assay to assess the frequency of FGFR1 amplification and assessed the optimal cutoff level of amplification for clinical application. RESULTS: In a training cohort of 203 NSCLCs, we established that a 3.5-fold amplification optimally divided patients into groups with different survival rates with a clear threshold level. Those with FGFR1 amplification levels above 3.5-fold had an inferior survival. These data were confirmed in a validation cohort of 142 NSCLC. After adjusting for age, sex, performance status, stage, and histology, patients with FGFR1 amplification levels above 3.5 fold had a hazard ratio of 2.91 (95% CI- 1.14, 7.41; pvalue-0.025 for death in the validation cohort. The rates of FGFR1 amplification using the cutoff level of 3.5 were 5.1% in squamous cell and 4.1% in adenocarcinomas. There was a non-significant trend towards higher amplifications rates in heavy smokers (> 15 pack-years of cigarette consumption as compared to light smokers. DISCUSSION: Our data suggest that a 3.5-fold amplification of FGFR1 is of clinical importance in NSCLC. Our cutpoint analysis showed a clear threshold effect for the impact of FGFR1 amplification on patients' survival, which can be used as an initial guide for patient selection in trials assessing efficacy of novel FGFR inhibitors.

  12. Rolling circle amplification of metazoan mitochondrialgenomes

    Energy Technology Data Exchange (ETDEWEB)

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


    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.

  13. Complex community of nitrite-dependent anaerobic methane oxidation bacteria in coastal sediments of the Mai Po wetland by PCR amplification of both 16S rRNA and pmoA genes. (United States)

    Chen, Jing; Zhou, Zhichao; Gu, Ji-Dong


    In the present work, both 16S rRNA and pmoA gene-based PCR primers were employed successfully to study the diversity and distribution of n-damo bacteria in the surface and lower layer sediments at the coastal Mai Po wetland. The occurrence of n-damo bacteria in both the surface and subsurface sediments with high diversity was confirmed in this study. Unlike the two other known n-damo communities from coastal areas, the pmoA gene-amplified sequences in the present work clustered not only with some freshwater subclusters but also within three newly erected marine subclusters mostly, indicating the unique niche specificity of n-damo bacteria in this wetland. Results suggested vegetation affected the distribution and community structures of n-damo bacteria in the sediments and n-damo could coexist with sulfate-reducing methanotrophs in the coastal ecosystem. Community structures of the Mai Po n-damo bacteria based on 16S rRNA gene were different from those of either the freshwater or the marine. In contrast, structures of the Mai Po n-damo communities based on pmoA gene grouped with the marine ones and were clearly distinguished from the freshwater ones. The abundance of n-damo bacteria at this wetland was quantified using 16S rRNA gene PCR primers to be 2.65-6.71 × 10(5) copies/g dry sediment. Ammonium and nitrite strongly affected the community structures and distribution of n-damo bacteria in the coastal Mai Po wetland sediments.

  14. Advances in Chemical Amplification Resist Systems (United States)

    Ito, Hiroshi


    The chemical amplification concept proposed in 1982 to boost resist sensitivities is now well accepted by the lithography community, which stems not only from high sensitivities that chemical amplification resist systems can offer but also from additional benefits of high contrasts and unexpectedly high resolution capabilities. The design flexibility and versatility that the use of acid as a catalytic species offers are another attractive feature of chemical amplification, giving rise to a birth of an entire family of advanced resist systems. Manufacture and prototype fabrication of DRAM’s by deep UV lithography have been accomplished with use of chemical amplification resists. However, some process problems uniquely associated with chemical amplification resists have surfaced recently, which include their latent image instability due to their sensitivity toward minute amounts of air-borne contaminants. This paper reviews recent advances made in our laboratory in the field of chemical amplification resist systems and discusses 1) influence of residual casting solvent on absorption of NMP by polymer films, 2) effects of polymer end groups on resist sensitivity, and 3) new imaging mechanisms based on acid-catalyzed dehydration.

  15. Complementary RNA amplification methods enhance microarray identification of transcripts expressed in the C. elegans nervous system

    Directory of Open Access Journals (Sweden)

    Levy Shawn


    Full Text Available Abstract Background DNA microarrays provide a powerful method for global analysis of gene expression. The application of this technology to specific cell types and tissues, however, is typically limited by small amounts of available mRNA, thereby necessitating amplification. Here we compare microarray results obtained with two different methods of RNA amplification to profile gene expression in the C. elegans larval nervous system. Results We used the mRNA-tagging strategy to isolate transcripts specifically from C. elegans larval neurons. The WT-Ovation Pico System (WT-Pico was used to amplify 2 ng of pan-neural RNA to produce labeled cDNA for microarray analysis. These WT-Pico-derived data were compared to microarray results obtained with a labeled aRNA target generated by two rounds of In Vitro Transcription (IVT of 25 ng of pan-neural RNA. WT-Pico results in a higher fraction of present calls than IVT, a finding consistent with the proposal that DNA-DNA hybridization results in lower mismatch signals than the RNA-DNA heteroduplexes produced by IVT amplification. Microarray data sets from these samples were compared to a reference profile of all larval cells to identify transcripts with elevated expression in neurons. These results were validated by the high proportion of known neuron-expressed genes detected in these profiles and by promoter-GFP constructs for previously uncharacterized genes in these data sets. Together, the IVT and WT-Pico methods identified 2,173 unique neuron-enriched transcripts. Only about half of these transcripts (1,044, however, are detected as enriched by both IVT and WT-Pico amplification. Conclusion We show that two different methods of RNA amplification, IVT and WT-Pico, produce valid microarray profiles of gene expression in the C. elegans larval nervous system with a low rate of false positives. However, our results also show that each method of RNA amplification detects a unique subset of bona fide neural


    Institute of Scientific and Technical Information of China (English)

    刘霄卉; 于立新; 刘畅; 周建华; 马学恩


    Objective:To clone and sequenceJSRV infection in sheep and healthy sheep Kras, Nros, Kras gene first and second exon and to study the mutation of the gene. Methods: In this study, the H - ras, N - ras, K-ras gene first and second exon DNA was extracted from healthy sheep and sheep infected with JSRV lung tissue and amplified with PCR. The PCR product was cloned into pMD -18T vector plasmid. Than the recorabinant plasmid was identified and sequenced. Results:Compared with other species, ras gene had little difference among species and highly homology with the cattle ras gene. The U-ras, K-ras gene cloned was identical in the first and second exon region sequence, while the N - ras had a A - V difference in the 81th amino acid. There were no mutation ras gene between the two cases of JSRV infection in sheep and healthy sheep neither in nucleic acid level nor in the amino acid level. Conclusion : The cloning and sequencing of the first and second exon of the sheep ras gene laid a good foundation for further study.%对JSRV感染羊及健康绵羊H-ras、N-ras、K-ras基因第一、二外显子区克隆并测序,研究其变异状况.方法:本研究从JSRV感染羊及健康绵羊肺组织中提取基因组DNA,应用PCR技术扩增Hras、Nras、Kras基因第一、二外显子序列,并通过T-A克隆的方法将其克隆入pMD - 18T载体中,鉴定为阳性的重组质粒进行序列测定分析.结果:与其他物种ras基因相比,所克隆的ras基因与其他物种之间的差异率较小,基本保守,且与牛的ras基因同源性最高.所克隆的H -ras、K-ras基因在第一、二外显子区域序列完全相同;N-ras第81位氨基酸A-V的不同;但试验中检测的2例JSRV感染羊与健康绵羊的ras无论是在核酸水平上,还是在氨基酸水平上均未发现突变.结论:克隆测序了绵羊ras基因第一、二外显子,为下一步实验奠定了基础.

  17. Chum-RNA allows preparation of a high-quality cDNA library from a single-cell quantity of mRNA without PCR amplification



    Linear RNA amplification using T7 RNA polymerase is useful in genome-wide analysis of gene expression using DNA microarrays, but exponential amplification using polymerase chain reaction (PCR) is still required for cDNA library preparation from single-cell quantities of RNA. We have designed a small RNA molecule called chum-RNA that has enabled us to prepare a single-cell cDNA library after four rounds of T7-based linear amplification, without using PCR amplification. Chum-RNA drove cDNA synt...

  18. Post-amplification Klenow fragment treatment alleviates PCR bias caused by partially single-stranded amplicons

    NARCIS (Netherlands)

    Egert, M.G.G.; Friedrich, M.W.


    Partially single-stranded amplicons, formed during PCR amplification of single and mixed templates, are a potential source of bias in genetic diversity studies. The analysis of 16S rRNA gene diversity in mixed template samples by the fingerprinting technique terminal restriction fragment length poly

  19. Identification of porcine Pneumocystis carinii as a genetically distinct organism by DNA amplification

    DEFF Research Database (Denmark)

    Wakefield, A. E.; Keely, S. P.; Stringer, J. R.


    DNA was amplified from lung samples from three piglets infected with Pneumocystis carinii, using oligonucleotide primers designed to the P. carinii mitochondrial large subunit ribosomal RNA gene. The nucleotide sequence of the amplification product was determined and indicated lack of sequence...

  20. Amplification of the polymerase gene of norovirus and a study of its genetic variation%诺如病毒聚合酶基因的扩增及其遗传变异研究

    Institute of Scientific and Technical Information of China (English)

    杜雄伟; 李叶; 肇惠君


    Objective To detect the pathogen and trace its origin. Methods The polymerase gene sequence was obtained by RT-PCR, and its genetic variation was studied based on molecular biological software and Internet databases. Results The pathogen is G II -4 genotype norovirus and its polymerase gene was conserved. The polymerase gene sequence had 99 % similarity to a strain that appeared in Japan at the same time. The virus strain may have originated from Japan. Conclusion Norovirus is a major cause of acute epidemic non-bacterial gastroenteritis in adults and children worldwide and is highly prevalent in shellfish, so its prevalence should be strictly monitored and its presence should be detected in imported/exported aquatic products to avoid transnational communication.%目的 检测该病原,并溯源. 方法 通过反转录聚合酶链反应(RT-PCR)、测序获得聚合酶基因序列,分子生物学分析软件及互联网数据库进行遗传变异研究. 结果 该病原为GⅡ-4亚型诺如病毒,其聚合酶基因非常保守,与同期日本爆发的诺如病毒毒株序列相似性达99%,可能来源于日本. 结论 诺如病毒感染引起的急性胃肠炎是影响全世界成人和儿童健康的重要因素,该病毒可以在贝类中富集,因此严格监测国内疫情、检测水产品进出境以避免该病的跨国传播.

  1. One New Method of Nucleic Acid Amplification-Loop-mediated Isothermal Amplification of DNA

    Institute of Scientific and Technical Information of China (English)

    Xue-en FANG; Jian LI; Qin CHEN


    Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method, which amplifies DNA with high specificity, sensitivity, rapidity and efficiency under isothermal conditions using a set of four specially designed primers and a Bst DNA polymerase with strand displacement activity. The basic principle, characteristics, development of LAMP and its applications are summarized in this article.

  2. CGH, cDNA and Tissue Microarray Analyses Implicate FGFR2 Amplification in a Small Subset of Breast Tumors

    Directory of Open Access Journals (Sweden)

    Mervi Heiskanen


    Full Text Available Multiple regions of the genome are often amplified during breast cancer development and progression, as evidenced in a number of published studies by comparative genomic hybridization (CGH. However, only relatively few target genes for such amplifications have been identified. Here, we indicate how small‐scale commercially available cDNA and CGH microarray formats combined with the tissue microarray technology enable rapid identification of putative amplification target genes as well as analysis of their clinical significance. According to CGH, the SUM‐52 breast cancer cell line harbors several high‐level DNA amplification sites, including the 10q26 chromosomal region where the fibroblast growth factor receptor 2 (FGFR2 gene has been localized. High level amplification of FGFR2 in SUM‐52 was identified using CGH analysis on a microarray of BAC clones. A cDNA microarray survey of 588 genes showed >40‐fold overexpression of FGFR2. Finally, a tissue microarray based FISH analysis of 750 uncultured primary breast cancers demonstrated in vivo amplification of the FGFR2 gene in about 1% of the tumors. In conclusion, three consecutive microarray (CGH, cDNA and tissue experiments revealed high‐level amplification and overexpression of the FGFR2 in a breast cancer cell line, but only a low frequency of involvement in primary breast tumors. Applied to a genomic scale with larger arrays, this strategy should facilitate identification of the most important target genes for cytogenetic rearrangements, such as DNA amplification sites detected by conventional CGH. Figures on‐4/heiskanen.htm

  3. Development of a nested polymerase chain reaction for amplification of a sequence of the p57 gene of Renibacterium salmoninarum that provides a highly sensitive method for detection of the bacterium in salmonid kidney (United States)

    Chase, D.M.; Pascho, R.J.


    Nucleic acid-based assays have shown promise for diagnosing Renibacterium salmoninarum in tissues and body fluids of salmonids. DeVelopment of a nested polymerase chain reaction (PCR) method to detect a 320 bp DNA segment of the gene encoding the p57 protein of R. salmoninarum is described. Whereas a conventional PCR for a 383 bp segment of the p57 gene reliably detected 1000 R. salmoninarum cells per reaction in kidney tissue, the nested PCR detected as few as 10 R. salmoninarum per reaction in kidney tissue. Two DNA extraction methods for the nested PCR were compared and the correlation between replicate samples was generally higher in samples extracted by the QIAamp system compared with those extracted by the phenol/chloroform method. The specificity of the nested PCR was confirmed by testing DNA extracts of common bacterial fish pathogens and a panel of bacterial species reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA) and the fluorescent antibody test (FAT) for R. salmoninarum. Kidney samples from 74 naturally infected chinook Salmon were examined by the nested PCR, the ELISA, and the FAT, and the detected prevalences of R. salmoninarum were 61, 47, and 43%, respectively.

  4. 鸡Toll样受体3基因全长的扩增及生物信息学分析%Amplification and bioinformatics analysis of the full-length chicken toil-like receptor 3 gene

    Institute of Scientific and Technical Information of China (English)

    甘珊珊; 何秀苗; 韦平


    In this study,the full-length chicken toll-like receptor 3 gene(TLR3) was amplified from the PBLs of Guangxi Sanhuang chicken by reverse transcription-polymerase chain reaction(RT-PCR). Sequence analysis and structure prediction of TLR3 gene were carried out. In results, the full-length TLR3 gene was 3 036 bp in size and shared from 96. 5% to 99. 7% homology with the other TLR3 sequences. The amino acid sequences showed that the mutations mainly happened in 5 end of extracellular region(from 1 to 173 amino acids). TLR3 shared a low homology from 68. 0% to 74. 8% with Bos taurus, Mus musculus ,Ovis aries ,Suis scrofa ,Equus caballus ,Oryctolagus,Pan trogldytes ,Homo sapiens and Gorilla. In the phyloge-netic tree,TLR3s of avian,mammals and rats were divided into two clades. The avian TLR3 belongs to one clade and the mammals and rats TLR3 belong to another clade. Structure prediction showed that TLR3 was a transmembrane protein whose extracellular region showed a horseshoe-shaped solenoid structure with fifteen leucine-rich repeats,consisted of a lot of α-helix on outside and β-sheet on the concave surface inside and they arranged parallelly and alternately. These results suggested the chicken TLR3 gene was distinct in dimentional structure,which might serve as a basis for further study on the function of TLR3 gene.%利用RT-PCR方法从广西三黄鸡外周血淋巴白细胞中扩增出鸡Toll样受体3基因(TLR3),进而对其进行序列分析和结构预测.结果显示,扩增出的TLR3基因全长为3036 bp,与GenBank中鸡TLR3序列同源性达96.5%~99.7%,突变主要集中在胞外区前173个氨基酸;与牛、鼠、绵羊、猪、马、家兔、黑猩猩、人、大猩猩的氨基酸同源性较低,为68.0%~74.8%;禽类TLR3与其他动物类及人类的TIR3分属于两大不同分支,处于不同进化位置;结构预测表明,GX-sh-chTLR3具有明显的跨膜蛋白结构,胞外区蛋白是由外侧多个α螺旋和内侧凹陷的多个β折叠构成

  5. Identification of alternative transcripts using rapid amplification of cDNA ends (RACE). (United States)

    Yeku, Oladapo; Scotto-Lavino, Elizabeth; Frohman, Michael A


    Many organisms, including humans, have many more proteins than are actually coded for by their genes. This discrepancy is partially explained by the existence of alternative transcripts produced by the same gene. Multiple isoforms of the same gene sometimes perform completely different functions, and as such, knowing the sequence of one of the transcripts is not enough. Rapid Amplification of cDNA Ends (RACE) provides an inexpensive and powerful tool to quickly identify alternative transcripts of a gene when the partial or complete sequence of only one transcript is known. In the following sections, we outline details for rapid amplification of 5' and 3' cDNA ends using the "New Race" technique.

  6. High-density SNP arrays improve detection of HER2 amplification and polyploidy in breast tumors

    DEFF Research Database (Denmark)

    Hansen, Thomas V. O.; Vikesaa, Jonas; Buhl, Sine S


    ) arrays can provide additional diagnostic power to assess HER2 gene status. METHODS: DNA from 65 breast tumor samples previously diagnosed by HER2 IHC and FISH analysis were blinded and examined for HER2 copy number variation employing SNP array analysis. RESULTS: SNP array analysis identified 24 (37......%) samples with selective amplification or imbalance of the HER2 region in the q-arm of chromosome 17. In contrast, only 15 (23%) tumors were found to have HER2 amplification by IHC and FISH analysis. In total, there was a discrepancy in 19 (29%) samples between SNP array and IHC/FISH analysis. In 12...

  7. Time varying arctic climate change amplification

    Energy Technology Data Exchange (ETDEWEB)

    Chylek, Petr [Los Alamos National Laboratory; Dubey, Manvendra K [Los Alamos National Laboratory; Lesins, Glen [DALLHOUSIE U; Wang, Muyin [NOAA/JISAO


    During the past 130 years the global mean surface air temperature has risen by about 0.75 K. Due to feedbacks -- including the snow/ice albedo feedback -- the warming in the Arctic is expected to proceed at a faster rate than the global average. Climate model simulations suggest that this Arctic amplification produces warming that is two to three times larger than the global mean. Understanding the Arctic amplification is essential for projections of future Arctic climate including sea ice extent and melting of the Greenland ice sheet. We use the temperature records from the Arctic stations to show that (a) the Arctic amplification is larger at latitudes above 700 N compared to those within 64-70oN belt, and that, surprisingly; (b) the ratio of the Arctic to global rate of temperature change is not constant but varies on the decadal timescale. This time dependence will affect future projections of climate changes in the Arctic.

  8. Amplification, Redundancy, and Quantum Chernoff Information (United States)

    Zwolak, Michael; Riedel, C. Jess; Zurek, Wojciech H.


    Amplification was regarded, since the early days of quantum theory, as a mysterious ingredient that endows quantum microstates with macroscopic consequences, key to the "collapse of the wave packet," and a way to avoid embarrassing problems exemplified by Schrödinger's cat. Such a bridge between the quantum microworld and the classical world of our experience was postulated ad hoc in the Copenhagen interpretation. Quantum Darwinism views amplification as replication, in many copies, of the information about quantum states. We show that such amplification is a natural consequence of a broad class of models of decoherence, including the photon environment we use to obtain most of our information. This leads to objective reality via the presence of robust and widely accessible records of selected quantum states. The resulting redundancy (the number of copies deposited in the environment) follows from the quantum Chernoff information that quantifies the information transmitted by a typical elementary subsystem of the environment.

  9. On Arbitrary Phases in Quantum Amplitude Amplification

    CERN Document Server

    Hoyer, P


    We consider the use of arbitrary phases in quantum amplitude amplification which is a generalization of quantum searching. We prove that the phase condition in amplitude amplification is given by $\\tan(\\phi/2)=\\tan(\\phi/2)(1-2a)$, where $\\phi$ and $\\phi$ are the phases used and where $a$ is the success probability of the given algorithm. Thus the choice of phases depends nontrivially and nonlinearly on the success probability. Utilizing this condition, we give methods for constructing quantum algorithms that succeed with certainty and for implementing arbitrary rotations. We also conclude that phase errors of order up to $\\frac{1}{\\sqrt{a}}$ can be tolerated in amplitude amplification.

  10. Detection of gene amplification in MYCN, C-MYC, MYCL1, ERBB2, EGFR, AKT2, and human papilloma virus in samples from cervical smear normal cytology, intraepithelial cervical neoplasia (CIN I, II, III, and cervical cancer

    Directory of Open Access Journals (Sweden)

    Dabeiba Adriana García


    Full Text Available Introducción: El cáncer cervical es el segundo cáncer más importante en mujeres a nivel mundial y es la segunda causa de muerte por cáncer en mujeres. Se ha demostrado que el proceso de carcinogénesis cervical presenta componentes tanto genéticos como epigenéticos y medio ambientales. En la actualidad, hay gran interés en la búsqueda de marcadores moleculares asociados con la progresión de esta enfermedad, uno de los posibles mecanismos y que además está poco estudiado en cáncer cervical es la amplificación génica de algunos oncogenes como la familia MYC, EGFR y AKT entre otros. Objetivos: Detectar la amplificación génica de MYCN, C-MYC, MYCL1, ERBB2, EGFR y AKT2 además de la presencia del virus de papiloma humano en cepillados cervicales en mujeres con citología normal o con neoplasia intraepitelial cervical (NIC I, II y III o con cáncer cervical. Métodos: Se genotipificó mediante reverse line blot (RLB el virus de papiloma humano (VPH y se determinó el estado de amplificación génica de los genes mencionados mediante PCR en tiempo real utilizando sondas taqman. Resultados: El VPH se encontró presente en 4% de las pacientes con citología normal, en 48% en NIC I, 63.6% en NIC II, 64% en NIC III y 70.8% en cáncer cervical. Los genes MYCN, MYCL1 y ERBB2 mostraron mayor amplificación en lesiones de alto grado y cáncer con diferencias estadísticamente significativas  a las lesiones de bajo grado y citología normal, en 39.1%, 34.7% y 30.4% respectivamente. Además, se encontraron amplificados los genes C-MYC, EGFR y AKT2, en muestras de pacientes con cáncer cervical, en 12%, 18% y 13% respectivamente. Sin embargo, no se observaron diferencias estadísticamente significativas con respecto a las lesiones de alto y bajo grado y citología normal. Conclusión: En las lesiones de alto grado como en cáncer cervical, se encuentra mayor prevalencia del virus al igual que se detectan mayor cantidad de alteraciones gen

  11. 小鼠GM-CSF基因的扩增及在HEK293T细胞中的分泌表达%Amplification of mouse GM-CSF gene and its expression in HEK293T cells

    Institute of Scientific and Technical Information of China (English)

    李楠; 张峰; 仲飞


    粒细胞-巨噬细胞集落刺激因子( GM-CSF)是机体免疫系统的重要细胞因子,具有生物佐剂作用,为研究GM-CSF的生物佐剂作用,本试验通过RT-PCR方法从小鼠脾脏细胞中扩增小鼠GM-CSF的cDNA,并将其插入到pcDNA3.1质粒中,构建成GM-CSF真核表达载体pcDNA3.1-GMCSF,并在真核细胞进行了瞬时表达.结果表明,本研究扩增的小鼠GM-CSF基因序列与GenBank序列完全一致,表达载体经脂质体介导转染HEK293T细胞,表达产物经western-blot检测,证明GM-CSF能够在HEK293T细胞中进行分泌表达.这为今后研究GM-CSF在动物疫苗,特别是DNA疫苗中的生物佐剂作用创造了必要的条件.%Granulocyte and macrophage colony-stimulating factor (GM-CSF) is an important cy-tokine in animal immune system and is also a biological adjuvant. To express GM-CSF in a secretory manner in eukaryotic cells, the mouse GM-CSF cDNA was amplified with RT-PCR from mouse spleen and was inserted into pcDNA3. 1A expression vector to construct its eukaryotic expression vector, pcDNA3. 1-GMCSF. The transient expression of GM-CSF was performed in HEK293T cells transfected via liposome mediation. The results showed that the amplified GM-CSF gene sequence was consistent with that published in GenBank. The recombi-nant mouse GM-CSF was detected by Western-blot in the culture medium of the transfected HEK293T cells, which indicates that the GM-CSF gene could be expressed and secreted in the cells. Those results provide the necessary conditions for further study on the GM-CSF adjuvant functions in animal vaccine, especially in animal DNA vaccine.

  12. Amplificação dos genes que codificam a endotelina-1 e seus receptores em valvas mitrais reumáticas Amplificación de los genes que codifican la endotelina-1 y sus receptores en válvulas mitrales reumáticas Amplification of the genes that codify endothelin-1 and its receptors in rheumatic mitral valves

    Directory of Open Access Journals (Sweden)

    Edmilson Bastos de Moura


    Full Text Available FUNDAMENTO: As cardiopatias são doenças de alta prevalência, sendo a cardite reumática uma doença de grande relevância em países em desenvolvimento. As alterações em câmaras cardíacas esquerdas se associam à disfunção endotelial, com aumento dos níveis de endotelina-1 (ET-1 e consequências sobre a circulação pulmonar, muitas vezes determinando a hipertensão pulmonar (HP. No entanto, a presença de ET-1 e seus receptores na própria valva mitral, promovendo alterações vasculares pulmonares e aumentando a deformação valvar reumática, ainda é um assunto não abordado na literatura. OBJETIVO: Determinar, mediante técnicas moleculares, a expressão dos genes da endotelina e dos seus receptores em valvas mitrais reumáticas. MÉTODOS: 27 pacientes submetidos à troca valvar mitral tiveram seu tecido valvar analisado, a fim de determinar a presença de genes de ET-1 e seus receptores A e B. Foram feitas análises histológica e molecular das valvas (divididas em fragmentos M1, M2 e M3 e colhidos dados clínicos e epidemiológicos dos pacientes. Foram divididos em três grupos: valvopatia mitral, mitroaórtica e pacientes reoperados. RESULTADOS: O estudo mostrou a manifestação do gene da ET-1 em 40,7% dos espécimes e de seu receptor A em todas as amostras, com manifestação minoritária do gene do receptor B (22,2%. CONCLUSÃO: Todos os pacientes expressaram a presença do gene do receptor A. Não houve diferença estatística quanto à gravidade da doença, expressa em classe funcional, e aos subgrupos estudados (valvopatas mitrais, mitroaórticos e pacientes reoperados, ou quanto à expressão dos genes da ET-1 e seus receptores entre os subgrupos estudados (valvopatas mitrais, mitroaórticos e pacientes reoperados.FUNDAMENTO: Las cardiopatías son enfermedades de alta prevalencia, siendo la carditis reumática una enfermedad de gran relevancia en países en desarrollo. Las alteraciones en cámaras cardíacas izquierdas

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

    Directory of Open Access Journals (Sweden)

    Ben Beheshti


    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.

  14. Continuous phase amplification with a Sagnac interferometer

    CERN Document Server

    Starling, David J; Williams, Nathan S; Jordan, Andrew N; Howell, John C


    We describe a weak value inspired phase amplification technique in a Sagnac interferometer. We monitor the relative phase between two paths of a slightly misaligned interferometer by measuring the average position of a split-Gaussian mode in the dark port. Although we monitor only the dark port, we show that the signal varies linearly with phase and that we can obtain similar sensitivity to balanced homodyne detection. We derive the source of the amplification both with classical wave optics and as an inverse weak value.

  15. Effective Privacy Amplification for Secure Classical Communications

    CERN Document Server

    Horvath, Tamas; Scheuer, Jacob


    We study the effectiveness of privacy amplification for classical key-distribution schemes. We find that, unlike quantum key distribution schemes, the high fidelity of the raw key in classical systems allow the users to always sift a secure shorter key, given that they have an upper bound of eavesdropper probability to correctly guess the exchanged key-bits. We establish the number of privacy amplification iterations needed to achieve information leak of 10^-8 in several classical systems and highlight the inherent tradeoff between the number of iterations and the security of the raw key.

  16. Parametric Amplification For Detecting Weak Optical Signals (United States)

    Hemmati, Hamid; Chen, Chien; Chakravarthi, Prakash


    Optical-communication receivers of proposed type implement high-sensitivity scheme of optical parametric amplification followed by direct detection for reception of extremely weak signals. Incorporates both optical parametric amplification and direct detection into optimized design enhancing effective signal-to-noise ratios during reception in photon-starved (photon-counting) regime. Eliminates need for complexity of heterodyne detection scheme and partly overcomes limitations imposed on older direct-detection schemes by noise generated in receivers and by limits on quantum efficiencies of photodetectors.

  17. 豹源猫杯状病毒ORF2基因的扩增、克隆及原核表达%Amplification, Clone and Prokaryotic Expression of ORF2 Gene of Feline Calicivirus Isolated from Leopard

    Institute of Scientific and Technical Information of China (English)

    刘秋艳; 应瑛; 王一鸣; 陈小庆; 赵艳丽; 胡桂学


    In order to prepare the antigen for serological detection of Feline calicivirus, the primers were designed in according to the gene sequence of 1 FCV isolated from a leopard preserved in our laboratory. The 1 734 nucleotides in open reading frame 2 (ORF2) were amplified and cloned into expression vector pET-28a to construct recombinant plasmid named pET-28a-FCV1 734. The recom-binant plasmid was transformed into E. coli. The expression product induced by IPTG was determined by SDS-PAGE and Western blot. The result proved that the expressed protein had good reactogenicity.%为制备猫杯状病毒血清学检测抗原,本研究根据实验室保存的1株豹源猫杯状病毒基因序列设计引物,扩增了开放阅读框2(ORF2)中1734 bp的核苷酸序列,将其克隆至表达载体pET-28a上,构建了pET-28a-FCV1734重组质粒。将该质粒转化大肠杆菌,诱导表达产物经SDS-PAGE和Western blot检测,证明所表达的蛋白具有良好的反应原性。

  18. A dual amplification fluorescent strategy for sensitive detection of DNA methyltransferase activity based on strand displacement amplification and DNAzyme amplification. (United States)

    Cui, Wanling; Wang, Lei; Jiang, Wei


    DNA methyltransferase (MTase) plays a critical role in many biological processes and has been regarded as a predictive cancer biomarker and a therapeutic target in cancer treatment. Sensitive detection of DNA MTase activity is essential for early cancer diagnosis and therapeutics. Here, we developed a dual amplification fluorescent strategy for sensitive detection of DNA MTase activity based on strand displacement amplification (SDA) and DNAzyme amplification. A trifunctional double-stranded DNA (dsDNA) probe was designed including a methylation site for DNA MTase recognition, a complementary sequence of 8-17 DNAzyme for synthesizing DNAzyme, and a nicking site for nicking enzyme cleavage. Firstly, the trifunctional dsDNA probe was methylated by DNA MTase to form the methylated dsDNA. Subsequently, HpaII restriction endonuclease specifically cleaved the residue of unmethylated dsDNA. Next, under the action of polymerase and nicking enzyme, the methylared dsDNA initiated SDA, releasing numbers of 8-17 DNAzymes. Finally, the released 8-17 DNAzymes triggered DNAzyme amplification reaction to induce a significant fluorescence enhancement. This strategy could detect DNA MTase activity as low as 0.0082U/mL. Additionally, the strategy was successfully applied for evaluating the inhibitions of DNA MTase using two anticancer drugs, 5-azacytidine and 5-aza-2'-deoxycytidine. The results indicate the proposed strategy has a potential application in early cancer diagnosis and therapeutics.

  19. Paralog-specific primers for the amplification of nuclear Loci in tetraploid barbels (barbus: cypriniformes). (United States)

    Gante, Hugo F; Alves, Maria Judite; Dowling, Thomas E


    Thirty paralog-specific primers were developed, following an intron-primed exon-crossing strategy, for S7 and growth hormone genes in Barbus (subgenera Barbus and Luciobarbus). We found that paralog-specific amplification requires the use of only one paralog-specific primer, allowing their simultaneous use with universal exon-primed intron-crossing primers of broad taxonomic applicability. This hybrid annealing strategy guarantees both specificity and generality of amplification reactions and represents a step forward in the amplification of duplicated nuclear loci in polyploid organisms and members of multigene families. Assays of several representative taxa identified high levels of segregating single nucleotide polymorphisms (SNPs) and nucleotide diversity within each of these subgenera. Additionally, several insertions-deletions (indels) that are diagnostic across species are found in intronic regions. Therefore, these primers provide a reliable source of valuable nuclear SNP and indel data for population and species level studies of barbels, such as applied conservation and basic evolutionary studies.

  20. Determination of DQB1 alleles using PCR amplification and allele-specific primers. (United States)

    Lepage, V; Ivanova, R; Loste, M N; Mallet, C; Douay, C; Naoumova, E; Charron, D


    Molecular genotyping of HLA class II genes is commonly carried out using polymerase chain reaction (PCR) in combination with sequence-specific oligotyping (PCR-SSO) or a combination of the PCR and restriction fragment length polymorphism methods (PCR-RFLP). However, the identification of the DQB1 type by PCR-SSO and PCR-RFLP is very time-consuming which is disadvantageous for the typing of cadaveric organ donors. We have developed a DQB1 typing method using PCR in combination with allele-specific amplification (PCR-ASA), which allows the identification of the 17 most frequent alleles in one step using seven amplification mixtures. PCR allele-specific amplification HLA-DQB1 typing is easy to perform, and the results are easy to interpret in routine clinical practice. The PCR-ASA method is therefore better suited to DQB1 typing for organ transplantation than other methods.

  1. Whole Genome Amplification in Genomic Analysis of Single Circulating Tumor Cells. (United States)

    Gasch, Christin; Pantel, Klaus; Riethdorf, Sabine


    Investigation of the genome of organisms is one of the major basics in molecular biology to understand the complex organization of cells. While genomic DNA can easily be isolated from tissues or cell cultures of plant, animal or human origin, DNA extraction from single cells is still challenging. Here, we describe three techniques for the amplification of genomic DNA of fixed single circulating tumor cells (CTC) isolated from blood of cancer patients. This amplification is aimed to increase DNA amounts from those of one cell to yields sufficient for different DNA analyses such as mutational analysis including next-generation sequencing, array-comparative genome hybridization (CGH), and quantitative measurement of gene amplifications. Molecular analysis of CTC as liquid biopsy can be used to identify therapeutic targets in personalized medicine directed, e.g. against human epidermal growth factor receptor 2 (HER2) or epidermal growth factor receptor (EGFR) and to stratify the patients to those therapies.

  2. ASAP: Amplification, sequencing & annotation of plastomes

    Directory of Open Access Journals (Sweden)

    Folta Kevin M


    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

  3. Genotypic analysis of cutaneous T-cell lymphoma: a comparative study of Southern blot analysis with polymerase chain reaction amplification of the T-cell receptor-gamma gene. (United States)

    Curcó, N; Servitje, O; Llucià, M; Bertran, J; Limón, A; Carmona, M; Romagosa, V; Peyrí, J


    The diagnosis of early cutaneous T-cell lymphoma (CTCL) is a difficult point in dermatology. Recently, Southern blot analysis (SBA) and polymerase chain reaction (PCR) have been used to detect clonality in initial lesions in which clinical and histological findings are unspecific. Forty-one samples from 25 patients with CTCL were investigated for the presence of T-cell receptor-gamma gene rearrangement using a nested PCR technique and analysed by polyacrylamide gel electrophoresis (PAGE). Conventional SBA was also performed on 28 samples from 20 of these patients. In addition, 20 samples corresponding to patients with large plaque parapsoriasis (LPP), cutaneous B-cell lymphoma (CBCL) and eczema were analysed by PCR in the same way as were the CTCL specimens. Most of the CTCL specimens (81%) showed clonality on PCR analysis. Among patients with mycosis fungoides, 71% of initial patch lesions and 100% of plaques and tumours showed clonal disease. Clonality could be detected in three of four histologically negative post-treatment lesions. Clonal rearrangement was detected in one of three patients with LPP and in three of 10 patients with CBCL. None of the samples corresponding to patients with eczema showed positive results. SBA was significantly less sensitive than PCR in detecting clonality in CTCL patients (42% among early disease and 60% among advanced cases). The results indicate that this PCR/PAGE technique is a reliable and useful method for the detection of clonality in early skin lesions of CTCL patients and probably in the identification of silent extracutaneous involvement.

  4. PCR bias in amplification of androgen receptor alleles, a trinucleotide repeat marker used in clonality studies. (United States)

    Mutter, G L; Boynton, K A


    Trinucleotide CAG repeats in the X-linked human androgen receptor gene (HUMARA) have proved a useful means of determining X chromosome haplotypes, and when combined with methylation analysis of nearby cytosine residues permits identification of non-random X inactivation in tumors of women. Co-amplification of two alleles in a heterozygote generates PCR products which differ in the number of CAG units, and thus their melting and secondary structure characteristics. We have shown that under optimal conditions amplification efficiency of two HUMARA alleles is near-equivalent, generating PCR products in a ratio proportional to that of the genomic template. In contrast, reduction of template quantity, damage of template by ultraviolet irradiation or addition of monovalent salts (sodium chloride, sodium acetate or ammonium acetate) produces highly variable imbalances of allelic PCR products, with a strong tendency to preferentially amplify lower molecular weight alleles. Variability and biasing was diminished by substitution of 7-deaza-2'-dGTP for dGTP during amplification, an intervention which reduces stability of intramolecular and intermolecular GC base pairing. We conclude that DNA which is scanty, damaged or salt contaminated may display amplification bias of GC-rich PCR targets, potentially confounding accurate interpretation or reproducibility of assays which require co-amplification of alleles.

  5. Gene (United States)

    U.S. Department of Health & Human Services — Gene integrates information from a wide range of species. A record may include nomenclature, Reference Sequences (RefSeqs), maps, pathways, variations, phenotypes,...

  6. Intelligence amplification framework for enhancing scheduling processes

    NARCIS (Netherlands)

    Dobrkovic, Andrej; Liu, Luyao; Iacob, Maria-Eugenia; Hillegersberg, van Jos


    The scheduling process in a typical business environment consists of predominantly repetitive tasks that have to be completed in limited time and often containing some form of uncertainty. The intelligence amplification is a symbiotic relationship between a human and an intelligent agent. This partn

  7. Desert Amplification in a Warming Climate (United States)

    Zhou, Liming


    Here I analyze the observed and projected surface temperature anomalies over land between 50°S-50°N for the period 1950–2099 by large-scale ecoregion and find strongest warming consistently and persistently seen over driest ecoregions such as the Sahara desert and the Arabian Peninsula during various 30-year periods, pointing to desert amplification in a warming climate. This amplification enhances linearly with the global mean greenhouse gases(GHGs) radiative forcing and is attributable primarily to a stronger GHGs-enhanced downward longwave radiation forcing reaching the surface over drier ecoregions as a consequence of a warmer and thus moister atmosphere in response to increasing GHGs. These results indicate that desert amplification may represent a fundamental pattern of global warming associated with water vapor feedbacks over land in low- and mid- latitudes where surface warming rates depend inversely on ecosystem dryness. It is likely that desert amplification might involve two types of water vapor feedbacks that maximize respectively in the tropical upper troposphere and near the surface over deserts, with both being very dry and thus extremely sensitive to changes of water vapor.

  8. Intelligence amplification framework for enhancing scheduling processes

    NARCIS (Netherlands)

    Dobrkovic, Andrej; Liu, Luyao; Iacob, Maria Eugenia; van Hillegersberg, Jos


    The scheduling process in a typical business environment consists of predominantly repetitive tasks that have to be completed in limited time and often containing some form of uncertainty. The intelligence amplification is a symbiotic relationship between a human and an intelligent agent. This

  9. Social amplification of risk: a conceptual framework

    Energy Technology Data Exchange (ETDEWEB)

    Kasperson, R.E.; Renn, O.; Slovic, P.; Brown, H.S.; Emel, J.; Goble, R.; Kasperson, J.X.; Ratick, S.


    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.

  10. Amplification of Mitochondrial DNA for detection of Plasmodiumvivax in Balochistan. (United States)

    Shahwani, Muhammad Naeem; Nisar, Samia; Aleem, Abdul; Panezai, Marina; Afridi, Sarwat; Malik, Shaukat Iqbal


    To access a new step using PCR to amplify the targeted mtDNA sequence for detecting specifically Plasmodium vivax and its co-infections, false positive and false negative results with Plasmodium falciparum. In this study we have standardized a new technical approach in which the target mitochondrial DNA sequence (mtDNA) was amplified by using a PCR technique as a tool to detect Plasmodium spp. Species specific primers were designed to hybridize with cytochrome c oxidase gene of P. vivax (cox I) and P. falciparum (cox III). Two hundred blood samples were collected on the basis of clinical symptoms which were initially examined through microscopic analysis after preparing Giemsa stained thick and thin blood smears. Afterwards genomic DNA was extracted from all samples and was then subjected to PCR amplification by using species specific primers and amplified segments were sequenced for confirmation of results. One-hundred and thirty-two blood samples were detected as positive for malaria by PCR, out of which 64 were found to be positive by PCR and 53 by both microscopy and PCR for P.vivax infection. Nine samples were found to be false negative, one P.vivax mono infection was declared as co infection by PCR and 3 samples identified as having P.falciparum gametes were confirmed as P.vivax by PCR amplification. Sensitivity and specificity were found to be 85% and 92% respectively. Results obtained through PCR method were comparatively better and reliable than microscopy.

  11. The research of combining high resolution melting with multiplex ligation-dependent probe amplification technology in the mutation scanning for PAH gene%联合应用高分辨率熔解曲线和多重连接依赖探针扩增技术对PAH基因突变筛查研究

    Institute of Scientific and Technical Information of China (English)

    季春燕; 孙莉莉; 曹丽华; 胡煜; 黄宏; 王述森; 罗阳


    目的 联合应用高分辨率熔解曲线(high resolution melting,HRM)和多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测苯丙酮尿症(phenylketonuria,PKU)患者基因突变,并探讨两种技术联合运用对于突变筛查的价值.方法 采用HRM和MLPA技术对26例临床诊断为PKU的患者苯丙氨酸羟化酶基因(phenylalanine hydroxylase,PAH)进行检测,并通过测序验证;针对未报道的基因改变进行聚合酶链反应-限制性片段长度多态性分析.结果 在52个等位基因中检测到44个突变等位基因(共21种不同突变),包括第4外显子拷贝数的增加,总检出率达84.62%(44/52),其中c.584_585insA和IVS10+ 1G>T突变在国内外未见报道.此外,R243Q(25%)突变为中国最常见的突变种类.结论 应用HRM和MLPA技术相对提高了PAH基因突变筛查的检出率,检测结果丰富了基因突变数据库,并为临床诊断与产前诊断提供实验室依据.%Objective To explore the value of combining high resolution melting (HRM) with multiplex ligation-dependent probe amplification ( MLPA ) for detecting mutations underlying phenylketonuria.Methods HRM was used for detecting small mutations in phenylalanine hydroxylase gene (PAH) of 26 phenylketonuria patients.The results were verified with DNA sequencing.MLPA was used for detecting potential deletions/duplications in the PAH gene. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was performed for additional potential mutations.Results A total of 21 mutations were found in 44/52 alleles (84.62%),which included a dupEx4.Among the 21 types of mutation,19 were reported previously,and the remaining two were novel mutations:c.584_585insA and IVS10+1G>T.In addition,the mutation of R243Q (25%) was the most common type in China.Conclusion The study showed that the combination of HRM and MLPA could increase the detection rate for mutation in PKU.The study

  12. A new evolutionary theory deduced mathematically from entropy amplification

    Institute of Scientific and Technical Information of China (English)


    A new evolutionary theory which is able to unite the present evolutionary debates is deduced mathematically from the principle of entropy amplification.It suggests that the extensive evolution is driven by the amplification of entropy,or microscopic diversity,and the biological evolution is driven by the amplification of biodiversity.Forming high hierarchies is the most important way for the amplification and brings out spontaneously three kinds of selection.This theory has some positive cultural meanings.

  13. Low Prevalence of TP53 Mutations and MDM2 Amplifications in Pediatric Rhabdomyosarcoma

    Directory of Open Access Journals (Sweden)

    Simona Ognjanovic


    Full Text Available The tumor suppressor gene TP53 is the most commonly mutated gene in human cancer. The reported prevalence of mutations in rhabdomyosarcoma (RMS varies widely, with recent larger studies suggesting that TP53 mutations in pediatric RMS may be extremely rare. Overexpression of MDM2 also attenuates p53 function. We have performed TP53 mutation/MDM2 amplification analyses in the largest series analyzed thus far, including DNA isolated from 37 alveolar and 38 embryonal RMS tumor samples obtained from the Cooperative Human Tissue Network (CHTN. Available samples were frozen tumor tissues (N=48 and histopathology slides. TP53 mutations in exons 4–9 were analyzed by direct sequencing in all samples, and MDM2 amplification analysis was performed by differential PCR on a subset of 22 samples. We found only one sample (1/75, 1.3% carrying a TP53 mutation at codon 259 (p.D259Y and no MDM2 amplification. Two SNPs in the TP53 pathway, associated with accelerated tumor onset in germline TP53 mutation carriers, (TP53 SNP72 (rs no. 1042522 and MDM2 SNP309 (rs no. 2279744, were not found to confer earlier tumor onset. In conclusion, we confirm the extremely low prevalence of TP53 mutations/MDM2 amplifications in pediatric RMS (1.33% and 0%, respectively. The possible inactivation of p53 function by other mechanisms thus remains to be elucidated.

  14. Detection of APC gene germline mutation in Chinese familial adenomatous polyposis by direct sequencing in combination with multiplex ligation-dependent probe amplification%直接测序联合多重连接依赖探针扩增法检测家族性腺瘤性息肉病APC基因胚系突变

    Institute of Scientific and Technical Information of China (English)

    金鹏; 崔伟佳; 盛剑秋; 付蕾; 安贺娟; 李爱琴; 张明智; 韩英; 李世荣


    目的 研究中国家族性腺瘤性息肉病(FAP)患者APC基因胚系突变的特点.方法 对来自北京、河北、河南、安徽、内蒙古、山西、福建等地区的14个FAP家系先证者用直接测序法进行APC基因突变检测,对突变检测阴性者应用多重连接依赖探针扩增(MLPA)技术进行APE基因大片段缺失检测.结果 14例先证者中9例(64.3%)检测出APC基因微小突变,其中移码突变6例,剪接区突变2例,无义突变1例;2例(14.3%)检测出APC基因大片段缺失,微小突变与大片段缺失的总检出率为78.6%.c.2336-2337insT、c.3923-3929delAAGAAAA、c.532-2A>T和c.4179-4180GAdelinsT等4个微小突变和外显子11、10A缺失、外显子15 start缺失等2个大片段缺失为首次报道.结论 中国FAP患者APC基因的胚系突变类型多样,以移码突变居多,突变位点以第15外显子居多;直接测序法联合MLPA法检测大片段缺失可提高APC基因突变的检出率.%Objective To investigate the characteristics of APC gene germline mutation in Chinese patients with familial adenomatous polyposis ( FAP). Methods The genomic DNA was extracted from peripheral venous blood drawn from probands of 14 Chinese FAP families from Beijing, Hebei, Henan,Anhui, Inner Mongolia, Shanxi and Fujian. The APC gene was amplified by PCR and underwent direct sequencing. Large fragment deletion was detected by multiplex ligation-dependent probe amplification (MLPA) only in micromutation-negative samples found by sequencing. Results APC gene micromutations were found in 9 probands and the mieromutation detection rate was 64. 3%, including 6 frameshift mutations, 2 splicing mutations and 1 nonsense mutation. Large fragment deletions of APC gene were detected in 2 probands ( 14. 3% ). The total mutation detection rate of micromutation and large fragment deletion was 78. 6%. Four novel micmromutations and 2 novel large fragment deletions were found, including c. 2336-2337insT, c. 3923-3929delAAGAAAA, c

  15. Amplification of the 20q chromosomal arm occurs early in tumorigenic transformation and may initiate cancer.

    Directory of Open Access Journals (Sweden)

    Yuval Tabach

    Full Text Available Duplication of chromosomal arm 20q occurs in prostate, cervical, colon, gastric, bladder, melanoma, pancreas and breast cancer, suggesting that 20q amplification may play a causal role in tumorigenesis. According to an alternative view, chromosomal imbalance is mainly a common side effect of cancer progression. To test whether a specific genomic aberration might serve as a cancer initiating event, we established an in vitro system that models the evolutionary process of early stages of prostate tumor formation; normal prostate cells were immortalized by the over-expression of human telomerase catalytic subunit hTERT, and cultured for 650 days till several transformation hallmarks were observed. Gene expression patterns were measured and chromosomal aberrations were monitored by spectral karyotype analysis at different times. Several chromosomal aberrations, in particular duplication of chromosomal arm 20q, occurred early in the process and were fixed in the cell populations, while other aberrations became extinct shortly after their appearance. A wide range of bioinformatic tools, applied to our data and to data from several cancer databases, revealed that spontaneous 20q amplification can promote cancer initiation. Our computational model suggests that 20q amplification induced deregulation of several specific cancer-related pathways including the MAPK pathway, the p53 pathway and Polycomb group factors. In addition, activation of Myc, AML, B-Catenin and the ETS family transcription factors was identified as an important step in cancer development driven by 20q amplification. Finally we identified 13 "cancer initiating genes", located on 20q13, which were significantly over-expressed in many tumors, with expression levels correlated with tumor grade and outcome suggesting that these genes induce the malignant process upon 20q amplification.

  16. Plasmonic Terahertz Amplification in Graphene-Based Asymmetric Hyperbolic Metamaterial

    Directory of Open Access Journals (Sweden)

    Igor Nefedov


    Full Text Available We propose and theoretically explore terahertz amplification, based on stimulated generation of plasmons in graphene asymmetric hyperbolic metamaterials (AHMM, strongly coupled to terahertz radiation. In contrast to the terahertz amplification in resonant nanocavities, AHMM provides a wide-band THz amplification without any reflection in optically thin graphene multilayers.

  17. Isothermal DNA amplification in bioanalysis: strategies and applications. (United States)

    Kim, Joonyul; Easley, Christopher J


    Isothermal DNA amplification is an alternative to PCR-based amplification for point-of-care diagnosis. Since the early 1990s, the approach has been refined into a simple, rapid and cost-effective tool by means of several distinct strategies. Input signals have been diversified from DNA to RNA, protein or small organic molecules by translating these signals into input DNA before amplification, thus allowing assays on various classes of biomolecules. In situ detection of single biomolecules has been achieved using an isothermal method, leveraging localized signal amplification in an intact specimen. A few pioneering studies to develop a homogenous isothermal protein assay have successfully translated structure-switching of a probe upon target binding into input DNA for isothermal amplification. In addition to the detection of specific targets, isothermal methods have made whole-genome amplification of single cells possible owing to the unbiased, linear nature of the amplification process as well as the large size of amplified products given by ϕ29 DNA polymerase. These applications have been devised with the four isothermal amplification strategies covered in this review: strand-displacement amplification, rolling circle amplification, helicase-dependent amplification and recombinase polymerase amplification.

  18. Bioanalytical applications of isothermal nucleic acid amplification techniques. (United States)

    Deng, Huimin; Gao, Zhiqiang


    The most popular in vitro nucleic acid amplification techniques like polymerase chain reaction (PCR) including real-time PCR are costly and require thermocycling, rendering them unsuitable for uses at point-of-care. Highly efficient in vitro nucleic acid amplification techniques using simple, portable and low-cost instruments are crucial in disease diagnosis, mutation detection and biodefense. Toward this goal, isothermal amplification techniques that represent a group of attractive in vitro nucleic acid amplification techniques for bioanalysis have been developed. Unlike PCR where polymerases are easily deactivated by thermally labile constituents in a sample, some of the isothermal nucleic acid amplification techniques, such as helicase-dependent amplification and nucleic acid sequence-based amplification, enable the detection of bioanalytes with much simplified protocols and with minimal sample preparations since the entire amplification processes are performed isothermally. This review focuses on the isothermal nucleic acid amplification techniques and their applications in bioanalytical chemistry. Starting off from their amplification mechanisms and significant properties, the adoption of isothermal amplification techniques in bioanalytical chemistry and their future perspectives are discussed. Representative examples illustrating the performance and advantages of each isothermal amplification technique are discussed along with some discussion on the advantages and disadvantages of each technique.

  19. Plasmonic Terahertz Amplification in Graphene-Based Asymmetric Hyperbolic Metamaterial


    Igor Nefedov; Leonid Melnikov


    We propose and theoretically explore terahertz amplification, based on stimulated generation of plasmons in graphene asymmetric hyperbolic metamaterials (AHMM), strongly coupled to terahertz radiation. In contrast to the terahertz amplification in resonant nanocavities, AHMM provides a wide-band THz amplification without any reflection in optically thin graphene multilayers.

  20. Quantitation of viral load using real-time amplification techniques

    NARCIS (Netherlands)

    Niesters, H G


    Real-time PCR amplification techniques are currently used to determine the viral load in clinical samples for an increasing number of targets. Real-time PCR reduces the time necessary to generate results after amplification. In-house developed PCR and nucleic acid sequence-based amplification (NASBA

  1. Pure invasive micropapillary carcinoma of the male breast: report of a rare case with C-MYC amplification. (United States)

    Trépant, Anne-Laure; Hoorens, Anne; Noël, Jean-Christophe


    Male breast cancer is rare. The most common histological subtypes include invasive carcinoma "of no special type" and papillary carcinoma. Other variants, including pure micropapillary carcinoma, have been described as well but are extremely rare. Pure micropapillary carcinoma has been recently characterized by a C-MYC gene amplification in women. We report here, occurring in a 73-year-old man, the first case of pure micropapillary carcinoma with amplification of the C-MYC gene. Copyright © 2014 Elsevier GmbH. All rights reserved.

  2. Next-generation repeat-free FISH probes for DNA amplification in glioblastoma in vivo: Improving patient selection to MDM2-targeted inhibitors. (United States)

    Brunelli, Matteo; Eccher, Albino; Cima, Luca; Trippini, Tobia; Pedron, Serena; Chilosi, Marco; Barbareschi, Mattia; Scarpa, Aldo; Pinna, Giampietro; Cabrini, Giulio; Pilotto, Sara; Carbognin, Luisa; Bria, Emilio; Tortora, Giampaolo; Fioravanzo, Adele; Schiavo, Nicola; Meglio, Mario; Sava, Teodoro; Belli, Laura; Martignoni, Guido; Ghimenton, Claudio


    A next-generation FISH probe mapping to the MDM2 locus-specific region has recently been designed. The level of MDM2 gene amplification (high versus low) may allow selection of patients for cancer treatment with MDM2 inhibitors and may predict their responsiveness. We investigated the spectrum of MDM2 gene alterations using the new probes in vivo after visualizing single neoplastic cells in situ from a series of glioblastomas. Signals from next-generation repeat-free FISH interphase probes were identified in tissue microarrays that included 3 spots for each of the 48 cases. The murine double minutes (MDM2)-specific DNA probe and the satellite enumeration probe for chromosome 12 were used. Three cases (6%) showed more than 25 signals (high gene amplification), and 7 (15%) showed 3-10 signals (gains); among these, 4 cases (8%) had an equal number of MDM2 and centromeric signals on chromosome 12 (polyploidy). Genomic heterogeneity was observed only in 3 cases with low gene amplification. In our series, 6% of glioblastomas exhibited high MDM2 amplification (in vivo) with a pattern related to the known double minutes/chromothripsis phenomenon (in situ), and only cases with low amplification showed genomic heterogeneity. We concluded that the rate of MDM2 gene amplification can be a useful predictive biomarker to improve patient selection.

  3. Genetic improvement of α-amylase producing Bacillus licheniformis by homolog-mediated α-amylase gene amplification%通过同源介导α-淀粉酶基因扩增改良地衣芽孢杆菌α-淀粉酶生产菌株

    Institute of Scientific and Technical Information of China (English)

    牛丹丹; 石贵阳; 王正祥


    地衣芽孢杆菌高温α-淀粉酶(BLA)是淀粉水解与生物加工过程中重要关键酶制剂之一.为了进一步提高地衣芽孢杆菌高温α-淀粉酶生产菌株的生产性能,本研究构建了一种含有地衣芽孢杆菌高温α-淀粉酶编码基因amyL的整合性重组质粒pBL-amyL.将重组质粒pBL-amyL转化入BLA工业生产菌株Bacillus licheniformis B0204,再在卡那霉素存在下介导其B.1icheniformis B0204染色体中的同源整合与高温α-淀粉酶编码基因amyL的扩增,由此获得了携带多个amyL拷贝的转化子.对转化子的amyL拷贝数及其BLA发酵水平分别用荧光实时定量PCR及摇瓶发酵试验进行评价与鉴定.与出发菌株B0204相比,含2~5倍amyL拷贝数的重组菌的BLA的合成水平显著提高.其中,重组菌REBL18生产BLA的水平提高了89.2%.%Bacillus licheniformis α-amylase (BLA) is one of the most important enzymes involved in starch hydrolysis and many biotechnological processes. To improve the BLA productivity, an integrative plasmid pBL-amyL carrying amyL gene encoding a thermophilic α-amylase of B. licheniformis was constructed and transformed into B. licheniformis B0204, an industrial α-amylase producer. The transformants harboring different copies of amyL were developed on kanamycin by using homolog-mediated chromosomal amplification of α-amylase gene. The recombinants with different multiple copies of amyL integrated in the chromosome were identified by real-time PCR and evaluated by shake-flask fermentation. Recombinants harboring 2-5 multiple copies of amyL produced more α-amylase comparison to the parental strain B0204.

  4. Relationshap between human papillomavirus DNA testing and human telomerase RNA gene amplification detection in esophageal cancer tissue%食管癌组织人乳头瘤病毒感染与人端粒酶RNA基因表达的关系

    Institute of Scientific and Technical Information of China (English)

    王新帅; 陈强; 王玉峰; 高社干; 张广平; 冯笑山


    Objective To study the relationshap between human papillomavirus (HPV) DNA testing and human telomerase (hTERC) RNA gene amplification detection in esophageal cancer tissue.Methods HPV DNA was tested by polymerase chain reaction (PCR) in 82 cases of esophageal cancer tissues.The fluorescence signals of hTERC in the esophageal cancer were detected by using interphase fluorescence in situ hybridization ( FISH ).Results There was HPV infection in esophageal cancer tissue,and the infection rate was about 43.7%.Mixed type was predominant,including the HPV 16 and 18.The mutation rate of hTERC in esophageal cancer tissue was about 80.1%,and there was a positive correlation between the HPV infection and mutation of hTERC (P<0.05 ).Conclusion HPV infection and mutation of hTERC in the esophageal cancer may be important factors of pathogenesis.The mutation of hTERC gene may plav an important role in the development of sequamous cell carcinoma.%目的 探讨食管癌组织中人乳头瘤病毒(HPV)感染与人端粒酶RNA(hTERC)基因表达的关系.方法 82例经病理确诊的食管癌患者组织标本,聚合酶链反应(PCR)技术结合等离子谐振技术检测食管癌组织中HPV感染,荧光原位杂交技术检测食管癌组织中hTERC基因的表达.结果 食管癌组织中存在HPV感染,感染率为43.7%,主要以混合型为主,包括高危亚型HPV16、18;食管癌组织中hTERC 基因的表达率为80.1%;HPV感染与hTERC基因表达关系密切(x2=9.834,P<0.05).结论 食管癌组织中HPV感染与hTERC基因表达是食管癌发病的重要因素,它可能通过hTERC基因途径引起癌变结果.

  5. Digital Microfluidics for Nucleic Acid Amplification

    Directory of Open Access Journals (Sweden)

    Beatriz Coelho


    Full Text Available Digital Microfluidics (DMF has emerged as a disruptive methodology for the control and manipulation of low volume droplets. In DMF, each droplet acts as a single reactor, which allows for extensive multiparallelization of biological and chemical reactions at a much smaller scale. DMF devices open entirely new and promising pathways for multiplex analysis and reaction occurring in a miniaturized format, thus allowing for healthcare decentralization from major laboratories to point-of-care with accurate, robust and inexpensive molecular diagnostics. Here, we shall focus on DMF platforms specifically designed for nucleic acid amplification, which is key for molecular diagnostics of several diseases and conditions, from pathogen identification to cancer mutations detection. Particular attention will be given to the device architecture, materials and nucleic acid amplification applications in validated settings.

  6. Amplification of postwildfire peak flow by debris (United States)

    Kean, J. W.; McGuire, L. A.; Rengers, F. K.; Smith, J. B.; Staley, D. M.


    In burned steeplands, the peak depth and discharge of postwildfire runoff can substantially increase from the addition of debris. Yet methods to estimate the increase over water flow are lacking. We quantified the potential amplification of peak stage and discharge using video observations of postwildfire runoff, compiled data on postwildfire peak flow (Qp), and a physically based model. Comparison of flood and debris flow data with similar distributions in drainage area (A) and rainfall intensity (I) showed that the median runoff coefficient (C = Qp/AI) of debris flows is 50 times greater than that of floods. The striking increase in Qp can be explained using a fully predictive model that describes the additional flow resistance caused by the emergence of coarse-grained surge fronts. The model provides estimates of the amplification of peak depth, discharge, and shear stress needed for assessing postwildfire hazards and constraining models of bedrock incision.

  7. Parametric nanomechanical amplification at very high frequency. (United States)

    Karabalin, R B; Feng, X L; Roukes, M L


    Parametric resonance and amplification are important in both fundamental physics and technological applications. Here we report very high frequency (VHF) parametric resonators and mechanical-domain amplifiers based on nanoelectromechanical systems (NEMS). Compound mechanical nanostructures patterned by multilayer, top-down nanofabrication are read out by a novel scheme that parametrically modulates longitudinal stress in doubly clamped beam NEMS resonators. Parametric pumping and signal amplification are demonstrated for VHF resonators up to approximately 130 MHz and provide useful enhancement of both resonance signal amplitude and quality factor. We find that Joule heating and reduced thermal conductance in these nanostructures ultimately impose an upper limit to device performance. We develop a theoretical model to account for both the parametric response and nonequilibrium thermal transport in these composite nanostructures. The results closely conform to our experimental observations, elucidate the frequency and threshold-voltage scaling in parametric VHF NEMS resonators and sensors, and establish the ultimate sensitivity limits of this approach.

  8. Gravito-magnetic amplification in cosmology

    CERN Document Server

    Tsagas, Christos G


    Magnetic fields interact with gravitational waves in various ways. We consider the coupling between the Weyl and the Maxwell fields in cosmology and study the effects of the former on the latter. The approach is fully analytical and the results are gauge-invariant. We show that the nature and the outcome of the gravito-magnetic interaction depends on the electric properties of the cosmic medium. When the conductivity is high, gravitational waves reduce the standard (adiabatic) decay rate of the B-field, leading to its superadiabatic amplification. In poorly conductive environments, on the other hand, Weyl-curvature distortions can result into the resonant amplification of large-scale cosmological magnetic fields. Driven by the gravitational waves, these B-fields oscillate with an amplitude that is found to diverge when the wavelengths of the two sources coincide. We present technical and physical aspects of the gravito-magnetic interaction and discuss its potential implications.

  9. Internal entanglement amplification by external interactions



    We propose a scheme to control the level of entanglement between two fixed spin-1/2 systems by interaction with a third particle. For specific designs, entanglement is shown to be "pumped" into the system from the surroundings even when the spin-spin interaction within the system is small or nonexistent. The effect of the external particle on the system is introduced by including a dynamic spinor in the Hamiltonian. Controlled amplification of the internal entanglement to its maximum value is...

  10. A mechanism for ramified rolling circle amplification

    Directory of Open Access Journals (Sweden)

    Smith James H


    Full Text Available Abstract Background Amplification of single-stranded DNA circles has wide utility for a variety of applications. The two-primer ramified rolling circle amplification (RAM reaction provides exponential DNA amplification under isothermal conditions, creating a regular laddered series of double-stranded DNA products. However, the molecular mechanism of the RAM reaction remains unexplained. Results A RAM reaction model predicts exponential accumulation of a double-stranded DNA product size series, and product-size ratios, that are consistent with observed RAM reaction products. The mechanism involves generation of a series of increasing size intermediate templates; those templates produce RAM products and recursively generate smaller intermediate templates. The model allows prediction of the number of rounds of circular template replication. Real-time RAM reaction data are consistent with the model. Analysis of RAM reaction products shows exponential growth limitation consistent with the model's predictions. Conclusions The model provides a rationale for the observed products of the RAM reaction, and the molecular yield among those products. Experimental results are consistent with the model.

  11. Nucleic acid amplification using microfluidic systems. (United States)

    Chang, Chen-Min; Chang, Wen-Hsin; Wang, Chih-Hung; Wang, Jung-Hao; Mai, John D; Lee, Gwo-Bin


    In the post-human-genome-project era, the development of molecular diagnostic techniques has advanced the frontiers of biomedical research. Nucleic-acid-based technology (NAT) plays an especially important role in molecular diagnosis. However, most research and clinical protocols still rely on the manual analysis of individual samples by skilled technicians which is a time-consuming and labor-intensive process. Recently, with advances in microfluidic designs, integrated micro total-analysis-systems have emerged to overcome the limitations of traditional detection assays. These microfluidic systems have the capability to rapidly perform experiments in parallel and with a high-throughput which allows a NAT analysis to be completed in a few hours or even a few minutes. These features have a significant beneficial influence on many aspects of traditional biological or biochemical research and this new technology is promising for improving molecular diagnosis. Thus, in the foreseeable future, microfluidic systems developed for molecular diagnosis using NAT will become an important tool in clinical diagnosis. One of the critical issues for NAT is nucleic acid amplification. In this review article, recent advances in nucleic acid amplification techniques using microfluidic systems will be reviewed. Different approaches for fast amplification of nucleic acids for molecular diagnosis will be highlighted.

  12. Using multiplex PCR amplification and typing kits for the analysis of DNA evidence in a serial killer case. (United States)

    Hochmeister, M N; Budowle, B; Eisenberg, A; Borer, U V; Dirnhofer, R


    Analysis of DNA evidence in a serial killer case was performed using the AmpliType HLA-DQ alpha-, AmpliType PM-, and the GenePrint STR Multiplex System PCR Amplification Kits. In addition, a sex typing procedure using the X-Y homologous gene amelogenin was carried out. DNA profiles from a single hair with attached sheath material, recovered from underneath the seat cover of the suspect's car seat were compared with DNA profiles derived from reference head hairs from a homicide victim. From the evidentiary sample only 9 ng of human DNA could be recovered. In a sample, where the quantity of DNA becomes a critical issue a powerful route is the simultaneous amplification of several loci (multiplex PCR). This is the first report where commercially available multiplex PCR amplification and typing kits have been introduced for the analysis of DNA evidence in a serial killer case and the analysis has been admitted in court.

  13. Prognostic impact of HER-2 Subclonal Amplification in breast cancer. (United States)

    Di Oto, Enrico; Brandes, Alba A; Cucchi, Maria C; Foschini, Maria P


    The presence of a limited number of cells with HER-2 amplification (Subclonal Amplification) in breast carcinomas is occasionally encountered, but its prognostic impact is poorly known. The purpose of this study is to evaluate the prognostic impact of HER-2 Subclonal Amplification in a retrospective series of breast cancers. Accordingly, 81 consecutive breast carcinomas showing HER-2 Subclonal Amplification were obtained from the histology files (case series). These cases were subdivided into two groups: (a) those cases in which the HER-2 Subclonal Amplification was consonant to the accepted criteria for amplification, showing clusters of amplified cells, and (b) those cases with rare HER-2 Subclonal Amplification that did not reflect the accepted criteria for amplification, showing scattered amplified cells only. The incidence of metastases and late recurrences of the case series was compared with a series composed of 109 consecutive cases, being HER-2 homogeneous (comprising 14 Amplified and 95 Non-Amplified cases), matched for grade and stage (control series). It appeared that cases showing Subclonal Amplification had an incidence of metastases intermediate between the cases Amplified and Non-Amplified. Specifically, Subclonal Amplification with clustered cells had a lower incidence of metastases than Amplified cases (12.9 versus 21.4%). On the contrary, Subclonal Amplification with scattered cells showed an incidence of metastases higher than Non-Amplified cases (14 versus 9.47%). In addition, patients Subclonal Amplification with clustered cells, who were treated with the specific monoclonal antibody, had a lower incidence of metastases than patients showing Subclonal Amplification with scattered cells, who did not receive target therapy. These data, together with those recently published, indicate that Subclonal Amplification has an impact on prognosis and should be taken into consideration to correctly plan the treatment of breast cancer patients.

  14. PCR bias in amplification of androgen receptor alleles, a trinucleotide repeat marker used in clonality studies.


    Mutter, G L; Boynton, K A


    Trinucleotide CAG repeats in the X-linked human androgen receptor gene (HUMARA) have proved a useful means of determining X chromosome haplotypes, and when combined with methylation analysis of nearby cytosine residues permits identification of non-random X inactivation in tumors of women. Co-amplification of two alleles in a heterozygote generates PCR products which differ in the number of CAG units, and thus their melting and secondary structure characteristics. We have shown that under opt...

  15. Cross-kingdom amplification using Bacteria-specific primers: Complications for studies of coral microbial ecology (United States)

    Galkiewicz, J.P.; Kellogg, C.A.


    PCR amplification of pure bacterial DNA is vital to the study of bacterial interactions with corals. Commonly used Bacteria-specific primers 8F and 27F paired with the universal primer 1492R amplify both eukaryotic and prokaryotic rRNA genes. An alternative primer set, 63F/1542R, is suggested to resolve this problem. Copyright ?? 2008, American Society for Microbiology. All Rights Reserved.

  16. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. (United States)

    Folmer, O; Black, M; Hoeh, W; Lutz, R; Vrijenhoek, R


    We describe "universal" DNA primers for polymerase chain reaction (PCR) amplification of a 710-bp fragment of the mitochondrial cytochrome c oxidase subunit I gene (COI) from 11 invertebrate phyla: Echinodermata, Mollusca, Annelida, Pogonophora, Arthropoda, Nemertinea, Echiura, Sipuncula, Platyhelminthes, Tardigrada, and Coelenterata, as well as the putative phylum Vestimentifera. Preliminary comparisons revealed that these COI primers generate informative sequences for phylogenetic analyses at the species and higher taxonomic levels.

  17. Ligase chain reaction amplification for sensitive electrochemiluminescent detection of single nucleotide polymorphisms

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ying; Yang, Mengli; Xiang, Yun, E-mail:; Yuan, Ruo, E-mail:; Chai, Yaqin


    Graphical abstract: -- Highlights: •Ligase chain reaction amplification (LCR) is employed to sensitively detect single nucleotide polymorphisms. •During LCR, the mutant target gene is recycled and duplicated exponentially to achieve dramatic signal amplification. •The method shows a selectivity factor of 10{sup 3} toward the mutant target gene against the interfering wild target gene. -- Abstract: Single nucleotide polymorphisms are the most common type of genetic variations among human beings and can serve as biomarkers for various types of diseases. In this work, based on ligase chain reaction amplification for the production of massive hemin/G-quadruplex DNAzymes to quench the electrochemiluminescent (ECL) emission of quantum dots (QDs), a universal and sensitive single nucleotide polymorphism detection method is described. During the ligase chain reaction process, the mutant K-ras target gene is recycled and exponentially duplicated, leading to the attachment of numerous G-rich sequences on the QD-embedded sensing surface. Upon the addition of the assistant sequences and hemin, numerous hemin/G-quadruplex DNAzymes are formed, which consume the dissolved oxygen in the detection buffer and result in significant quenching of QD ECL emission for sensitive single nucleotide polymorphism determination. The developed method shows a linear range of 50 fM to 50 pM and an estimated detection limit of 45 fM for the mutant K-ras gene. The proposed strategy also exhibits high selectivity towards the mutant K-ras gene against the co-existence of 10{sup 3}-fold excess of the wild-type K-ras gene, which makes our method a useful addition to the alternatives for single nucleotide polymorphism monitoring.

  18. Typing for HLA-DPB1*03 and HLA-DPB1*06 using allele-specific DNA in vitro amplification and allele-specific oligonucleotide probes. Detection of "new" DPB1*06 variants

    DEFF Research Database (Denmark)

    Fugger, L; Morling, N; Ryder, L P;


    DP gene typing using in vitro DNA amplification combined with sequence-specific oligonucleotide probes has recently been reported. The resulting DNA amplification was specific for the HLA-DPB locus. Typing for the individual DPB alleles was exclusively dependent on the hybridizations of the probe...

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

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


    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.

  20. Diverse Roles of Axonemal Dyneins in Drosophila Auditory Neuron Function and Mechanical Amplification in Hearing. (United States)

    Karak, Somdatta; Jacobs, Julie S; Kittelmann, Maike; Spalthoff, Christian; Katana, Radoslaw; Sivan-Loukianova, Elena; Schon, Michael A; Kernan, Maurice J; Eberl, Daniel F; Göpfert, Martin C


    Much like vertebrate hair cells, the chordotonal sensory neurons that mediate hearing in Drosophila are motile and amplify the mechanical input of the ear. Because the neurons bear mechanosensory primary cilia whose microtubule axonemes display dynein arms, we hypothesized that their motility is powered by dyneins. Here, we describe two axonemal dynein proteins that are required for Drosophila auditory neuron function, localize to their primary cilia, and differently contribute to mechanical amplification in hearing. Promoter fusions revealed that the two axonemal dynein genes Dmdnah3 (=CG17150) and Dmdnai2 (=CG6053) are expressed in chordotonal neurons, including the auditory ones in the fly's ear. Null alleles of both dyneins equally abolished electrical auditory neuron responses, yet whereas mutations in Dmdnah3 facilitated mechanical amplification, amplification was abolished by mutations in Dmdnai2. Epistasis analysis revealed that Dmdnah3 acts downstream of Nan-Iav channels in controlling the amplificatory gain. Dmdnai2, in addition to being required for amplification, was essential for outer dynein arms in auditory neuron cilia. This establishes diverse roles of axonemal dyneins in Drosophila auditory neuron function and links auditory neuron motility to primary cilia and axonemal dyneins. Mutant defects in sperm competition suggest that both dyneins also function in sperm motility.

  1. 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.

  2. Yoctomole electrochemical genosensing of Ebola virus cDNA by rolling circle and circle to circle amplification. (United States)

    Carinelli, S; Kühnemund, M; Nilsson, M; Pividori, M I


    This work addresses the design of an Ebola diagnostic test involving a simple, rapid, specific and highly sensitive procedure based on isothermal amplification on magnetic particles with electrochemical readout. Ebola padlock probes were designed to detect a specific L-gene sequence present in the five most common Ebola species. Ebola cDNA was amplified by rolling circle amplification (RCA) on magnetic particles. Further re-amplification was performed by circle-to-circle amplification (C2CA) and the products were detected in a double-tagging approach using a biotinylated capture probe for immobilization on magnetic particles and a readout probe for electrochemical detection by square-wave voltammetry on commercial screen-printed electrodes. The electrochemical genosensor was able to detect as low as 200 ymol, corresponding to 120 cDNA molecules of L-gene Ebola virus with a limit of detection of 33 cDNA molecules. The isothermal double-amplification procedure by C2CA combined with the electrochemical readout and the magnetic actuation enables the high sensitivity, resulting in a rapid, inexpensive, robust and user-friendly sensing strategy that offers a promising approach for the primary care in low resource settings, especially in less developed countries. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Multiplexed Recombinase Polymerase Amplification Assay To Detect Intestinal Protozoa. (United States)

    Crannell, Zachary; Castellanos-Gonzalez, Alejandro; Nair, Gayatri; Mejia, Rojelio; White, A Clinton; Richards-Kortum, Rebecca


    This work describes a proof-of-concept multiplex recombinase polymerase amplification (RPA) assay with lateral flow readout that is capable of simultaneously detecting and differentiating DNA from any of the diarrhea-causing protozoa Giardia, Cryptosporidium, and Entamoeba. Together, these parasites contribute significantly to the global burden of diarrheal illness. Differential diagnosis of these parasites is traditionally accomplished via stool microscopy. However, microscopy is insensitive and can miss up to half of all cases. DNA-based diagnostics such as polymerase chain reaction (PCR) are far more sensitive; however, they rely on expensive thermal cycling equipment, limiting their availability to centralized reference laboratories. Isothermal DNA amplification platforms, such as the RPA platform used in this study, alleviate the need for thermal cycling equipment and have the potential to broaden access to more sensitive diagnostics. Until now, multiplex RPA assays have not been developed that are capable of simultaneously detecting and differentiating infections caused by different pathogens. We developed a multiplex RPA assay to detect the presence of DNA from Giardia, Cryptosporidium, and Entamoeba. The multiplex assay was characterized using synthetic DNA, where the limits-of-detection were calculated to be 403, 425, and 368 gene copies per reaction of the synthetic Giardia, Cryptosporidium, and Entamoeba targets, respectively (roughly 1.5 orders of magnitude higher than for the same targets in a singleplex RPA assay). The multiplex assay was also characterized using DNA extracted from live parasites spiked into stool samples where the limits-of-detection were calculated to be 444, 6, and 9 parasites per reaction for Giardia, Cryptosporidium, and Entamoeba parasites, respectively. This proof-of-concept assay may be reconfigured to detect a wide variety of targets by re-designing the primer and probe sequences.

  4. Indirect imaging of cardiac-specific transgene expression using a bidirectional two-step transcriptional amplification strategy

    DEFF Research Database (Denmark)

    Chen, I Y; Gheysens, O; Ray, S


    Transcriptional targeting for cardiac gene therapy is limited by the relatively weak activity of most cardiac-specific promoters. We have developed a bidirectional plasmid vector, which uses a two-step transcriptional amplification (TSTA) strategy to enhance the expression of two optical reporter...

  5. Predicting responsiveness to sorafenib: can the determination of FGF3/FGF4 amplifications enrich for clinical benefit? (United States)

    Harding, James J; Abou-Alfa, Ghassan K


    A small proportion of patients with advanced hepatocellular carcinoma harbor FGF3/4 gene amplifications in an 11q13 amplicon. A recent report suggests that the presence of this alteration might enrich for sensitivity to sorafenib. Further, there is growing evidence that interference with the FGF/FGFR axis has therapeutic potential in patients with advanced hepatocellular carcinoma.

  6. Identification of porcine Pneumocystis carinii as a genetically distinct organism by DNA amplification

    DEFF Research Database (Denmark)

    Wakefield, A E; Keely, S P; Stringer, J R


    DNA was amplified from lung samples from three piglets infected with Pneumocystis carinii, using oligonucleotide primers designed to the P. carinii mitochondrial large subunit ribosomal RNA gene. The nucleotide sequence of the amplification product was determined and indicated lack of sequence...

  7. Whole Genome Amplification from Blood Spot Samples. (United States)

    Sørensen, Karina Meden


    Whole genome amplification is an invaluable technique when working with DNA extracted from blood spots, as the DNA obtained from this source often is too limited for extensive genetic analysis. Two techniques that amplify the entire genome are common. Here, both are described with focus on the benefits and drawbacks of each system. However, in order to obtain the best possible WGA result the quality of input DNA extracted from the blood spot is essential, but also time consumption, flexibility in format and elution volume and price of the technology are factors influencing system choice. Here, three DNA extraction techniques are described and the above aspects are compared between the systems.

  8. Amplification Without Inversion in Semiconductor Quantum Dot (United States)

    Hajibadali, A.; Abbasian, K.; Rostami, A.

    In this paper, we have realized amplification without inversion (AWI) in quantum dot (QD). A Y-type four-level system of InxGa1-xN quantum dot has been obtained and investigated for AWI. It has been shown that, with proper setting of control fields' amplitude, we can obtain reasonable gain. With proper setting of phase difference of control fields and probe field, we can obtain considerable gain in resonant wavelength. We have designed this system by solving the Schrödinger-Poisson equations for InxGa1-xN quantum dot in GaN substrate, self-consistently.

  9. Amplification of curvature perturbations in cyclic cosmology (United States)

    Zhang, Jun; Liu, Zhi-Guo; Piao, Yun-Song


    We analytically and numerically show that through the cycles with nonsingular bounce, the amplitude of curvature perturbation on a large scale will be amplified and the power spectrum will redden. In some sense, this amplification will eventually destroy the homogeneity of the background, which will lead to the ultimate end of cycles of the global universe. We argue that for the model with increasing cycles, it might be possible that a fissiparous multiverse will emerge after one or several cycles, in which the cycles will continue only at corresponding local regions.

  10. Amplification Effects and Unconventional Monetary Policies

    Directory of Open Access Journals (Sweden)



    Full Text Available Global financial crises trigger off amplification effects, which allow relatively small shocks to propagate through the whole financial system. For this reason, the range of Central banks policies is now widening beyond conventional monetary policies and lending of last resort. The aim of this paper is to establish a rule for this practice. The model is based on the formalization of funding conditions in various types of markets. We conduct a comprehensive analysis of the “unconventional monetary policies”, and especially quantify government bonds purchases by the Central bank.

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

    Directory of Open Access Journals (Sweden)

    Xue-feng CAO


    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

  12. Advances in isothermal amplification: novel strategies inspired by biological processes. (United States)

    Li, Jia; Macdonald, Joanne


    Nucleic acid amplification is an essential process in biological systems. The in vitro adoption of this process has resulted in powerful techniques that underpin modern molecular biology. The most common tool is polymerase chain reaction (PCR). However, the requirement for a thermal cycler has somewhat limited applications of this classic nucleic acid amplification technique. Isothermal amplification, on the other hand, obviates the use of a thermal cycler because reactions occur at a single temperature. Isothermal amplification methods are diverse, but all have been developed from an understanding of natural nucleic acid amplification processes. Here we review current isothermal amplification methods as classified by their enzymatic mechanisms. We compare their advantages, disadvantages, efficiencies, and applications. Finally, we mention some new developments associated with this technology, and consider future possibilities in molecular engineering and recombinant technologies that may develop from an appreciation of the molecular biology of natural systems.

  13. 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)


    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.

  14. Amplification of fibroblast growth factor receptor-1 in breast cancer and the effects of brivanib alaninate. (United States)

    Shiang, Christine Y; Qi, Yuan; Wang, Bailiang; Lazar, Vladimir; Wang, Jing; Fraser Symmans, W; Hortobagyi, Gabriel N; Andre, Fabrice; Pusztai, Lajos


    Fibroblast growth factor receptor-1 (FGFR-1) is amplified in 10% of human breast cancers. The goal of this study was to test the correlation between FGFR-1 amplification and expression and sensitivity to brivanib, an FGFR-1 small molecule inhibitor, in breast cancer cell lines in vitro. Using CGH array and gene expression profiling, FGFR-1 DNA copy number, mRNA, and protein expression were measured in 21 cell lines and correlated with growth inhibition by brivanib. We examined FGFR-1 autophosphorylation and kinase activity, as well as phosphorylation of downstream signaling molecules in response to bFGF and brivanib exposure. CAMA, MDA-MB-361, and HCC38 cells had FGFR-1 amplification and protein overexpression. Brivanib GI(50) values were significantly lower in the gene amplified (15.17 μM, n = 3) compared to normal copy number (69.09 μM, n = 11) or FGFR-1 deleted (76.14 μM, n = 7) cells (P = 0.0107). Among nonamplified cells, there was no correlation between FGFR-1 mRNA or protein expression levels and brivanib sensitivity. Two of three FGFR-1 amplified cells were sensitive to bFGF-induced growth stimulation, which was blocked by brivanib. In cells with amplified FGFR-1, brivanib decreased receptor autophosphorylation, inhibited bFGF-induced tyrosine kinase activity, and reduced phosphorylation of ERK and AKT. Breast cancer cell lines with FGFR-1 gene amplification and protein overexpression are more sensitive to growth inhibition by brivanib than nonamplified cells. These findings suggest that FGFR-1 amplification or protein overexpression in breast cancers may be an indicator for brivanib treatment, where it may have direct anti-proliferative effects in addition to its' anti-angiogenic effects.

  15. Mechanism of seasonal Arctic sea ice evolution and Arctic amplification


    Kim, Kwang-Yul; Hamlington, Benjamin D.; Na, Hanna; Kim, Jinju


    Sea ice loss is proposed as a primary reason for the Arctic amplification, although the physical mechanism of the Arctic amplification and its connection with sea ice melting is still in debate. In the present study, monthly ERA-Interim reanalysis data are analyzed via cyclostationary empirical orthogonal function analysis to understand the seasonal mechanism of sea ice loss in the Arctic Ocean and the Arctic amplification. While sea ice loss is widespread over much of the p...

  16. Space Optical Communications Using Laser Beam Amplification (United States)

    Agrawal, Govind


    The Space Optical Communications Using Laser Beam Amplification (SOCLBA) project will provide a capability to amplify a laser beam that is received in a modulating retro-reflector (MRR) located in a satellite in low Earth orbit. It will also improve the pointing procedure between Earth and spacecraft terminals. The technology uses laser arrays to strengthen the reflected laser beam from the spacecraft. The results of first year's work (2014) show amplification factors of 60 times the power of the signal beam. MMRs are mirrors that reflect light beams back to the source. In space optical communications, a high-powered laser interrogator beam is directed from the ground to a satellite. Within the satellite, the beam is redirected back to ground using the MMR. In the MMR, the beam passes through modulators, which encode a data signal onto the returning beam. MMRs can be used in small spacecraft for optical communications. The SOCLBA project is significant to NASA and small spacecraft due to its application to CubeSats for optical data transmission to ground stations, as well as possible application to spacecraft for optical data transmission.

  17. Experimental noiseless linear amplification using weak measurements (United States)

    Ho, Joseph; Boston, Allen; Palsson, Matthew; Pryde, Geoff


    The viability of quantum communication schemes rely on sending quantum states of light over long distances. However, transmission loss can degrade the signal strength, adding noise. Heralded noiseless amplification of a quantum signal can provide a solution by enabling longer direct transmission distances and by enabling entanglement distillation. The central idea of heralded noiseless amplification—a conditional modification of the probability distribution over photon number of an optical quantum state—is suggestive of a parallel with weak measurement: in a weak measurement, learning partial information about an observable leads to a conditional back-action of a commensurate size. Here we experimentally investigate the application of weak, or variable-strength, measurements to the task of heralded amplification, by using a quantum logic gate to weakly couple a small single-optical-mode quantum state (the signal) to an ancilla photon (the meter). The weak measurement is carried out by choosing the measurement basis of the meter photon and, by conditioning on the meter outcomes, the signal is amplified. We characterise the gain of the amplifier as a function of the measurement strength, and use interferometric methods to show that the operation preserves the coherence of the signal.

  18. Magnetic Field Amplification in Young Galaxies

    CERN Document Server

    Schober, Jennifer; Klessen, Ralf S


    The Universe at present is highly magnetized, with fields of the order of a few 10^-5 G and coherence lengths larger than 10 kpc in typical galaxies like the Milky Way. We propose that the magnetic field was amplified to this values already during the formation and the early evolution of the galaxies. Turbulence in young galaxies is driven by accretion as well as by supernova (SN) explosions of the first generation of stars. The small-scale dynamo can convert the turbulent kinetic energy into magnetic energy and amplify very weak primordial magnetic seed fields on short timescales. The amplification takes place in two phases: in the kinematic phase the magnetic field grows exponentially, with the largest growth on the smallest non-resistive scale. In the following non-linear phase the magnetic energy is shifted towards larger scales until the dynamo saturates on the turbulent forcing scale. To describe the amplification of the magnetic field quantitatively we model the microphysics in the interstellar medium ...

  19. Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues

    Directory of Open Access Journals (Sweden)

    Watson Spencer K


    Full Text Available Abstract Background Formalin-fixed paraffin-embedded (FFPE tissues represent the largest source of archival biological material available for genomic studies of human cancer. Therefore, it is desirable to develop methods that enable whole genome amplification (WGA using DNA extracted from FFPE tissues. Multiple-strand Displacement Amplification (MDA is an isothermal method for WGA that uses the large fragment of Bst DNA polymerase. To date, MDA has been feasible only for genomic DNA isolated from fresh or snap-frozen tissue, and yields a representational distortion of less than threefold. Results We amplified genomic DNA of five FFPE samples of normal human lung tissue with the large fragment of Bst DNA polymerase. Using quantitative PCR, the copy number of 7 genes was evaluated in both amplified and original DNA samples. Four neuroblastoma xenograft samples derived from cell lines with known N-myc gene copy number were also evaluated, as were 7 samples of non-small cell lung cancer (NSCLC tumors with known Skp2 gene amplification. In addition, we compared the array comparative genomic hybridization (CGH-based genome profiles of two NSCLC samples before and after Bst MDA. A median 990-fold amplification of DNA was achieved. The DNA amplification products had a very high molecular weight (> 23 Kb. When the gene content of the amplified samples was compared to that of the original samples, the representational distortion was limited to threefold. Array CGH genome profiles of amplified and non-amplified FFPE DNA were similar. Conclusion Large fragment Bst DNA polymerase is suitable for WGA of DNA extracted from FFPE tissues, with an expected maximal representational distortion of threefold. Amplified DNA may be used for the detection of gene copy number changes by quantitative realtime PCR and genome profiling by array CGH.

  20. Loop-mediated isothermal amplification for detection of nucleic acids. (United States)

    Tanner, Nathan A; Evans, Thomas C


    Sequence-specific isothermal nucleic acid amplification techniques are ideally suited for use in molecular diagnostic applications because they do not require thermal cycling equipment and the reactions are typically fast. One of the most widely cited isothermal techniques is termed loop-mediated isothermal amplification (LAMP). This protocol allows amplification times as fast as 5 to 10 min. Furthermore, various methodologies to detect amplification have been applied to LAMP to increase its utility for the point-of-care market. Basic LAMP protocols are provided herein for detection of specific DNA and RNA targets, along with a method to perform multiplex LAMP reactions, permitting even greater flexibility from this powerful technique.

  1. Evaluation of MDM2 and CDK4 amplification by real-time PCR on paraffin wax-embedded material: a potential tool for the diagnosis of atypical lipomatous tumours/well-differentiated liposarcomas. (United States)

    Hostein, I; Pelmus, M; Aurias, A; Pedeutour, F; Mathoulin-Pélissier, S; Coindre, J M


    Atypical lipomatous tumours/well-differentiated liposarcomas and dedifferentiated liposarcomas are characterized by 12q13-15 region amplification. In contrast, this molecular event has not been reported in benign lipomas. Within the 12q13-15 chromosomal region, the MDM2, SAS, HMGA2, and CDK4 genes are the most frequent targets of amplification. A series of lipomas (36 cases) and liposarcomas (48 cases) was analysed for MDM2 and CDK4 gene amplification by real-time PCR. MDM2 and CDK4 gene amplification was detected in 2.8% and 5.6% of lipomas and 98.2% and 82.4% of liposarcomas, respectively. Moreover, co-amplification of the two genes as well as a higher-level amplification was observed more frequently in dedifferentiated liposarcomas than in atypical lipomatous tumours/well-differentiated liposarcomas. Real-time PCR proved to be a fast and reliable method to characterize lipomas and liposarcomas by quantification of MDM2 and CDK4 gene amplification. It is applicable to paraffin wax-embedded tissues and could be useful when histological diagnosis is difficult.

  2. Simple and inexpensive three-step rapid amplification of cDNA 5′ ends using 5′ phosphorylated primers


    Dallmeier, Kai; Neyts, Johan


    Rapid amplification of cDNA 5′ ends (5′-RACE) is routinely used for the sequence analysis of the upstream noncoding regions of cellular mRNAs; however, it represents a tedious and cost-intensive procedure. By employing 5′ phosphorylated gene-specific primers for first-strand cDNA synthesis, we cut short the previously established reverse ligation and amplification protocol of Mandl and coworkers (BioTechniques, 1991, vol. 10, pp. 484–486) to a streamlined three-step procedure that no longer d...

  3. Seismic Wave Amplification in 3D Alluvial Basins: 3D/1D Amplification Ratios from Fast Multipole BEM Simulations

    CERN Document Server

    Fajardo, Kristel C Meza; Chaillat, Stéphanie; Lenti, Luca


    In this work, we study seismic wave amplification in alluvial basins having 3D standard geometries through the Fast Multipole Boundary Element Method in the frequency domain. We investigate how much 3D amplification differs from the 1D (horizontal layering) case. Considering incident fields of plane harmonic waves, we examine the relationships between the amplification level and the most relevant physical parameters of the problem (impedance contrast, 3D aspect ratio, vertical and oblique incidence of plane waves). The FMBEM results show that the most important parameters for wave amplification are the impedance contrast and the so-called equivalent shape ratio. Using these two parameters, we derive simple rules to compute the fundamental frequency for various 3D basin shapes and the corresponding 3D/1D amplification factor for 5% damping. Effects on amplification due to 3D basin asymmetry are also studied and incorporated in the derived rules.

  4. 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.


    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.

  5. Allele-specific amplification in cancer revealed by SNP array analysis.

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    Thomas LaFramboise


    Full Text Available Amplification, deletion, and loss of heterozygosity of genomic DNA are hallmarks of cancer. In recent years a variety of studies have emerged measuring total chromosomal copy number at increasingly high resolution. Similarly, loss-of-heterozygosity events have been finely mapped using high-throughput genotyping technologies. We have developed a probe-level allele-specific quantitation procedure that extracts both copy number and allelotype information from single nucleotide polymorphism (SNP array data to arrive at allele-specific copy number across the genome. Our approach applies an expectation-maximization algorithm to a model derived from a novel classification of SNP array probes. This method is the first to our knowledge that is able to (a determine the generalized genotype of aberrant samples at each SNP site (e.g., CCCCT at an amplified site, and (b infer the copy number of each parental chromosome across the genome. With this method, we are able to determine not just where amplifications and deletions occur, but also the haplotype of the region being amplified or deleted. The merit of our model and general approach is demonstrated by very precise genotyping of normal samples, and our allele-specific copy number inferences are validated using PCR experiments. Applying our method to a collection of lung cancer samples, we are able to conclude that amplification is essentially monoallelic, as would be expected under the mechanisms currently believed responsible for gene amplification. This suggests that a specific parental chromosome may be targeted for amplification, whether because of germ line or somatic variation. An R software package containing the methods described in this paper is freely available at

  6. Detection of HIV cDNA Point Mutations with Rolling-Circle Amplification Arrays

    Directory of Open Access Journals (Sweden)

    Zhongwei Wu


    Full Text Available In this paper we describe an isothermal rolling-circle amplification (RCA protocol to detect gene point mutations on chips. The method is based on an allele-specific oligonucleotide circularization mediated by a special DNA ligase. The probe is circularized when perfect complementary sequences between the probe oligonucleotide and HIV cDNA gene. Mismatches around the ligation site can prevent probe circularization. The circularized probe (C-probe can be amplified by rolling circle amplification to generate multimeric singlestranded DNA (ssDNA under isothermal conditions. There are four sequence regions to bind respectively with fluorescent probe, RCA primer, solid probe and HIV cDNA template in the C-probe which we designed. These ssDNA products are hybridized with fluorescent probes and solid probes which are immobilized on a glass slide composing a regular microarray pattern. The fluorescence signals can be monitored by a scanner in the presence of HIV cDNA templates, whereas the probes cannot be circularized and signal of fluorescence cannot be found. The RCA array has capability of high-throughput detection of the point mutation and the single-nucleotide polymorphism (SNP.The development of C-probe-based technologies offers a promising prospect for situ detection, microarray, molecular diagnosis, single nucleotide polymorphism, and whole genome amplification.

  7. Massive Amplification at an Unselected Locus Accompanies Complex Chromosomal Rearrangements in Yeast

    Directory of Open Access Journals (Sweden)

    Agnès Thierry


    Full Text Available Gene amplification has been observed in different organisms in response to environmental constraints, such as limited nutrients or exposure to a variety of toxic compounds, conferring them with specific phenotypic adaptations via increased expression levels. However, the presence of multiple gene copies in natural genomes has generally not been found in the absence of specific functional selection. Here, we show that the massive amplification of a chromosomal locus (up to 880 copies per cell occurs in the absence of any direct selection, and is associated with low-order amplifications of flanking segments in complex chromosomal alterations. These results were obtained from mutants with restored phenotypes that spontaneously appeared from genetically engineered strains of the yeast Saccharomyces cerevisiae suffering from severe fitness reduction. Grossly extended chromosomes (macrotene were formed, with complex structural alterations but sufficient stability to propagate unchanged over successive generations. Their detailed molecular analysis, including complete genome sequencing, identification of sequence breakpoints, and comparisons between mutants, revealed novel mechanisms causing their formation, whose combined action underlies the astonishing dynamics of eukaryotic chromosomes and their consequences.

  8. RNA amplification of bromodeoxyuridine labeled newborn neurons in the monkey hippocampus. (United States)

    Counts, Scott E; Chen, Er-Yun; Ginsberg, Stephen D; Kordower, Jeffrey H; Mufson, Elliott J


    Neurogenesis has been demonstrated in the adult mammalian hippocampus by the immunohistochemical identification of cells co-labeled with the neuronal marker NeuN and bromodeoxyuridine (BrdU), a marker for DNA synthesis. Whether these newly born neurons exhibit a genetic signature similar to that of existing hippocampal cells remains unknown. Recent advances in single cell RNA amplification techniques provide a unique method for profiling the mRNA complement of cells developed during adult neurogenesis. Standard protocols for identifying BrdU-positive cells requires an acid denaturation step that may preclude the amplification of cellular RNA for expression analysis. We first tested whether the BrdU reaction product was visible in monkey hippocampal tissue following treatment with dilutions of HCl (2-0.2 M) or citric acid (1.0-0.1 M). BrdU-labeled cells were visible only in tissue sections treated with 2 M HCl. RNA amplification was not compromised in cells dual-labeled for BrdU and NeuN using the 2 M HCl acid denaturation step. These cells express mRNAs encoding a wide variety of functional protein subclasses including glutamate receptors. The present study demonstrates for the first time that BrdU immunohistochemisty is compatable with gene array technology in the primate hippocampus to evaluate subclasses of genes in newborn neurons.

  9. Detection of HIV cDNA point mutations with rolling-circle amplification arrays. (United States)

    Wu, Lingwei; Liu, Quanjun; Wu, Zhongwei; Lu, Zuhong


    In this paper we describe an isothermal rolling-circle amplification (RCA) protocol to detect gene point mutations on chips. The method is based on an allele-specific oligonucleotide circularization mediated by a special DNA ligase. The probe is circularized when perfect complementary sequences between the probe oligonucleotide and HIV cDNA gene. Mismatches around the ligation site can prevent probe circularization. The circularized probe (C-probe) can be amplified by rolling circle amplification to generate multimeric singlestranded DNA (ssDNA) under isothermal conditions. There are four sequence regions to bind respectively with fluorescent probe, RCA primer, solid probe and HIV cDNA template in the C-probe which we designed. These ssDNA products are hybridized with fluorescent probes and solid probes which are immobilized on a glass slide composing a regular microarray pattern. The fluorescence signals can be monitored by a scanner in the presence of HIV cDNA templates, whereas the probes cannot be circularized and signal of fluorescence cannot be found. The RCA array has capability of high-throughput detection of the point mutation and the single-nucleotide polymorphism (SNP).The development of C-probe-based technologies offers a promising prospect for situ detection, microarray, molecular diagnosis, single nucleotide polymorphism, and whole genome amplification.

  10. GMO detection in food and feed through screening by visual loop-mediated isothermal amplification assays. (United States)

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


    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.

  11. Nuclease-free target recycling signal amplification for ultrasensitive multiplexing DNA biosensing. (United States)

    Zhao, Zhihan; Chen, Shixing; Wang, Jianbang; Su, Jing; Xu, Jiaqiang; Mathur, Sanjay; Fan, Chunhai; Song, Shiping


    Ultrasensitive biosensing technologies without gene amplification held great promise for direct detection of DNA. Herein we report a novel biosensing method, combining target recycling signal-amplification strategy and a homemade electrochemical device. Especially, the target recycling was achieved by a strand displacement process, no needing the help of any nucleases. In the presence of target DNA, the recycling system could be activated to generate a cascade of assembly steps with three hairpin DNA segments. Each recycling process were accompanied by a disassembly step that the last hairpin DNA segment displaces target DNA from the complex at the end of each circulation, freeing targets to activate the self-assembly of more trefoil DNA structures. This biosensing method could detect target DNA at aM level and can distinguish target DNA from interfering DNAs, demonstrating its high sensitivity and high selectivity. Importantly, the biosensing method could work well with serum samples. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Use of signal-mediated amplification of RNA technology (SMART) to detect marine cyanophage DNA. (United States)

    Hall, M J; Wharam, S D; Weston, A; Cardy, D L N; Wilson, W H


    Here, we describe the application of an isothermal nucleic acid amplification assay, signal-mediated amplification of RNA technology (SMART), to detect DNA extracted from marine cyanophages known to infect unicellular cyanobacteria from the genus Synechococcus. The SMART assay is based on the target-dependent production of multiple copies of an RNA signal, which is measured by an enzyme-linked oligosorbent assay. SMART was able to detect both synthetic oligonucleotide targets and genomic cyanophage DNA using probes designed against the portal vertex gene (g20). Specific signals were obtained for each cyanophage strain (S-PM2 and S-BnMI). Nonspecific genomic DNA did not produce false signals or inhibit the detection of a specific target. In addition, we found that extensive purification of target DNA may not be required since signals were obtained from crude cyanophage lysates. This is the first report of the SMART assay being used to discriminate between two similar target sequences.

  13. Lysis of a Single Cyanobacterium for Whole Genome Amplification

    Directory of Open Access Journals (Sweden)

    Richard N. Zare


    Full Text Available Bacterial species from natural environments, exhibiting a great degree of genetic diversity that has yet to be characterized, pose a specific challenge to whole genome amplification (WGA from single cells. A major challenge is establishing an effective, compatible, and controlled lysis protocol. We present a novel lysis protocol that can be used to extract genomic information from a single cyanobacterium of Synechocystis sp. PCC 6803 known to have multilayer cell wall structures that resist conventional lysis methods. Simple but effective strategies for releasing genomic DNA from captured cells while retaining cellular identities for single-cell analysis are presented. Successful sequencing of genetic elements from single-cell amplicons prepared by multiple displacement amplification (MDA is demonstrated for selected genes (15 loci nearly equally spaced throughout the main chromosome.


    Institute of Scientific and Technical Information of China (English)

    WANG Wei王伟; Marianne Ifversen; ZHAO Chun-ting赵春亭; WANG Hong-yi汪洪毅; ZHAO Hong-guo赵洪国


    Objective: To establish a method to improve the detection of disseminated tumor cells in bone marrow and peripheral blood samples of neuroblastoma patients and analysis of cytogenetic aberration. Methods: Immunofluorescent staining was performed using a cocktail of primary monoclonal neuroblastoma antibodies (14.G2a, 5.1H11). Fluorescence in situ hybridization was applied with fluorescent probes specific for Nmyc genes afterwards. A novel computer assisted scanning system for automatic search, image analysis and repositioning of these positive cells was developed. Fifty-six bone marrow and peripheral blood samples from 7 patients were evaluated by this method. Results: Fluorescence in situ hybridization can be combined with immunofluorescent staining in detecting Nmyc amplification in neuroblastoma patients. Fluorescence in situ hybridization results correlated well with data obtained by conventional cytogenetic procedures. Conclusion: The technique described allows search of tumor cells in the bone marrow as well as detection of Nmyc amplification in interphase nuclei.

  15. A simple molecular beacon with duplex-specific nuclease amplification for detection of microRNA. (United States)

    Li, Yingcun; Zhang, Jiangyan; Zhao, Jingjing; Zhao, Likun; Cheng, Yongqiang; Li, Zhengping


    MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene activity, promoting or inhibiting cell proliferation, migration and apoptosis. Abnormal expression of miRNAs is associated with many diseases. Therefore, it is essential to establish a simple, rapid and sensitive miRNA detection method. In this paper, based on a simple molecular beacon (MB) and duplex-specific nuclease (DSN), we developed a target recycling amplification method for miRNA detection. By controlling the number of stem bases to 5, the MB probe used in this method can be prevented from hydrolysis by DSN without special modification. This assay is direct and simple to quantitatively detect miRNA with high sensitivity and specificity. The MB probe design provides a new strategy for nuclease-based amplification reaction.

  16. Broadening and Amplification of an Infrared Femtosecond Pulse for Optical Parametric Chirped-Pulse Amplification