A multi-sample based method for identifying common CNVs in normal human genomic structure using high-resolution aCGH data.

Directory of Open Access Journals (Sweden)

Chihyun Park

Full Text Available BACKGROUND: It is difficult to identify copy number variations (CNV in normal human genomic data due to noise and non-linear relationships between different genomic regions and signal intensity. A high-resolution array comparative genomic hybridization (aCGH containing 42 million probes, which is very large compared to previous arrays, was recently published. Most existing CNV detection algorithms do not work well because of noise associated with the large amount of input data and because most of the current methods were not designed to analyze normal human samples. Normal human genome analysis often requires a joint approach across multiple samples. However, the majority of existing methods can only identify CNVs from a single sample. METHODOLOGY AND PRINCIPAL FINDINGS: We developed a multi-sample-based genomic variations detector (MGVD that uses segmentation to identify common breakpoints across multiple samples and a k-means-based clustering strategy. Unlike previous methods, MGVD simultaneously considers multiple samples with different genomic intensities and identifies CNVs and CNV zones (CNVZs; CNVZ is a more precise measure of the location of a genomic variant than the CNV region (CNVR. CONCLUSIONS AND SIGNIFICANCE: We designed a specialized algorithm to detect common CNVs from extremely high-resolution multi-sample aCGH data. MGVD showed high sensitivity and a low false discovery rate for a simulated data set, and outperformed most current methods when real, high-resolution HapMap datasets were analyzed. MGVD also had the fastest runtime compared to the other algorithms evaluated when actual, high-resolution aCGH data were analyzed. The CNVZs identified by MGVD can be used in association studies for revealing relationships between phenotypes and genomic aberrations. Our algorithm was developed with standard C++ and is available in Linux and MS Windows format in the STL library. It is freely available at: http://embio.yonsei.ac.kr/~Park/mgvd.php.

Small sample whole-genome amplification

Science.gov (United States)

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

2005-11-01

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

Super-resolution imaging of a 2.5 kb non-repetitive DNA in situ in the nuclear genome using molecular beacon probes

Science.gov (United States)

Ni, Yanxiang; Cao, Bo; Ma, Tszshan; Niu, Gang; Huo, Yingdong; Huang, Jiandong; Chen, Danni; Liu, Yi; Yu, Bin; Zhang, Michael Q; Niu, Hanben

2017-01-01

High-resolution visualization of short non-repetitive DNA in situ in the nuclear genome is essential for studying looping interactions and chromatin organization in single cells. Recent advances in fluorescence in situ hybridization (FISH) using Oligopaint probes have enabled super-resolution imaging of genomic domains with a resolution limit of 4.9 kb. To target shorter elements, we developed a simple FISH method that uses molecular beacon (MB) probes to facilitate the probe-target binding, while minimizing non-specific fluorescence. We used three-dimensional stochastic optical reconstruction microscopy (3D-STORM) with optimized imaging conditions to efficiently distinguish sparsely distributed Alexa-647 from background cellular autofluorescence. Utilizing 3D-STORM and only 29–34 individual MB probes, we observed 3D fine-scale nanostructures of 2.5 kb integrated or endogenous unique DNA in situ in human or mouse genome, respectively. We demonstrated our MB-based FISH method was capable of visualizing the so far shortest non-repetitive genomic sequence in 3D at super-resolution. DOI: http://dx.doi.org/10.7554/eLife.21660.001 PMID:28485713

High quality copy number and genotype data from FFPE samples using Molecular Inversion Probe (MIP) microarrays

Energy Technology Data Exchange (ETDEWEB)

Wang, Yuker; Carlton, Victoria E.H.; Karlin-Neumann, George; Sapolsky, Ronald; Zhang, Li; Moorhead, Martin; Wang, Zhigang C.; Richardson, Andrea L.; Warren, Robert; Walther, Axel; Bondy, Melissa; Sahin, Aysegul; Krahe, Ralf; Tuna, Musaffe; Thompson, Patricia A.; Spellman, Paul T.; Gray, Joe W.; Mills, Gordon B.; Faham, Malek

2009-02-24

A major challenge facing DNA copy number (CN) studies of tumors is that most banked samples with extensive clinical follow-up information are Formalin-Fixed Paraffin Embedded (FFPE). DNA from FFPE samples generally underperforms or suffers high failure rates compared to fresh frozen samples because of DNA degradation and cross-linking during FFPE fixation and processing. As FFPE protocols may vary widely between labs and samples may be stored for decades at room temperature, an ideal FFPE CN technology should work on diverse sample sets. Molecular Inversion Probe (MIP) technology has been applied successfully to obtain high quality CN and genotype data from cell line and frozen tumor DNA. Since the MIP probes require only a small ({approx}40 bp) target binding site, we reasoned they may be well suited to assess degraded FFPE DNA. We assessed CN with a MIP panel of 50,000 markers in 93 FFPE tumor samples from 7 diverse collections. For 38 FFPE samples from three collections we were also able to asses CN in matched fresh frozen tumor tissue. Using an input of 37 ng genomic DNA, we generated high quality CN data with MIP technology in 88% of FFPE samples from seven diverse collections. When matched fresh frozen tissue was available, the performance of FFPE DNA was comparable to that of DNA obtained from matched frozen tumor (genotype concordance averaged 99.9%), with only a modest loss in performance in FFPE. MIP technology can be used to generate high quality CN and genotype data in FFPE as well as fresh frozen samples.

Highly sensitive polymerase chain reaction-free quantum dot-based quantification of forensic genomic DNA

International Nuclear Information System (INIS)

Tak, Yu Kyung; Kim, Won Young; Kim, Min Jung; Han, Eunyoung; Han, Myun Soo; Kim, Jong Jin; Kim, Wook; Lee, Jong Eun; Song, Joon Myong

2012-01-01

Highlights: ► Genomic DNA quantification were performed using a quantum dot-labeled Alu sequence. ► This probe provided PCR-free determination of human genomic DNA. ► Qdot-labeled Alu probe-hybridized genomic DNAs had a 2.5-femtogram detection limit. ► Qdot-labeled Alu sequence was used to assess DNA samples for human identification. - Abstract: Forensic DNA samples can degrade easily due to exposure to light and moisture at the crime scene. In addition, the amount of DNA acquired at a criminal site is inherently limited. This limited amount of human DNA has to be quantified accurately after the process of DNA extraction. The accurately quantified extracted genomic DNA is then used as a DNA template in polymerase chain reaction (PCR) amplification for short tandem repeat (STR) human identification. Accordingly, highly sensitive and human-specific quantification of forensic DNA samples is an essential issue in forensic study. In this work, a quantum dot (Qdot)-labeled Alu sequence was developed as a probe to simultaneously satisfy both the high sensitivity and human genome selectivity for quantification of forensic DNA samples. This probe provided PCR-free determination of human genomic DNA and had a 2.5-femtogram detection limit due to the strong emission and photostability of the Qdot. The Qdot-labeled Alu sequence has been used successfully to assess 18 different forensic DNA samples for STR human identification.

Whole genomic DNA probe for detection of Porphyromonas endodontalis.

Science.gov (United States)

Nissan, R; Makkar, S R; Sela, M N; Stevens, R

2000-04-01

The purpose of the present study was to develop a DNA probe for Porphyromonas endodontalis. Pure cultures of P. endodontalis were grown in TYP medium, in an anaerobic chamber. DNA was extracted from the P. endodontalis and labeled using the Genius System by Boehringer Mannheim. The labeled P. endodontalis DNA was used in dot-blot hybridization reactions with homologous (P. endodontalis) and unrelated bacterial samples. To determine specificity, strains of 40 other oral bacterial species (e.g. Porphyromonas gingivalis, Porphyromonas asaccharolytica, and Prevotella intermedia) were spotted and reacted with the P. endodontalis DNA probe. None of the panel of 40 oral bacteria hybridized with the P. endodontalis probe, whereas the blot of the homologous organism showed a strong positive reaction. To determine the sensitivity of the probe, dilutions of a P. endodontalis suspension of known concentration were blotted onto a nylon membrane and reacted with the probe. The results of our investigation indicate that the DNA probe that we have prepared specifically detects only P. endodontalis and can detect at least 3 x 10(4) cells.

Specification for soil multisensor and soil sampling cone penetrometer probes

International Nuclear Information System (INIS)

Iwatate, D.F.

1997-01-01

Specification requirements for engineering, fabrication, and performance of cone penetrometer (CP) soil multisensor and sampling probes (CP-probes) which are required to support contract procurement for services are presented. The specification provides a documented technical basis of quality assurance that is required to use the probes in an operating Hanford tank farm. The documentation cited in this specification will be incorporated into an operational fielding plan that will address all activities associated with the use of the CP-probes. The probes discussed in this specification support the Hanford Tanks Initiative AX-104 Tank Plume Characterization Sub-task. The probes will be used to interrogate soils and vadose zone surrounding tank AX-104

Single point aerosol sampling: Evaluation of mixing and probe performance in a nuclear stack

Energy Technology Data Exchange (ETDEWEB)

Rodgers, J.C.; Fairchild, C.I.; Wood, G.O. [Los Alamos National Laboratory, NM (United States)] [and others

1995-02-01

Alternative Reference Methodologies (ARMs) have been developed for sampling of radionuclides from stacks and ducts that differ from the methods required by the U.S. EPA. The EPA methods are prescriptive in selection of sampling locations and in design of sampling probes whereas the alternative methods are performance driven. Tests were conducted in a stack at Los Alamos National Laboratory to demonstrate the efficacy of the ARMs. Coefficients of variation of the velocity tracer gas, and aerosol particle profiles were determined at three sampling locations. Results showed numerical criteria placed upon the coefficients of variation by the ARMs were met at sampling stations located 9 and 14 stack diameters from flow entrance, but not at a location that is 1.5 diameters downstream from the inlet. Experiments were conducted to characterize the transmission of 10 {mu}m aerodynamic equivalent diameter liquid aerosol particles through three types of sampling probes. The transmission ratio (ratio of aerosol concentration at the probe exit plane to the concentration in the free stream) was 107% for a 113 L/min (4-cfm) anisokinetic shrouded probe, but only 20% for an isokinetic probe that follows the EPA requirements. A specially designed isokinetic probe showed a transmission ratio of 63%. The shrouded probe performance would conform to the ARM criteria; however, the isokinetic probes would not.

PROBES, POPULATIONS, SAMPLES, MEASUREMENTS AND RELATIONS IN STEREOLOGY

Directory of Open Access Journals (Sweden)

Robert T Dehoff

2011-05-01

Full Text Available This summary paper provides an overview of the content of stereology. The typical problem at hand centers around some three dimensional object that has an internal structure that determines its function, performance, or response. To understand and quantify the geometry of that structure it is necessary to probe it with geometric entities: points, lines, planes volumes, etc. Meaningful results are obtained only if the set of probes chosen for use in the assessment is drawn uniformly from the population of such probes for the structure as a whole. This requires an understanding of the population of each kind of probe. Interaction of the probes with the structure produce geometric events which are the focus of stereological measurements. In almost all applications the measurement that is made is a simple count of the number of these events. Rigorous application of these requirements for sample design produce unbiased estimates of geometric properties of features in the structure no matter how complex are the features or what their arrangement in space. It is this assumption-free characteristic of the methodology that makes it a powerful tool for characterizing the internal structure of three dimensional objects.

Lanthanide complexes as luminogenic probes to measure sulfide levels in industrial samples

International Nuclear Information System (INIS)

Thorson, Megan K.; Ung, Phuc; Leaver, Franklin M.; Corbin, Teresa S.; Tuck, Kellie L.; Graham, Bim; Barrios, Amy M.

2015-01-01

A series of lanthanide-based, azide-appended complexes were investigated as hydrogen sulfide-sensitive probes. Europium complex 1 and Tb complex 3 both displayed a sulfide-dependent increase in luminescence, while Tb complex 2 displayed a decrease in luminescence upon exposure to NaHS. The utility of the complexes for monitoring sulfide levels in industrial oil and water samples was investigated. Complex 3 provided a sensitive measure of sulfide levels in petrochemical water samples (detection limit ∼ 250 nM), while complex 1 was capable of monitoring μM levels of sulfide in partially refined crude oil. - Highlights: • Lanthanide–azide based sulfide sensors were synthesized and characterized. • The probes have excitation and emission profiles compatible with sulfide-contaminated samples from the petrochemical industry. • A terbium-based probe was used to measure the sulfide concentration in oil refinery wastewater. • A europium-based probe had compatibility with partially refined crude oil samples.

Lanthanide complexes as luminogenic probes to measure sulfide levels in industrial samples

Energy Technology Data Exchange (ETDEWEB)

Thorson, Megan K. [Department of Medicinal Chemistry, University of Utah College of Pharmacy, Salt Lake City, UT 84108 (United States); Ung, Phuc [Monash Institute of Pharmaceutical Sciences, Monash University, Victoria 3052 (Australia); Leaver, Franklin M. [Water & Energy Systems Technology, Inc., Kaysville, UT 84037 (United States); Corbin, Teresa S. [Quality Services Laboratory, Tesoro Refining and Marketing, Salt Lake City, UT 84103 (United States); Tuck, Kellie L., E-mail: kellie.tuck@monash.edu [School of Chemistry, Monash University, Victoria 3800 (Australia); Graham, Bim, E-mail: bim.graham@monash.edu [Monash Institute of Pharmaceutical Sciences, Monash University, Victoria 3052 (Australia); Barrios, Amy M., E-mail: amy.barrios@utah.edu [Department of Medicinal Chemistry, University of Utah College of Pharmacy, Salt Lake City, UT 84108 (United States)

2015-10-08

A series of lanthanide-based, azide-appended complexes were investigated as hydrogen sulfide-sensitive probes. Europium complex 1 and Tb complex 3 both displayed a sulfide-dependent increase in luminescence, while Tb complex 2 displayed a decrease in luminescence upon exposure to NaHS. The utility of the complexes for monitoring sulfide levels in industrial oil and water samples was investigated. Complex 3 provided a sensitive measure of sulfide levels in petrochemical water samples (detection limit ∼ 250 nM), while complex 1 was capable of monitoring μM levels of sulfide in partially refined crude oil. - Highlights: • Lanthanide–azide based sulfide sensors were synthesized and characterized. • The probes have excitation and emission profiles compatible with sulfide-contaminated samples from the petrochemical industry. • A terbium-based probe was used to measure the sulfide concentration in oil refinery wastewater. • A europium-based probe had compatibility with partially refined crude oil samples.

New approaches to nanoparticle sample fabrication for atom probe tomography

International Nuclear Information System (INIS)

Felfer, P.; Li, T.; Eder, K.; Galinski, H.; Magyar, A.P.; Bell, D.C.; Smith, G.D.W.; Kruse, N.; Ringer, S.P.; Cairney, J.M.

2015-01-01

Due to their unique properties, nano-sized materials such as nanoparticles and nanowires are receiving considerable attention. However, little data is available about their chemical makeup at the atomic scale, especially in three dimensions (3D). Atom probe tomography is able to answer many important questions about these materials if the challenge of producing a suitable sample can be overcome. In order to achieve this, the nanomaterial needs to be positioned within the end of a tip and fixed there so the sample possesses sufficient structural integrity for analysis. Here we provide a detailed description of various techniques that have been used to position nanoparticles on substrates for atom probe analysis. In some of the approaches, this is combined with deposition techniques to incorporate the particles into a solid matrix, and focused ion beam processing is then used to fabricate atom probe samples from this composite. Using these approaches, data has been achieved from 10–20 nm core–shell nanoparticles that were extracted directly from suspension (i.e. with no chemical modification) with a resolution of better than ±1 nm. - Highlights: • Samples for APT of nanoparticles were fabricated from particle powders and dispersions. • Electrophoresis was suitable for producing samples from dispersions. • Powder lift-out was successfully producing samples from particle agglomerates. • Dispersion application/coating delivered the highest quality results.

New approaches to nanoparticle sample fabrication for atom probe tomography

Energy Technology Data Exchange (ETDEWEB)

Felfer, P., E-mail: peter.felfer@sydney.edu.au [School for Aerospace, Mechanical and Mechatronic Engineering/Australian Centre for Microscopy and Microanalysis, The University of Sydney, NSW 2006 (Australia); Li, T. [School for Aerospace, Mechanical and Mechatronic Engineering/Australian Centre for Microscopy and Microanalysis, The University of Sydney, NSW 2006 (Australia); Materials Department, The University of Oxford, Oxford (United Kingdom); Eder, K. [School for Aerospace, Mechanical and Mechatronic Engineering/Australian Centre for Microscopy and Microanalysis, The University of Sydney, NSW 2006 (Australia); Galinski, H. [School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138 (United States); Magyar, A.P.; Bell, D.C. [School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138 (United States); Center for Nanoscale Systems, Harvard University, Cambridge, MA 02138 (United States); Smith, G.D.W. [Materials Department, The University of Oxford, Oxford (United Kingdom); Kruse, N. [Chemical Physics of Materials (Catalysis-Tribology), Université Libre de Bruxelles, Campus Plaine, CP 243, 1050 Brussels (Belgium); Ringer, S.P.; Cairney, J.M. [School for Aerospace, Mechanical and Mechatronic Engineering/Australian Centre for Microscopy and Microanalysis, The University of Sydney, NSW 2006 (Australia)

2015-12-15

Due to their unique properties, nano-sized materials such as nanoparticles and nanowires are receiving considerable attention. However, little data is available about their chemical makeup at the atomic scale, especially in three dimensions (3D). Atom probe tomography is able to answer many important questions about these materials if the challenge of producing a suitable sample can be overcome. In order to achieve this, the nanomaterial needs to be positioned within the end of a tip and fixed there so the sample possesses sufficient structural integrity for analysis. Here we provide a detailed description of various techniques that have been used to position nanoparticles on substrates for atom probe analysis. In some of the approaches, this is combined with deposition techniques to incorporate the particles into a solid matrix, and focused ion beam processing is then used to fabricate atom probe samples from this composite. Using these approaches, data has been achieved from 10–20 nm core–shell nanoparticles that were extracted directly from suspension (i.e. with no chemical modification) with a resolution of better than ±1 nm. - Highlights: • Samples for APT of nanoparticles were fabricated from particle powders and dispersions. • Electrophoresis was suitable for producing samples from dispersions. • Powder lift-out was successfully producing samples from particle agglomerates. • Dispersion application/coating delivered the highest quality results.

Targeted Capture and High-Throughput Sequencing Using Molecular Inversion Probes (MIPs).

Science.gov (United States)

Cantsilieris, Stuart; Stessman, Holly A; Shendure, Jay; Eichler, Evan E

2017-01-01

Molecular inversion probes (MIPs) in combination with massively parallel DNA sequencing represent a versatile, yet economical tool for targeted sequencing of genomic DNA. Several thousand genomic targets can be selectively captured using long oligonucleotides containing unique targeting arms and universal linkers. The ability to append sequencing adaptors and sample-specific barcodes allows large-scale pooling and subsequent high-throughput sequencing at relatively low cost per sample. Here, we describe a "wet bench" protocol detailing the capture and subsequent sequencing of >2000 genomic targets from 192 samples, representative of a single lane on the Illumina HiSeq 2000 platform.

The international Genome sample resource (IGSR): A worldwide collection of genome variation incorporating the 1000 Genomes Project data.

Science.gov (United States)

Clarke, Laura; Fairley, Susan; Zheng-Bradley, Xiangqun; Streeter, Ian; Perry, Emily; Lowy, Ernesto; Tassé, Anne-Marie; Flicek, Paul

2017-01-04

The International Genome Sample Resource (IGSR; http://www.internationalgenome.org) expands in data type and population diversity the resources from the 1000 Genomes Project. IGSR represents the largest open collection of human variation data and provides easy access to these resources. IGSR was established in 2015 to maintain and extend the 1000 Genomes Project data, which has been widely used as a reference set of human variation and by researchers developing analysis methods. IGSR has mapped all of the 1000 Genomes sequence to the newest human reference (GRCh38), and will release updated variant calls to ensure maximal usefulness of the existing data. IGSR is collecting new structural variation data on the 1000 Genomes samples from long read sequencing and other technologies, and will collect relevant functional data into a single comprehensive resource. IGSR is extending coverage with new populations sequenced by collaborating groups. Here, we present the new data and analysis that IGSR has made available. We have also introduced a new data portal that increases discoverability of our data-previously only browseable through our FTP site-by focusing on particular samples, populations or data sets of interest. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Characterization of Actinomyces with genomic DNA fingerprints and rRNA gene probes.

Science.gov (United States)

Bowden, G; Johnson, J; Schachtele, C

1993-08-01

Cellular DNA from 25 Actinomyces naeslundii and Actinomyces viscosus strains belonging to the 7 taxonomic clusters of Fillery et al. (1978) and several unclustered strains was obtained by enzymatic and N-lauroylsarcosine/guanidine isothiocyanate treatment of whole cells, followed by extraction of the nucleic acid. The DNA samples were digested with restriction endonucleases BamHI or PvuII, and agarose gel electrophoresis was used to obtain DNA fingerprints. The DNA fragments were subjected to Southern blot hybridization with a digoxigenin-labeled cDNA probe transcribed from Escherichia coli 16S and 23S rRNA. The patterns of bands from genomic (DNA fingerprints) and rDNA fingerprints (ribotypes) were used for comparison between the taxonomic cluster strains and strains within clusters. Representative strains from each taxonomic cluster provided different BamHI DNA fingerprints and ribotype patterns with 3 to 9 distinct bands. Some strains within a cluster showed identical ribotype patterns with both endonucleases (A. naeslundii B120 and A. naeslundii B102 from cluster 3), while others showed the same pattern with BamHI but a different pattern with PvuII (A. naeslundii ATCC 12104 and 398A from cluster 5). A viscosus ATCC 15987 (cluster 7) and its parent strain T6 yielded identical fingerprint and ribotype patterns. The genomic diversity revealed by DNA fingerprinting and ribotyping demonstrates that these techniques, which do not require phenotypic expression, are suited for study of the oral ecology of the Actinomyces, and for epidemiological tracking of specific Actinomyces strains associated with caries lesions and sites of periodontal destruction.

Design, validation and annotation of transcriptome-wide oligonucleotide probes for the oligochaete annelid Eisenia fetida.

Directory of Open Access Journals (Sweden)

Ping Gong

Full Text Available High density oligonucleotide probe arrays have increasingly become an important tool in genomics studies. In organisms with incomplete genome sequence, one strategy for oligo probe design is to reduce the number of unique probes that target every non-redundant transcript through bioinformatic analysis and experimental testing. Here we adopted this strategy in making oligo probes for the earthworm Eisenia fetida, a species for which we have sequenced transcriptome-scale expressed sequence tags (ESTs. Our objectives were to identify unique transcripts as targets, to select an optimal and non-redundant oligo probe for each of these target ESTs, and to annotate the selected target sequences. We developed a streamlined and easy-to-follow approach to the design, validation and annotation of species-specific array probes. Four 244K-formatted oligo arrays were designed using eArray and were hybridized to a pooled E. fetida cRNA sample. We identified 63,541 probes with unsaturated signal intensities consistently above the background level. Target transcripts of these probes were annotated using several sequence alignment algorithms. Significant hits were obtained for 37,439 (59% probed targets. We validated and made publicly available 63.5K oligo probes so the earthworm research community can use them to pursue ecological, toxicological, and other functional genomics questions. Our approach is efficient, cost-effective and robust because it (1 does not require a major genomics core facility; (2 allows new probes to be easily added and old probes modified or eliminated when new sequence information becomes available, (3 is not bioinformatics-intensive upfront but does provide opportunities for more in-depth annotation of biological functions for target genes; and (4 if desired, EST orthologs to the UniGene clusters of a reference genome can be identified and selected in order to improve the target gene specificity of designed probes. This approach is

Specific single-cell isolation and genomic amplification of uncultured microorganisms

DEFF Research Database (Denmark)

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

2007-01-01

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

A multi-probe thermophoretic soot sampling system for high-pressure diffusion flames

Energy Technology Data Exchange (ETDEWEB)

Vargas, Alex M.; Gülder, Ömer L. [Institute for Aerospace Studies, University of Toronto, Toronto, Ontario M3H 5T6 (Canada)

2016-05-15

Optical diagnostics and physical probing of the soot processes in high pressure combustion pose challenges that are not faced in atmospheric flames. One of the preferred methods of studying soot in atmospheric flames is in situ thermophoretic sampling followed by transmission electron microscopy imaging and analysis for soot sizing and morphology. The application of this method of sampling to high pressures has been held back by various operational and mechanical problems. In this work, we describe a rotating disk multi-probe thermophoretic soot sampling system, driven by a microstepping stepper motor, fitted into a high-pressure chamber capable of producing sooting laminar diffusion flames up to 100 atm. Innovative aspects of the sampling system design include an easy and precise control of the sampling time down to 2.6 ms, avoidance of the drawbacks of the pneumatic drivers used in conventional thermophoretic sampling systems, and the capability to collect ten consecutive samples in a single experimental run. Proof of principle experiments were performed using this system in a laminar diffusion flame of methane, and primary soot diameter distributions at various pressures up to 10 atm were determined. High-speed images of the flame during thermophoretic sampling were recorded to assess the influence of probe intrusion on the flow field of the flame.

Homogeneous versus heterogeneous probes for microbial ecological microarrays.

Science.gov (United States)

Bae, Jin-Woo; Park, Yong-Ha

2006-07-01

Microbial ecological microarrays have been developed for investigating the composition and functions of microorganism communities in environmental niches. These arrays include microbial identification microarrays, which use oligonucleotides, gene fragments or microbial genomes as probes. In this article, the advantages and disadvantages of each type of probe are reviewed. Oligonucleotide probes are currently useful for probing uncultivated bacteria that are not amenable to gene fragment probing, whereas the functional gene fragments amplified randomly from microbial genomes require phylogenetic and hierarchical categorization before use as microbial identification probes, despite their high resolution for both specificity and sensitivity. Until more bacteria are sequenced and gene fragment probes are thoroughly validated, heterogeneous bacterial genome probes will provide a simple, sensitive and quantitative tool for exploring the ecosystem structure.

Rapid colorimetric assay for detection of Listeria monocytogenes in food samples using LAMP formation of DNA concatemers and gold nanoparticle-DNA probe complex

Science.gov (United States)

Wachiralurpan, Sirirat; Sriyapai, Thayat; Areekit, Supatra; Sriyapai, Pichapak; Augkarawaritsawong, Suphitcha; Santiwatanakul, Somchai; Chansiri, Kosum

2018-04-01

ABSTRACT Listeria monocytogenes is a major foodborne pathogen of global health concern. Herein, the rapid diagnosis of L. monocytogenes has been achieved using loop-mediated isothermal amplification (LAMP) based on the phosphatidylcholine-phospholipase C gene (plcB). Colorimetric detection was then performed through the formation of DNA concatemers and a gold nanoparticle/DNA probe complex (GNP/DNA probe). The overall detection process was accomplished within approximately 1 h with no need for complicated equipment. The limits of detection for L. monocytogenes in the forms of purified genomic DNA and pure culture were 800 fg and 2.82 CFU mL-1, respectively. No cross reactions were observed from closely related bacteria species. The LAMP-GNP/DNA probe assay was applied to the detection of 200 raw chicken meat samples and compared to routine standard methods. The data revealed that the specificity, sensitivity and accuracy were 100%, 90.20% and 97.50%, respectively. The present assay was 100% in conformity with LAMP-agarose gel electrophoresis assay. Five samples that were negative by both assays appeared to have the pathogen at below the level of detection. The assay can be applied as a rapid direct screening method for L. monocytogenes.

Analysis of non-contact and contact probe-to-sample thermal exchange for quantitative measurements of thin film and nanostructure thermal conductivity by the scanning hot probe method

Science.gov (United States)

Wilson, Adam A.

The ability to measure thermal properties of thin films and nanostructured materials is an important aspect of many fields of academic study. A strategy especially well-suited for nanoscale investigations of these properties is the scanning hot probe technique, which is unique in its ability to non-destructively interrogate the thermal properties with high resolution, both laterally as well as through the thickness of the material. Strategies to quantitatively determine sample thermal conductivity depend on probe calibration. State of the art calibration strategies assume that the area of thermal exchange between probe and sample does not vary with sample thermal conductivity. However, little investigation has gone into determining whether or not that assumption is valid. This dissertation provides a rigorous study into the probe-to-sample heat transfer through the air gap at diffusive distances for a variety of values of sample thermal conductivity. It is demonstrated that the thermal exchange radius and gap/contact thermal resistance varies with sample thermal conductivity as well as tip-to-sample clearance in non-contact mode. In contact mode, it is demonstrated that higher thermal conductivity samples lead to a reduction in thermal exchange radius for Wollaston probe tips. Conversely, in non-contact mode and in contact mode for sharper probe tips where air contributes the most to probe-to-sample heat transfer, the opposite trend occurs. This may be attributed to the relatively strong solid-to-solid conduction occurring between probe and sample for the Wollaston probes. A three-dimensional finite element (3DFE) model was developed to investigate how the calibrated thermal exchange parameters vary with sample thermal conductivity when calibrating the probe via the intersection method in non-contact mode at diffusive distances. The 3DFE model was then used to explore the limits of sensitivity of the experiment for a range of simulated experimental conditions. It

FDTD simulations and analysis of thin sample dielectric properties measurements using coaxial probes

Energy Technology Data Exchange (ETDEWEB)

Bringhurst, S.; Iskander, M.F.; White, M.J. [Univ. of Utah, Salt Lake City, UT (United States). Electrical Engineering Dept.

1996-12-31

A metallized ceramic probe has been designed for high temperature broadband dielectric properties measurements. The probe was fabricated out of an alumina tube and rod as the outer and inner conductors respectively. The alumina was metallized with a 3 mil layer of moly-manganese and then covered with a 0.5 mil protective layer of nickel plating. The probe has been used to make complex dielectric properties measurements over the complete frequency band from 500 MHz to 3 GHz, and for temperatures as high as 1,000 C. A 3D Finite-Difference Time-Domain (FDTD) code was used to help investigate the feasibility of this probe to measure the complex permittivity of thin samples. It is shown that by backing the material under test with a standard material of known dielectric constant, the complex permittivity of thin samples can be measured accurately using the developed FDTD algorithm. This FDTD procedure for making thin sample dielectric properties measurements will be described.

Probe depth matters in dermal microdialysis sampling of benzoic acid after topical application

DEFF Research Database (Denmark)

Holmgaard, R; Benfeldt, E; Bangsgaard, N

2012-01-01

-2 mm) and deep (>2 mm) positioning of the linear MD probe in the dermis of human abdominal skin, followed by topical application of 4 mg/ml of benzoic acid (BA) in skin chambers overlying the probes. Dialysate was sampled every hour for 12 h and analysed for BA content by high-performance liquid...... chromatography. Probe depth was measured by 20-MHz ultrasound scanning. The area under the time-versus-concentration curve (AUC) describes the drug exposure in the tissue during the experiment and is a relevant parameter to compare for the 3 dermal probe depths investigated. The AUC(0-12) were: superficial...... significantly different from each other (p value paper demonstrates that there is an inverse relationship between the depth of the probe in the dermis and the amount of drug sampled following topical penetration ex vivo. The result is of relevance to the in vivo situation, and it can...

Interim report on updated microarray probes for the LLNL Burkholderia pseudomallei SNP array

Energy Technology Data Exchange (ETDEWEB)

Gardner, S; Jaing, C

2012-03-27

The overall goal of this project is to forensically characterize 100 unknown Burkholderia isolates in the US-Australia collaboration. We will identify genome-wide single nucleotide polymorphisms (SNPs) from B. pseudomallei and near neighbor species including B. mallei, B. thailandensis and B. oklahomensis. We will design microarray probes to detect these SNP markers and analyze 100 Burkholderia genomic DNAs extracted from environmental, clinical and near neighbor isolates from Australian collaborators on the Burkholderia SNP microarray. We will analyze the microarray genotyping results to characterize the genetic diversity of these new isolates and triage the samples for whole genome sequencing. In this interim report, we described the SNP analysis and the microarray probe design for the Burkholderia SNP microarray.

Multiple biopsy probe sampling enabled minimally invasive electrical impedance tomography

International Nuclear Information System (INIS)

Shini, Mohanad; Rubinsky, Boris

2008-01-01

Biopsies are a reliable method for examining tissues and organs inside the body, in particular for detection of tumors. However, a single biopsy produces only limited information on the site from which it is taken. Therefore, tumor detection now employs multiple biopsy samplings to examine larger volumes of tissue. Nevertheless, even with multiple biopsies, the information remains discrete, while the costs of biopsy increase. Here we propose and evaluate the feasibility of using minimally invasive medical imaging as a means to overcome the limitations of discrete biopsy sampling. The minimally invasive medical imaging technique employs the biopsy probe as electrodes for measurements of electrical impedance tomography relevant data during each biopsy sampling. The data from multiple samplings are combined and used to produce an EIT image of the tissue. Two- and three-dimensional mathematical simulations confirm that the minimally invasive medical imaging technique can produce electrical impedance tomography images of the tissues between the biopsy probe insertion sites. We show that these images can detect tumors that would be missed with multiple biopsy samplings only, and that the technique may facilitate the detection of tumors with fewer biopsies, thereby reducing the cost of cancer detection

Human MLPA Probe Design (H-MAPD: a probe design tool for both electrophoresis-based and bead-coupled human multiplex ligation-dependent probe amplification assays

Directory of Open Access Journals (Sweden)

Hatchwell Eli

2008-09-01

Full Text Available Abstract Background Multiplex ligation-dependent probe amplification (MLPA is an efficient and reliable technique for gene dosage analysis. Currently MLPA can be conducted on two platforms: traditional electrophoresis-based, and FlexMAP bead-coupled. Since its introduction in 2002, MLPA has been rapidly adopted in both clinical and research situations. However, MLPA probe design is a time consuming process requiring many steps that address multiple criteria. There exist only one or two commercial software packages for traditional electrophoresis-based MLPA probe design. To our knowledge, no software is yet available that performs bead-coupled MLPA probe design. Results We have developed H-MAPD, a web-based tool that automates the generation and selection of probes for human genomic MLPA. The software performs physical-chemical property tests using UNAFold software, and uniqueness tests using the UCSC genome browser. H-MAPD supports both traditional electrophoresis-based assays, as well as FlexMAP bead-coupled MLPA. Conclusion H-MAPD greatly reduces the efforts for human genomic MLPA probe design. The software is written in Perl-CGI, hosted on a Linux server, and is freely available to non-commercial users.

Copy number variation in the bovine genome

DEFF Research Database (Denmark)

Fadista, João; Thomsen, Bo; Holm, Lars-Erik

2010-01-01

to genetic variation in cattle. Results We designed and used a set of NimbleGen CGH arrays that tile across the assayable portion of the cattle genome with approximately 6.3 million probes, at a median probe spacing of 301 bp. This study reports the highest resolution map of copy number variation...... in the cattle genome, with 304 CNV regions (CNVRs) being identified among the genomes of 20 bovine samples from 4 dairy and beef breeds. The CNVRs identified covered 0.68% (22 Mb) of the genome, and ranged in size from 1.7 to 2,031 kb (median size 16.7 kb). About 20% of the CNVs co-localized with segmental...... duplications, while 30% encompass genes, of which the majority is involved in environmental response. About 10% of the human orthologous of these genes are associated with human disease susceptibility and, hence, may have important phenotypic consequences. Conclusions Together, this analysis provides a useful...

Shared probe design and existing microarray reanalysis using PICKY

Directory of Open Access Journals (Sweden)

Chou Hui-Hsien

2010-04-01

Full Text Available Abstract Background Large genomes contain families of highly similar genes that cannot be individually identified by microarray probes. This limitation is due to thermodynamic restrictions and cannot be resolved by any computational method. Since gene annotations are updated more frequently than microarrays, another common issue facing microarray users is that existing microarrays must be routinely reanalyzed to determine probes that are still useful with respect to the updated annotations. Results PICKY 2.0 can design shared probes for sets of genes that cannot be individually identified using unique probes. PICKY 2.0 uses novel algorithms to track sharable regions among genes and to strictly distinguish them from other highly similar but nontarget regions during thermodynamic comparisons. Therefore, PICKY does not sacrifice the quality of shared probes when choosing them. The latest PICKY 2.1 includes the new capability to reanalyze existing microarray probes against updated gene sets to determine probes that are still valid to use. In addition, more precise nonlinear salt effect estimates and other improvements are added, making PICKY 2.1 more versatile to microarray users. Conclusions Shared probes allow expressed gene family members to be detected; this capability is generally more desirable than not knowing anything about these genes. Shared probes also enable the design of cross-genome microarrays, which facilitate multiple species identification in environmental samples. The new nonlinear salt effect calculation significantly increases the precision of probes at a lower buffer salt concentration, and the probe reanalysis function improves existing microarray result interpretations.

Sealed magic angle spinning nuclear magnetic resonance probe and process for spectroscopy of hazardous samples

Energy Technology Data Exchange (ETDEWEB)

Cho, Herman M.; Washton, Nancy M.; Mueller, Karl T.; Sears, Jr., Jesse A.; Townsend, Mark R.; Ewing, James R.

2016-06-14

A magic-angle-spinning (MAS) nuclear magnetic resonance (NMR) probe is described that includes double containment enclosures configured to seal and contain hazardous samples for analysis. The probe is of a modular design that ensures containment of hazardous samples during sample analysis while preserving spin speeds for superior NMR performance and convenience of operation.

Sample Preparation for Electron Probe Microanalysis-Pushing the Limits.

Science.gov (United States)

Geller, Joseph D; Engle, Paul D

2002-01-01

There are two fundamental considerations in preparing samples for electron probe microanalysis (EPMA). The first one may seem obvious, but we often find it is overlooked. That is, the sample analyzed should be representative of the population from which it comes. The second is a direct result of the assumptions in the calculations used to convert x-ray intensity ratios, between the sample and standard, to concentrations. Samples originate from a wide range of sources. During their journey to being excited under the electron beam for the production of x rays there are many possibilities for sample alteration. Handling can contaminate samples by adding extraneous matter. In preparation, the various abrasives used in sizing the sample by sawing, grinding and polishing can embed themselves. The most accurate composition of a contaminated sample is, at best, not representative of the original sample; it is misleading. Our laboratory performs EPMA analysis on customer submitted samples and prepares over 250 different calibration standards including pure elements, compounds, alloys, glasses and minerals. This large variety of samples does not lend itself to mass production techniques, including automatic polishing. Our manual preparation techniques are designed individually for each sample. The use of automated preparation equipment does not lend itself to this environment, and is not included in this manuscript. The final step in quantitative electron probe microanalysis is the conversion of x-ray intensities ratios, known as the "k-ratios," to composition (in mass fraction or atomic percent) and/or film thickness. Of the many assumptions made in the ZAF (where these letters stand for atomic number, absorption and fluorescence) corrections the localized geometry between the sample and electron beam, or takeoff angle, must be accurately known. Small angular errors can lead to significant errors in the final results. The sample preparation technique then becomes very

Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization

International Nuclear Information System (INIS)

Collomb, J.; Finance, C.; Alabouch, S.; Laporte, J.

1992-01-01

Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG 78), selected in the cDNA library, including a large portion of the nucleocapsid (N), matrix (M) and peplomeric (S) protein genes , were used as probes for a slot blot hybridization assay. Two probe labelling techniques were compared, radiolabelling with 32 P and enzymatic labelling through covalent linkage to peroxidase and chemiluminescence detection. The radioactive probe 174 detected as little as 1 to 3 pg of viral RNA, while the less sensitive enzymatic probe could not reveal more than 100 pg of RNA. No significant detection amplification was achieved when a mixture of the three probes was used. Probe 174 allowed specific identification for BECV. No hybridization was noticed either with rotaviruses or even with other antigenically unrelated members of the family Coronaviridae such as transmissible gastroenteritis virus. The test proved valid for detection of BECV in the supernatant of infected HRT-18 cells: genomic RNA could be detected after direct spotting of samples, but prior nucleic acid extraction after proteinase K treatment improved virus detection. BECV diagnosis in faecal samples using enzymatic probe was compared with conventional diagnostic methods. (authors)

Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization

Energy Technology Data Exchange (ETDEWEB)

Collomb, J; Finance, C; Alabouch, S [Lab. de Microbiologie Moleculaire, Faculte des Sciences Pharmaceutiques et Biologiques, Univ. de Nancy I, Nancy (France); Laporte, J [Station de Virologie et d' Immunologie Moleculaires, INRA, Jouy-en-Josas (France)

1992-01-01

Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG 78), selected in the cDNA library, including a large portion of the nucleocapsid (N), matrix (M) and peplomeric (S) protein genes , were used as probes for a slot blot hybridization assay. Two probe labelling techniques were compared, radiolabeled with [sup 32]P and enzymatic labeled through covalent linkage to peroxidase for chemiluminescence detection. The radioactive probe 174 detected as little as 1-3 pg of viral RNA, while the less sensitive enzymatic probe could not reveal more than 100 pg of RNA. No significant detection amplification was achieved when a mixture of the three probes was used. Probe 174 allowed specific identification for BECV. No hybridization was noticed either with rotaviruses or even with other antigenically unrelated members of the family Coronaviridae such as transmissible gastroenteritis virus. The test proved valid for detection of BECV in the supernatant of infected HRT-18 cells: genomic RNA could be detected after direct spotting of samples, but prior nucleic acid extraction after proteinase K treatment improved virus detection. BECV diagnosis in fecal samples using enzymatic probe was compared with conventional diagnostic methods. (authors).

Detection of hepatitis A virus by hybridization with single-stranded RNA probes

International Nuclear Information System (INIS)

Xi, J.; Estes, M.K.; Metcalf, T.G.

1987-01-01

An improved method of dot-blot hybridization to detect hepatitis A virus (HAV) was developed with single-stranded RNA (ssRNA) probes. Radioactive and nonradioactive ssRNA probes were generated by in vitro transcription of HAV templates inserted into the plasmid pGEM-1. 32 P-labeled ssRNA probes were at least eightfold more sensitive than the 32 P-labeled double-stranded cDNA counterparts, whereas biotin-labeled ssRNA probes showed a sensitivity comparable with that of the 32 P-labeled double-stranded cDNA counterparts. Hybridization of HAV with the ssRNA probes at high stringency revealed specific reactions with a high signal-to-noise ratio. The differential hybridization reactions seen with probes of positive and negative sense (compared with HAV genomic RNA) were used to detect HAV in clinical and field samples. A positive/negative ratio was introduced as an indicator that permitted an semiquantitative expression of a positive HAV reaction. Good agreement of this indicator was observed with normal stool samples and with HAV-seeded samples. By using this system, HAV was detected in estuarine and freshwater samples collected from a sewage-polluted bayou in Houston and a saltwater tributary of Galveston Bay

Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes

DEFF Research Database (Denmark)

Nielsen, Henrik Bjørn; Almeida, Mathieu; Juncker, Agnieszka

2014-01-01

of microbial genomes without the need for reference sequences. We demonstrate the method on data from 396 human gut microbiome samples and identify 7,381 co-abundance gene groups (CAGs), including 741 metagenomic species (MGS). We use these to assemble 238 high-quality microbial genomes and identify...

Detection of Helicobacter Pylori Genome with an Optical Biosensor Based on Hybridization of Urease Gene with a Gold Nanoparticles-Labeled Probe

Science.gov (United States)

Shahrashoob, M.; Mohsenifar, A.; Tabatabaei, M.; Rahmani-Cherati, T.; Mobaraki, M.; Mota, A.; Shojaei, T. R.

2016-05-01

A novel optics-based nanobiosensor for sensitive determination of the Helicobacter pylori genome using a gold nanoparticles (AuNPs)-labeled probe is reported. Two specific thiol-modified capture and signal probes were designed based on a single-stranded complementary DNA (cDNA) region of the urease gene. The capture probe was immobilized on AuNPs, which were previously immobilized on an APTES-activated glass, and the signal probe was conjugated to different AuNPs as well. The presence of the cDNA in the reaction mixture led to the hybridization of the AuNPs-labeled capture probe and the signal probe with the cDNA, and consequently the optical density of the reaction mixture (AuNPs) was reduced proportionally to the cDNA concentration. The limit of detection was measured at 0.5 nM.

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

Science.gov (United States)

Leichty, Aaron R; Brisson, Dustin

2014-10-01

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

Getting complete genomes from complex samples using nanopore sequencing

DEFF Research Database (Denmark)

Kirkegaard, Rasmus Hansen; Karst, Søren Michael; Albertsen, Mads

Short read sequencing and metagenomic binning workflows have made it possible to extract bacterial genome bins from environmental microbial samples containing hundreds to thousands of different species. However, these genome bins often do not represent complete genomes, as they are mostly...... fragmented, incomplete and often contaminated with foreign DNA and with no robust strategies to validate the quality. The value of these `draft genomes` have limited, lasting value to the scientific community, as gene synteny is broken and the uncertainty of what is missing. The genetic material most often...... missed is important multi-copy and/or conserved marker genes such as the 16S rRNA gene, as sequence micro-heterogeneity prevents assembly of these genes in the de novo assembly. We demonstrate that using nanopore long reads it is now possible to overcome these issues and make complete genomes from...

Getting complete genomes from complex samples using nanopore sequencing

DEFF Research Database (Denmark)

Kirkegaard, Rasmus Hansen; Karst, Søren Michael; Albertsen, Mads

Background Short read DNA sequencing and metagenomic binning workflows have made it possible to extract bacterial genome bins from environmental microbial samples containing hundreds to thousands of different species. However, these genome bins often do not represent complete genomes......, as they are mostly fragmented, incomplete and often contaminated with foreign DNA. The value of these `draft genomes` have limited, lasting value to the scientific community, as gene synteny is broken and there is some uncertainty of what is missing1. The genetic material most often missed is important multi......-copy and/or conserved marker genes such as the 16S rRNA gene, as sequence micro-heterogeneity prevents assembly of these genes in the de novo assembly. However, long read sequencing technologies are emerging promising an end to fragmented genome assemblies2. Experimental design We extracted DNA from a full...

Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes.

Science.gov (United States)

Nielsen, H Bjørn; Almeida, Mathieu; Juncker, Agnieszka Sierakowska; Rasmussen, Simon; Li, Junhua; Sunagawa, Shinichi; Plichta, Damian R; Gautier, Laurent; Pedersen, Anders G; Le Chatelier, Emmanuelle; Pelletier, Eric; Bonde, Ida; Nielsen, Trine; Manichanh, Chaysavanh; Arumugam, Manimozhiyan; Batto, Jean-Michel; Quintanilha Dos Santos, Marcelo B; Blom, Nikolaj; Borruel, Natalia; Burgdorf, Kristoffer S; Boumezbeur, Fouad; Casellas, Francesc; Doré, Joël; Dworzynski, Piotr; Guarner, Francisco; Hansen, Torben; Hildebrand, Falk; Kaas, Rolf S; Kennedy, Sean; Kristiansen, Karsten; Kultima, Jens Roat; Léonard, Pierre; Levenez, Florence; Lund, Ole; Moumen, Bouziane; Le Paslier, Denis; Pons, Nicolas; Pedersen, Oluf; Prifti, Edi; Qin, Junjie; Raes, Jeroen; Sørensen, Søren; Tap, Julien; Tims, Sebastian; Ussery, David W; Yamada, Takuji; Renault, Pierre; Sicheritz-Ponten, Thomas; Bork, Peer; Wang, Jun; Brunak, Søren; Ehrlich, S Dusko

2014-08-01

Most current approaches for analyzing metagenomic data rely on comparisons to reference genomes, but the microbial diversity of many environments extends far beyond what is covered by reference databases. De novo segregation of complex metagenomic data into specific biological entities, such as particular bacterial strains or viruses, remains a largely unsolved problem. Here we present a method, based on binning co-abundant genes across a series of metagenomic samples, that enables comprehensive discovery of new microbial organisms, viruses and co-inherited genetic entities and aids assembly of microbial genomes without the need for reference sequences. We demonstrate the method on data from 396 human gut microbiome samples and identify 7,381 co-abundance gene groups (CAGs), including 741 metagenomic species (MGS). We use these to assemble 238 high-quality microbial genomes and identify affiliations between MGS and hundreds of viruses or genetic entities. Our method provides the means for comprehensive profiling of the diversity within complex metagenomic samples.

Sample Preparation for Electron Probe Microanalysis—Pushing the Limits

Science.gov (United States)

Geller, Joseph D.; Engle, Paul D.

2002-01-01

There are two fundamental considerations in preparing samples for electron probe microanalysis (EPMA). The first one may seem obvious, but we often find it is overlooked. That is, the sample analyzed should be representative of the population from which it comes. The second is a direct result of the assumptions in the calculations used to convert x-ray intensity ratios, between the sample and standard, to concentrations. Samples originate from a wide range of sources. During their journey to being excited under the electron beam for the production of x rays there are many possibilities for sample alteration. Handling can contaminate samples by adding extraneous matter. In preparation, the various abrasives used in sizing the sample by sawing, grinding and polishing can embed themselves. The most accurate composition of a contaminated sample is, at best, not representative of the original sample; it is misleading. Our laboratory performs EPMA analysis on customer submitted samples and prepares over 250 different calibration standards including pure elements, compounds, alloys, glasses and minerals. This large variety of samples does not lend itself to mass production techniques, including automatic polishing. Our manual preparation techniques are designed individually for each sample. The use of automated preparation equipment does not lend itself to this environment, and is not included in this manuscript. The final step in quantitative electron probe microanalysis is the conversion of x-ray intensities ratios, known as the “k-ratios,” to composition (in mass fraction or atomic percent) and/or film thickness. Of the many assumptions made in the ZAF (where these letters stand for atomic number, absorption and fluorescence) corrections the localized geometry between the sample and electron beam, or takeoff angle, must be accurately known. Small angular errors can lead to significant errors in the final results. The sample preparation technique then becomes very

Using genomic DNA-based probe-selection to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species

Directory of Open Access Journals (Sweden)

Townsend Henrik J

2005-11-01

Full Text Available Abstract High-density oligonucleotide (oligo arrays are a powerful tool for transcript profiling. Arrays based on GeneChip® technology are amongst the most widely used, although GeneChip® arrays are currently available for only a small number of plant and animal species. Thus, we have developed a method to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species and tested the method by analysing the transcriptome of Brassica oleracea L., a species for which no GeneChip® array is available, using a GeneChip® array designed for Arabidopsis thaliana (L. Heynh. Genomic DNA from B. oleracea was labelled and hybridised to the ATH1-121501 GeneChip® array. Arabidopsis thaliana probe-pairs that hybridised to the B. oleracea genomic DNA on the basis of the perfect-match (PM probe signal were then selected for subsequent B. oleracea transcriptome analysis using a .cel file parser script to generate probe mask files. The transcriptional response of B. oleracea to a mineral nutrient (phosphorus; P stress was quantified using probe mask files generated for a wide range of gDNA hybridisation intensity thresholds. An example probe mask file generated with a gDNA hybridisation intensity threshold of 400 removed > 68 % of the available PM probes from the analysis but retained >96 % of available A. thaliana probe-sets. Ninety-nine of these genes were then identified as significantly regulated under P stress in B. oleracea, including the homologues of P stress responsive genes in A. thaliana. Increasing the gDNA hybridisation intensity thresholds up to 500 for probe-selection increased the sensitivity of the GeneChip® array to detect regulation of gene expression in B. oleracea under P stress by up to 13-fold. Our open-source software to create probe mask files is freely available http://affymetrix.arabidopsis.info/xspecies/ and may be used to facilitate transcriptomic analyses of a wide range of plant and animal

Detection of short repeated genomic sequences on metaphase chromosomes using padlock probes and target primed rolling circle DNA synthesis

Directory of Open Access Journals (Sweden)

Stougaard Magnus

2007-11-01

Full Text Available Abstract Background In situ detection of short sequence elements in genomic DNA requires short probes with high molecular resolution and powerful specific signal amplification. Padlock probes can differentiate single base variations. Ligated padlock probes can be amplified in situ by rolling circle DNA synthesis and detected by fluorescence microscopy, thus enhancing PRINS type reactions, where localized DNA synthesis reports on the position of hybridization targets, to potentially reveal the binding of single oligonucleotide-size probe molecules. Such a system has been presented for the detection of mitochondrial DNA in fixed cells, whereas attempts to apply rolling circle detection to metaphase chromosomes have previously failed, according to the literature. Methods Synchronized cultured cells were fixed with methanol/acetic acid to prepare chromosome spreads in teflon-coated diagnostic well-slides. Apart from the slide format and the chromosome spreading everything was done essentially according to standard protocols. Hybridization targets were detected in situ with padlock probes, which were ligated and amplified using target primed rolling circle DNA synthesis, and detected by fluorescence labeling. Results An optimized protocol for the spreading of condensed metaphase chromosomes in teflon-coated diagnostic well-slides was developed. Applying this protocol we generated specimens for target primed rolling circle DNA synthesis of padlock probes recognizing a 40 nucleotide sequence in the male specific repetitive satellite I sequence (DYZ1 on the Y-chromosome and a 32 nucleotide sequence in the repetitive kringle IV domain in the apolipoprotein(a gene positioned on the long arm of chromosome 6. These targets were detected with good efficiency, but the efficiency on other target sites was unsatisfactory. Conclusion Our aim was to test the applicability of the method used on mitochondrial DNA to the analysis of nuclear genomes, in particular as

An Optimized Set of Fluorescence In Situ Hybridization Probes for Detection of Pancreatobiliary Tract Cancer in Cytology Brush Samples.

Science.gov (United States)

Barr Fritcher, Emily G; Voss, Jesse S; Brankley, Shannon M; Campion, Michael B; Jenkins, Sarah M; Keeney, Matthew E; Henry, Michael R; Kerr, Sarah M; Chaiteerakij, Roongruedee; Pestova, Ekaterina V; Clayton, Amy C; Zhang, Jun; Roberts, Lewis R; Gores, Gregory J; Halling, Kevin C; Kipp, Benjamin R

2015-12-01

Pancreatobiliary cancer is detected by fluorescence in situ hybridization (FISH) of pancreatobiliary brush samples with UroVysion probes, originally designed to detect bladder cancer. We designed a set of new probes to detect pancreatobiliary cancer and compared its performance with that of UroVysion and routine cytology analysis. We tested a set of FISH probes on tumor tissues (cholangiocarcinoma or pancreatic carcinoma) and non-tumor tissues from 29 patients. We identified 4 probes that had high specificity for tumor vs non-tumor tissues; we called this set of probes pancreatobiliary FISH. We performed a retrospective analysis of brush samples from 272 patients who underwent endoscopic retrograde cholangiopancreatography for evaluation of malignancy at the Mayo Clinic; results were available from routine cytology and FISH with UroVysion probes. Archived residual specimens were retrieved and used to evaluate the pancreatobiliary FISH probes. Cutoff values for FISH with the pancreatobiliary probes were determined using 89 samples and validated in the remaining 183 samples. Clinical and pathologic evidence of malignancy in the pancreatobiliary tract within 2 years of brush sample collection was used as the standard; samples from patients without malignancies were used as negative controls. The validation cohort included 85 patients with malignancies (46.4%) and 114 patients with primary sclerosing cholangitis (62.3%). Samples containing cells above the cutoff for polysomy (copy number gain of ≥2 probes) were classified as positive in FISH with the UroVysion and pancreatobiliary probes. Multivariable logistic regression was used to estimate associations between clinical and pathology findings and results from FISH. The combination of FISH probes 1q21, 7p12, 8q24, and 9p21 identified cancer cells with 93% sensitivity and 100% specificity in pancreatobiliary tissue samples and were therefore included in the pancreatobiliary probe set. In the validation cohort of

High-throughput automated microfluidic sample preparation for accurate microbial genomics.

Science.gov (United States)

Kim, Soohong; De Jonghe, Joachim; Kulesa, Anthony B; Feldman, David; Vatanen, Tommi; Bhattacharyya, Roby P; Berdy, Brittany; Gomez, James; Nolan, Jill; Epstein, Slava; Blainey, Paul C

2017-01-27

Low-cost shotgun DNA sequencing is transforming the microbial sciences. Sequencing instruments are so effective that sample preparation is now the key limiting factor. Here, we introduce a microfluidic sample preparation platform that integrates the key steps in cells to sequence library sample preparation for up to 96 samples and reduces DNA input requirements 100-fold while maintaining or improving data quality. The general-purpose microarchitecture we demonstrate supports workflows with arbitrary numbers of reaction and clean-up or capture steps. By reducing the sample quantity requirements, we enabled low-input (∼10,000 cells) whole-genome shotgun (WGS) sequencing of Mycobacterium tuberculosis and soil micro-colonies with superior results. We also leveraged the enhanced throughput to sequence ∼400 clinical Pseudomonas aeruginosa libraries and demonstrate excellent single-nucleotide polymorphism detection performance that explained phenotypically observed antibiotic resistance. Fully-integrated lab-on-chip sample preparation overcomes technical barriers to enable broader deployment of genomics across many basic research and translational applications.

A Targeted Enrichment Strategy for Massively Parallel Sequencing of Angiosperm Plastid Genomes

Directory of Open Access Journals (Sweden)

Gregory W. Stull

2013-02-01

Full Text Available Premise of the study: We explored a targeted enrichment strategy to facilitate rapid and low-cost next-generation sequencing (NGS of numerous complete plastid genomes from across the phylogenetic breadth of angiosperms. Methods and Results: A custom RNA probe set including the complete sequences of 22 previously sequenced eudicot plastomes was designed to facilitate hybridization-based targeted enrichment of eudicot plastid genomes. Using this probe set and an Agilent SureSelect targeted enrichment kit, we conducted an enrichment experiment including 24 angiosperms (22 eudicots, two monocots, which were subsequently sequenced on a single lane of the Illumina GAIIx with single-end, 100-bp reads. This approach yielded nearly complete to complete plastid genomes with exceptionally high coverage (mean coverage: 717×, even for the two monocots. Conclusions: Our enrichment experiment was highly successful even though many aspects of the capture process employed were suboptimal. Hence, significant improvements to this methodology are feasible. With this general approach and probe set, it should be possible to sequence more than 300 essentially complete plastid genomes in a single Illumina GAIIx lane (achieving 50× mean coverage. However, given the complications of pooling numerous samples for multiplex sequencing and the limited number of barcodes (e.g., 96 available in commercial kits, we recommend 96 samples as a current practical maximum for multiplex plastome sequencing. This high-throughput approach should facilitate large-scale plastid genome sequencing at any level of phylogenetic diversity in angiosperms.

Genotyping-by-sequencing for Populus population genomics: an assessment of genome sampling patterns and filtering approaches.

Directory of Open Access Journals (Sweden)

Martin P Schilling

Full Text Available Continuing advances in nucleotide sequencing technology are inspiring a suite of genomic approaches in studies of natural populations. Researchers are faced with data management and analytical scales that are increasing by orders of magnitude. With such dramatic advances comes a need to understand biases and error rates, which can be propagated and magnified in large-scale data acquisition and processing. Here we assess genomic sampling biases and the effects of various population-level data filtering strategies in a genotyping-by-sequencing (GBS protocol. We focus on data from two species of Populus, because this genus has a relatively small genome and is emerging as a target for population genomic studies. We estimate the proportions and patterns of genomic sampling by examining the Populus trichocarpa genome (Nisqually-1, and demonstrate a pronounced bias towards coding regions when using the methylation-sensitive ApeKI restriction enzyme in this species. Using population-level data from a closely related species (P. tremuloides, we also investigate various approaches for filtering GBS data to retain high-depth, informative SNPs that can be used for population genetic analyses. We find a data filter that includes the designation of ambiguous alleles resulted in metrics of population structure and Hardy-Weinberg equilibrium that were most consistent with previous studies of the same populations based on other genetic markers. Analyses of the filtered data (27,910 SNPs also resulted in patterns of heterozygosity and population structure similar to a previous study using microsatellites. Our application demonstrates that technically and analytically simple approaches can readily be developed for population genomics of natural populations.

Sensitivity study of micro four-point probe measurements on small samples

DEFF Research Database (Denmark)

Wang, Fei; Petersen, Dirch Hjorth; Hansen, Torben Mikael

2010-01-01

probes than near the outer ones. The sensitive area is defined for infinite film, circular, square, and rectangular test pads, and convergent sensitivities are observed for small samples. The simulations show that the Hall sheet resistance RH in micro Hall measurements with position error suppression...

A workflow to preserve genome-quality tissue samples from plants in botanical gardens and arboreta1

Science.gov (United States)

Gostel, Morgan R.; Kelloff, Carol; Wallick, Kyle; Funk, Vicki A.

2016-01-01

Premise of the study: Internationally, gardens hold diverse living collections that can be preserved for genomic research. Workflows have been developed for genomic tissue sampling in other taxa (e.g., vertebrates), but are inadequate for plants. We outline a workflow for tissue sampling intended for two audiences: botanists interested in genomics research and garden staff who plan to voucher living collections. Methods and Results: Standard herbarium methods are used to collect vouchers, label information and images are entered into a publicly accessible database, and leaf tissue is preserved in silica and liquid nitrogen. A five-step approach for genomic tissue sampling is presented for sampling from living collections according to current best practices. Conclusions: Collecting genome-quality samples from gardens is an economical and rapid way to make available for scientific research tissue from the diversity of plants on Earth. The Global Genome Initiative will facilitate and lead this endeavor through international partnerships. PMID:27672517

Detection of G-Quadruplex Structures Formed by G-Rich Sequences from Rice Genome and Transcriptome Using Combined Probes.

Science.gov (United States)

Chang, Tianjun; Li, Weiguo; Ding, Zhan; Cheng, Shaofei; Liang, Kun; Liu, Xiangjun; Bing, Tao; Shangguan, Dihua

2017-08-01

Putative G-quadruplex (G4) forming sequences (PQS) are highly prevalent in the genome and transcriptome of various organisms and are considered as potential regulation elements in many biological processes by forming G4 structures. The formation of G4 structures highly depends on the sequences and the environment. In most cases, it is difficult to predict G4 formation by PQS, especially PQS containing G2 tracts. Therefore, the experimental identification of G4 formation is essential in the study of G4-related biological functions. Herein, we report a rapid and simple method for the detection of G4 structures by using a pair of complementary reporters, hemin and BMSP. This method was applied to detect G4 structures formed by PQS (DNA and RNA) searched in the genome and transcriptome of Oryza sativa. Unlike most of the reported G4 probes that only recognize part of G4 structures, the proposed method based on combined probes positively responded to almost all G4 conformations, including parallel, antiparallel, and mixed/hybrid G4, but did not respond to non-G4 sequences. This method shows potential for high-throughput identification of G4 structures in genome and transcriptome. Furthermore, BMSP was observed to drive some PQS to form more stable G4 structures or induce the G4 formation of some PQS that cannot form G4 in normal physiological conditions, which may provide a powerful molecular tool for gene regulation.

Assessing probe-specific dye and slide biases in two-color microarray data

Directory of Open Access Journals (Sweden)

Goldberg Zelanna

2008-07-01

Full Text Available Abstract Background A primary reason for using two-color microarrays is that the use of two samples labeled with different dyes on the same slide, that bind to probes on the same spot, is supposed to adjust for many factors that introduce noise and errors into the analysis. Most users assume that any differences between the dyes can be adjusted out by standard methods of normalization, so that measures such as log ratios on the same slide are reliable measures of comparative expression. However, even after the normalization, there are still probe specific dye and slide variation among the data. We define a method to quantify the amount of the dye-by-probe and slide-by-probe interaction. This serves as a diagnostic, both visual and numeric, of the existence of probe-specific dye bias. We show how this improved the performance of two-color array analysis for arrays for genomic analysis of biological samples ranging from rice to human tissue. Results We develop a procedure for quantifying the extent of probe-specific dye and slide bias in two-color microarrays. The primary output is a graphical diagnostic of the extent of the bias which called ECDF (Empirical Cumulative Distribution Function, though numerical results are also obtained. Conclusion We show that the dye and slide biases were high for human and rice genomic arrays in two gene expression facilities, even after the standard intensity-based normalization, and describe how this diagnostic allowed the problems causing the probe-specific bias to be addressed, and resulted in important improvements in performance. The R package LMGene which contains the method described in this paper has been available to download from Bioconductor.

Minimum detection limit and spatial resolution of thin-sample field-emission electron probe microanalysis

International Nuclear Information System (INIS)

Kubo, Yugo; Hamada, Kotaro; Urano, Akira

2013-01-01

The minimum detection limit and spatial resolution for a thinned semiconductor sample were determined by electron probe microanalysis (EPMA) using a Schottky field emission (FE) electron gun and wavelength dispersive X-ray spectrometry. Comparison of the FE-EPMA results with those obtained using energy dispersive X-ray spectrometry in conjunction with scanning transmission electron microscopy, confirmed that FE-EPMA is largely superior in terms of detection sensitivity. Thin-sample FE-EPMA is demonstrated as a very effective method for high resolution, high sensitivity analysis in a laboratory environment because a high probe current and high signal-to-noise ratio can be achieved. - Highlights: • Minimum detection limit and spatial resolution determined for FE-EPMA. • Detection sensitivity of FE-EPMA greatly superior to that of STEM-EDX. • Minimum detection limit and spatial resolution controllable by probe current

Report for Development of a Census Array and Evaluation of the Array to Detect Biothreat Agents and Environmental Samples for DHS

Energy Technology Data Exchange (ETDEWEB)

Jaing, C; Jackson, P

2011-04-14

The objective of this project is to provide DHS a comprehensive evaluation of the current genomic technologies including genotyping, Taqman PCR, multiple locus variable tandem repeat analysis (MLVA), microarray and high-throughput DNA sequencing in the analysis of biothreat agents from complex environmental samples. This report focuses on the design, testing and results of samples on the Census Array. We designed a Census/Detection Array to detect all sequenced viruses (including phage), bacteria (eubacteria), and plasmids. Family-specific probes were selected for all sequenced viral and bacterial complete genomes, segments, and plasmids. Probes were designed to tolerate some sequence variation to enable detection of divergent species with homology to sequenced organisms, and to be unique relative to the human genome. A combination of 'detection' probes with high levels of conservation within a family plus 'census' probes targeting strain/isolate specific regions enabled detection and taxonomic classification from the level of family down to the strain. The array has wider coverage of bacterial and viral targets based on more recent sequence data and more probes per target than other microbial detection/discovery arrays in the literature. We tested the array with purified bacterial and viral DNA/RNA samples, artificial mixes of known bacterial/viral samples, spiked DNA against complex background including BW aerosol samples and soil samples, and environmental samples to evaluate the array's sensitivity and forensic capability. The data were analyzed using our novel maximum likelihood software. For most of the organisms tested, we have achieved at least species level discrimination.

A direct detection of Escherichia coli genomic DNA using gold nanoprobes

Directory of Open Access Journals (Sweden)

Padmavathy

2012-02-01

Full Text Available Abstract Background In situation like diagnosis of clinical and forensic samples there exists a need for highly sensitive, rapid and specific DNA detection methods. Though conventional DNA amplification using PCR can provide fast results, it is not widely practised in diagnostic laboratories partially because it requires skilled personnel and expensive equipment. To overcome these limitations nanoparticles have been explored as signalling probes for ultrasensitive DNA detection that can be used in field applications. Among the nanomaterials, gold nanoparticles (AuNPs have been extensively used mainly because of its optical property and ability to get functionalized with a variety of biomolecules. Results We report a protocol for the use of gold nanoparticles functionalized with single stranded oligonucleotide (AuNP- oligo probe as visual detection probes for rapid and specific detection of Escherichia coli. The AuNP- oligo probe on hybridization with target DNA containing complementary sequences remains red whereas test samples without complementary DNA sequences to the probe turns purple due to acid induced aggregation of AuNP- oligo probes. The color change of the solution is observed visually by naked eye demonstrating direct and rapid detection of the pathogenic Escherichia coli from its genomic DNA without the need for PCR amplification. The limit of detection was ~54 ng for unamplified genomic DNA. The method requires less than 30 minutes to complete after genomic DNA extraction. However, by using unamplified enzymatic digested genomic DNA, the detection limit of 11.4 ng was attained. Results of UV-Vis spectroscopic measurement and AFM imaging further support the hypothesis of aggregation based visual discrimination. To elucidate its utility in medical diagnostic, the assay was validated on clinical strains of pathogenic Escherichia coli obtained from local hospitals and spiked urine samples. It was found to be 100% sensitive and proves to

Probe-diverse ptychography

Energy Technology Data Exchange (ETDEWEB)

Peterson, I., E-mail: isaac.russellpeterson@rmit.edu.au [ARC Centre of Excellence for Coherent X-ray Science, the University of Melbourne, School of Physics, Victoria 3010 (Australia); Harder, R. [Advanced Photon Source, Argonne National Laboratory, Argonne, IL 60439 (United States); Robinson, I.K. [Research Complex at Harwell, Didcot, Oxfordshire OX11 0DE (United Kingdom); London Centre for Nanotechnology, University College London, London WC1H 0AH (United Kingdom)

2016-12-15

We propose an extension of ptychography where the target sample is scanned separately through several probes with distinct amplitude and phase profiles and a diffraction image is recorded for each probe and each sample translation. The resulting probe-diverse dataset is used to iteratively retrieve high-resolution images of the sample and all probes simultaneously. The method is shown to yield significant improvement in the reconstructed sample image compared to the image obtained using the standard single-probe ptychographic phase-retrieval scheme.

High-density rhesus macaque oligonucleotide microarray design using early-stage rhesus genome sequence information and human genome annotations

Directory of Open Access Journals (Sweden)

Magness Charles L

2007-01-01

Full Text Available Abstract Background Until recently, few genomic reagents specific for non-human primate research have been available. To address this need, we have constructed a macaque-specific high-density oligonucleotide microarray by using highly fragmented low-pass sequence contigs from the rhesus genome project together with the detailed sequence and exon structure of the human genome. Using this method, we designed oligonucleotide probes to over 17,000 distinct rhesus/human gene orthologs and increased by four-fold the number of available genes relative to our first-generation expressed sequence tag (EST-derived array. Results We constructed a database containing 248,000 exon sequences from 23,000 human RefSeq genes and compared each human exon with its best matching sequence in the January 2005 version of the rhesus genome project list of 486,000 DNA contigs. Best matching rhesus exon sequences for each of the 23,000 human genes were then concatenated in the proper order and orientation to produce a rhesus "virtual transcriptome." Microarray probes were designed, one per gene, to the region closest to the 3' untranslated region (UTR of each rhesus virtual transcript. Each probe was compared to a composite rhesus/human transcript database to test for cross-hybridization potential yielding a final probe set representing 18,296 rhesus/human gene orthologs, including transcript variants, and over 17,000 distinct genes. We hybridized mRNA from rhesus brain and spleen to both the EST- and genome-derived microarrays. Besides four-fold greater gene coverage, the genome-derived array also showed greater mean signal intensities for genes present on both arrays. Genome-derived probes showed 99.4% identity when compared to 4,767 rhesus GenBank sequence tag site (STS sequences indicating that early stage low-pass versions of complex genomes are of sufficient quality to yield valuable functional genomic information when combined with finished genome information from

Open Probe fast GC-MS - combining ambient sampling ultra-fast separation and in-vacuum ionization for real-time analysis.

Science.gov (United States)

Keshet, U; Alon, T; Fialkov, A B; Amirav, A

2017-07-01

An Open Probe inlet was combined with a low thermal mass ultra-fast gas chromatograph (GC), in-vacuum electron ionization ion source and a mass spectrometer (MS) of GC-MS for obtaining real-time analysis with separation. The Open Probe enables ambient sampling via sample vaporization in an oven that is open to room air, and the ultra-fast GC provides ~30-s separation, while if no separation is required, it can act as a transfer line with 2 to 3-s sample transfer time. Sample analysis is as simple as touching the sample, pushing the sample holder into the Open Probe oven and obtaining the results in 30 s. The Open Probe fast GC was mounted on a standard Agilent 7890 GC that was coupled with an Agilent 5977A MS. Open Probe fast GC-MS provides real-time analysis combined with GC separation and library identification, and it uses the low-cost MS of GC-MS. The operation of Open Probe fast GC-MS is demonstrated in the 30-s separation and 50-s full analysis cycle time of tetrahydrocannabinol and cannabinol in Cannabis flower, sub 1-min analysis of trace trinitrotoluene transferred from a finger onto a glass surface, vitamin E in canola oil, sterols in olive oil, polybrominated flame retardants in plastics, alprazolam in Xanax drug pill and free fatty acids and cholesterol in human blood. The extrapolated limit of detection for pyrene is Open Probe fast GC-MS is demonstrated in the analysis of heroin in its street drug powder. The use of Open Probe with the fast GC acting as a transfer line is demonstrated in <10-s analysis without separation of ibuprofen and estradiol. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Application of synthetic DNA probes to the analysis of DNA sequence variants in man

International Nuclear Information System (INIS)

Wallace, R.B.; Petz, L.D.; Yam, P.Y.

1986-01-01

Oligonucleotide probes provide a tool to discriminate between any two alleles on the basis of hybridization. Random sampling of the genome with different oligonucleotide probes should reveal polymorphism in a certain percentage of the cases. In the hope of identifying polymorphic regions more efficiently, we chose to take advantage of the proposed hypermutability of repeated DNA sequences and the specificity of oligonucleotide hybridization. Since, under appropriate conditions, oligonucleotide probes require complete base pairing for hybridization to occur, they will only hybridize to a subset of the members of a repeat family when all members of the family are not identical. The results presented here suggest that oligonucleotide hybridization can be used to extend the genomic sequences that can be tested for the presence of RFLPs. This expands the tools available to human genetics. In addition, the results suggest that repeated DNA sequences are indeed more polymorphic than single-copy sequences. 28 references, 2 figures

Mass amplifying probe for sensitive fluorescence anisotropy detection of small molecules in complex biological samples.

Science.gov (United States)

Cui, Liang; Zou, Yuan; Lin, Ninghang; Zhu, Zhi; Jenkins, Gareth; Yang, Chaoyong James

2012-07-03

Fluorescence anisotropy (FA) is a reliable and excellent choice for fluorescence sensing. One of the key factors influencing the FA value for any molecule is the molar mass of the molecule being measured. As a result, the FA method with functional nucleic acid aptamers has been limited to macromolecules such as proteins and is generally not applicable for the analysis of small molecules because their molecular masses are relatively too small to produce observable FA value changes. We report here a molecular mass amplifying strategy to construct anisotropy aptamer probes for small molecules. The probe is designed in such a way that only when a target molecule binds to the probe does it activate its binding ability to an anisotropy amplifier (a high molecular mass molecule such as protein), thus significantly increasing the molecular mass and FA value of the probe/target complex. Specifically, a mass amplifying probe (MAP) consists of a targeting aptamer domain against a target molecule and molecular mass amplifying aptamer domain for the amplifier protein. The probe is initially rendered inactive by a small blocking strand partially complementary to both target aptamer and amplifier protein aptamer so that the mass amplifying aptamer domain would not bind to the amplifier protein unless the probe has been activated by the target. In this way, we prepared two probes that constitute a target (ATP and cocaine respectively) aptamer, a thrombin (as the mass amplifier) aptamer, and a fluorophore. Both probes worked well against their corresponding small molecule targets, and the detection limits for ATP and cocaine were 0.5 μM and 0.8 μM, respectively. More importantly, because FA is less affected by environmental interferences, ATP in cell media and cocaine in urine were directly detected without any tedious sample pretreatment. Our results established that our molecular mass amplifying strategy can be used to design aptamer probes for rapid, sensitive, and selective

Measurement of locus copy number by hybridisation with amplifiable probes

Science.gov (United States)

Armour, John A. L.; Sismani, Carolina; Patsalis, Philippos C.; Cross, Gareth

2000-01-01

Despite its fundamental importance in genome analysis, it is only recently that systematic approaches have been developed to assess copy number at specific genetic loci, or to examine genomic DNA for submicroscopic deletions of unknown location. In this report we show that short probes can be recovered and amplified quantitatively following hybridisation to genomic DNA. This simple observation forms the basis of a new approach to determining locus copy number in complex genomes. The power and specificity of multiplex amplifiable probe hybridisation is demonstrated by the simultaneous assessment of copy number at a set of 40 human loci, including detection of deletions causing Duchenne muscular dystrophy and Prader–Willi/Angelman syndromes. Assembly of other probe sets will allow novel, technically simple approaches to a wide variety of genetic analyses, including the potential for extension to high resolution genome-wide screens for deletions and amplifications. PMID:10606661

Measurement of locus copy number by hybridisation with amplifiable probes.

Science.gov (United States)

Armour, J A; Sismani, C; Patsalis, P C; Cross, G

2000-01-15

Despite its fundamental importance in genome analysis, it is only recently that systematic approaches have been developed to assess copy number at specific genetic loci, or to examine genomic DNA for submicro-scopic deletions of unknown location. In this report we show that short probes can be recovered and amplified quantitatively following hybridisation to genomic DNA. This simple observation forms the basis of a new approach to determining locus copy number in complex genomes. The power and specificity of multiplex amplifiable probe hybridisation is demonstrated by the simultaneous assessment of copy number at a set of 40 human loci, including detection of deletions causing Duchenne muscular dystrophy and Prader-Willi/Angelman syndromes. Assembly of other probe sets will allow novel, technically simple approaches to a wide variety of genetic analyses, including the potential for extension to high resolution genome-wide screens for deletions and amplifications.

Quantification of multielement-multilayer-samples in electron probe analysis

International Nuclear Information System (INIS)

Pfeiffer, A.

1995-03-01

The following dissertation presents the theoretical basis of analytical correction models and Monte Carlo simulations in the field of electron probe microanalysis to describe the excitation conditions of x-rays in a multilayer-multielement-sample. In this connection analyzing programs have been developed to make a quantitative investigation of heterogeneous samples possible. In the work the mathematical methods and formulas, which are mainly based on empirical and semiempirical findings, are described and their validity is discussed in detail. Especially the improvements of the 'multiple reflections'-model by August are compared with the Φ(ρz)-models by Pouchou, Merlet and Bastin. The calculations of depth distribution functions for characteristics and continuous fluorescence excitation result in a consistent and completeΦ(ρz)-model. This allows to analyze layered structures in great detail. Because of the increasing importance in electron probe microanalysis and as a reference method a Monte Carlo model is described. With this model electron trajectories and excitation conditions in arbitrary two dimensional geometries can be calculated. The validity of the analytical model is proven with a comprehensive comparison of results of new calculations to published data. To show an application of the programs and models in routine use in the industrial research and development, a quantitative analysis of a Co/Si system is made. In the conclusion of this dissertation some reflections upon investigations, which are based on this work and which should be made in future are outlined. (author)

Gene probes: principles and protocols

National Research Council Canada - National Science Library

Aquino de Muro, Marilena; Rapley, Ralph

2002-01-01

... of labeled DNA has allowed genes to be mapped to single chromosomes and in many cases to a single chromosome band, promoting significant advance in human genome mapping. Gene Probes: Principles and Protocols presents the principles for gene probe design, labeling, detection, target format, and hybridization conditions together with detailed protocols, accom...

Quantification of different Eubacterium spp. in human fecal samples with species-specific 16S rRNA-targeted oligonucleotide probes.

Science.gov (United States)

Schwiertz, A; Le Blay, G; Blaut, M

2000-01-01

Species-specific 16S rRNA-targeted, Cy3 (indocarbocyanine)-labeled oligonucleotide probes were designed and validated to quantify different Eubacterium species in human fecal samples. Probes were directed at Eubacterium barkeri, E. biforme, E. contortum, E. cylindroides (two probes), E. dolichum, E. hadrum, E. lentum, E. limosum, E. moniliforme, and E. ventriosum. The specificity of the probes was tested with the type strains and a range of common intestinal bacteria. With one exception, none of the probes showed cross-hybridization under stringent conditions. The species-specific probes were applied to fecal samples obtained from 12 healthy volunteers. E. biforme, E. cylindroides, E. hadrum, E. lentum, and E. ventriosum could be determined. All other Eubacterium species for which probes had been designed were under the detection limit of 10(7) cells g (dry weight) of feces(-1). The cell counts obtained are essentially in accordance with the literature data, which are based on colony counts. This shows that whole-cell in situ hybridization with species-specific probes is a valuable tool for the enumeration of Eubacterium species in feces.

Group normalization for genomic data.

Science.gov (United States)

Ghandi, Mahmoud; Beer, Michael A

2012-01-01

Data normalization is a crucial preliminary step in analyzing genomic datasets. The goal of normalization is to remove global variation to make readings across different experiments comparable. In addition, most genomic loci have non-uniform sensitivity to any given assay because of variation in local sequence properties. In microarray experiments, this non-uniform sensitivity is due to different DNA hybridization and cross-hybridization efficiencies, known as the probe effect. In this paper we introduce a new scheme, called Group Normalization (GN), to remove both global and local biases in one integrated step, whereby we determine the normalized probe signal by finding a set of reference probes with similar responses. Compared to conventional normalization methods such as Quantile normalization and physically motivated probe effect models, our proposed method is general in the sense that it does not require the assumption that the underlying signal distribution be identical for the treatment and control, and is flexible enough to correct for nonlinear and higher order probe effects. The Group Normalization algorithm is computationally efficient and easy to implement. We also describe a variant of the Group Normalization algorithm, called Cross Normalization, which efficiently amplifies biologically relevant differences between any two genomic datasets.

Teolenn: an efficient and customizable workflow to design high-quality probes for microarray experiments

Science.gov (United States)

Jourdren, Laurent; Duclos, Aurélie; Brion, Christian; Portnoy, Thomas; Mathis, Hugues; Margeot, Antoine; Le Crom, Stéphane

2010-01-01

Despite the development of new high-throughput sequencing techniques, microarrays are still attractive tools to study small genome organisms, thanks to sample multiplexing and high-feature densities. However, the oligonucleotide design remains a delicate step for most users. A vast array of software is available to deal with this problem, but each program is developed with its own strategy, which makes the choice of the best solution difficult. Here we describe Teolenn, a universal probe design workflow developed with a flexible and customizable module organization allowing fixed or variable length oligonucleotide generation. In addition, our software is able to supply quality scores for each of the designed probes. In order to assess the relevance of these scores, we performed a real hybridization using a tiling array designed against the Trichoderma reesei fungus genome. We show that our scoring pipeline correlates with signal quality for 97.2% of all the designed probes, allowing for a posteriori comparisons between quality scores and signal intensities. This result is useful in discarding any bad scoring probes during the design step in order to get high-quality microarrays. Teolenn is available at http://transcriptome.ens.fr/teolenn/. PMID:20176570

Assessment of the LV-C2 Stack Sampling Probe Location for Compliance with ANSI/HPS N13.1-1999

Energy Technology Data Exchange (ETDEWEB)

Glissmeyer, John A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Antonio, Ernest J. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Flaherty, Julia E. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

2015-09-01

This document reports on a series of tests conducted to assess the proposed air sampling location for the Hanford Tank Waste Treatment and Immobilization Plant (WTP) Low-Activity Waste (LAW) C2V (LV-C2) exhaust stack with respect to the applicable criteria regarding the placement of an air sampling probe. Federal regulations require that a sampling probe be located in the exhaust stack according to the criteria established by the American National Standards Institute/Health Physics Society (ANSI/HPS) N13.1-1999, Sampling and Monitoring Releases of Airborne Radioactive Substances from the Stack and Ducts of Nuclear Facilities. These criteria address the capability of the sampling probe to extract a sample that represents the effluent stream. The tests were conducted on the LV-C2 scale model system. Based on the scale model tests, the location proposed for the air sampling probe in the scale model stack meets the requirements of the ANSI/HPS N13.1-1999 standard for velocity uniformity, flow angle, gas tracer and particle tracer uniformity. Additional velocity uniformity and flow angle tests on the actual stack will be necessary during cold startup to confirm the validity of the scale model results in representing the actual stack.

Genomic Sequencing of Single Microbial Cells from Environmental Samples

Energy Technology Data Exchange (ETDEWEB)

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

2008-02-01

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

Nucleic acid probes as a diagnostic method for tick-borne hemoparasites of veterinary importance.

Science.gov (United States)

Figueroa, J V; Buening, G M

1995-03-01

An increased number of articles on the use of nucleic acid-based hybridization techniques for diagnostic purposes have been recently published. This article reviews nucleic acid-based hybridization as an assay to detect hemoparasite infections of economic relevance in veterinary medicine. By using recombinant DNA techniques, selected clones containing inserts of Anaplasma, Babesia, Cowdria or Theileria genomic DNA sequences have been obtained, and they are now available to be utilized as specific, highly sensitive DNA or RNA probes to detect the presence of the hemoparasite DNA in an infected animal. Either in an isotopic or non-isotopic detection system, probes have allowed scientists to test for--originally in samples collected from experimentally infected animals and later in samples collected in the field--the presence of hemoparasites during the prepatent, patent, convalescent, and chronic periods of the infection in the host. Nucleic acid probes have given researchers the opportunity to carry out genomic analysis of parasite DNA to differentiate hemoparasite species and to identify genetically distinct populations among and within isolates, strains and clonal populations. Prevalence of parasite infection in the tick vector can now be accomplished more specifically with the nucleic acid probes. Lately, with the advent of the polymerase chain reaction technique, small numbers of hemoparasites can be positively identified in the vertebrate host and tick vector. These techniques can be used to assess the veterinary epidemiological situation in a particular geographical region for the planning of control measures.

A Prototype Ice-Melting Probe for Collecting Biological Samples from Cryogenic Ice at Low Pressure

Science.gov (United States)

Davis, Ashley

2017-08-01

In the Solar System, the surface of an icy moon is composed of irregular ice formations at cryogenic temperatures (pumps. The device contains a heated conical probe with a central orifice, which is forced into surface ice and directs the meltwater upward into a reservoir. The force on the probe is proportional to the height of meltwater (pressure) obtained in the system and allows regulation of the melt rate and temperature of the sample. The device can collect 5-50 mL of meltwater from the surface of an ice block at 233-208 K with an environmental pressure of less than 10-2 atm while maintaining a sample temperature between 273 and 293 K. These conditions maintain most biological samples in a pristine state and maintain the integrity of most organisms' structure and function.

Genome Sequence of Legionella massiliensis, Isolated from a Cooling Tower Water Sample.

Science.gov (United States)

Pagnier, Isabelle; Croce, Olivier; Robert, Catherine; Raoult, Didier; La Scola, Bernard

2014-10-16

We present the draft genome sequence of Legionella massiliensis strain LegA(T), recovered from a cooling tower water sample, using an amoebal coculture procedure. The strain described here is composed of 4,387,007 bp, with a G+C content of 41.19%, and its genome has 3,767 protein-coding genes and 60 predicted RNA genes. Copyright © 2014 Pagnier et al.

Fluorographene as a Mass Spectrometry Probe for High-Throughput Identification and Screening of Emerging Chemical Contaminants in Complex Samples.

Science.gov (United States)

Huang, Xiu; Liu, Qian; Huang, Xiaoyu; Nie, Zhou; Ruan, Ting; Du, Yuguo; Jiang, Guibin

2017-01-17

Mass spectrometry techniques for high-throughput analysis of complex samples are of profound importance in many areas such as food safety, omics studies, and environmental health science. Here we report the use of fluorographene (FG) as a new mass spectrometry probe for high-throughput identification and screening of emerging chemical contaminants in complex samples. FG was facilely synthesized by one-step exfoliation of fluorographite. With FG as a matrix or probe in matrix-assisted or surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (MALDI- or SELDI-TOF MS), higher sensitivity (detection limits at ppt or subppt levels), and better reproducibility were achieved than with other graphene-based materials due to the unique chemical structure and self-assembly properties of FG. The method was validated with different types of real complex samples. By using FG as a SELDI probe, we could easily detect trace amount of bisphenol S in paper products and high-fat canned food samples. Furthermore, we have successfully identified and screened as many as 28 quaternary ammonium halides in sewage sludge samples collected from municipal wastewater treatment plants. These results demonstrate that FG probe is a powerful tool for high-throughput analysis of complex samples by MS.

Sex genes for genomic analysis in human brain: internal controls for comparison of probe level data extraction.

Directory of Open Access Journals (Sweden)

Ellis Steven P

2003-09-01

Full Text Available Abstract Background Genomic studies of complex tissues pose unique analytical challenges for assessment of data quality, performance of statistical methods used for data extraction, and detection of differentially expressed genes. Ideally, to assess the accuracy of gene expression analysis methods, one needs a set of genes which are known to be differentially expressed in the samples and which can be used as a "gold standard". We introduce the idea of using sex-chromosome genes as an alternative to spiked-in control genes or simulations for assessment of microarray data and analysis methods. Results Expression of sex-chromosome genes were used as true internal biological controls to compare alternate probe-level data extraction algorithms (Microarray Suite 5.0 [MAS5.0], Model Based Expression Index [MBEI] and Robust Multi-array Average [RMA], to assess microarray data quality and to establish some statistical guidelines for analyzing large-scale gene expression. These approaches were implemented on a large new dataset of human brain samples. RMA-generated gene expression values were markedly less variable and more reliable than MAS5.0 and MBEI-derived values. A statistical technique controlling the false discovery rate was applied to adjust for multiple testing, as an alternative to the Bonferroni method, and showed no evidence of false negative results. Fourteen probesets, representing nine Y- and two X-chromosome linked genes, displayed significant sex differences in brain prefrontal cortex gene expression. Conclusion In this study, we have demonstrated the use of sex genes as true biological internal controls for genomic analysis of complex tissues, and suggested analytical guidelines for testing alternate oligonucleotide microarray data extraction protocols and for adjusting multiple statistical analysis of differentially expressed genes. Our results also provided evidence for sex differences in gene expression in the brain prefrontal cortex

In situ optical sequencing and structure analysis of a trinucleotide repeat genome region by localization microscopy after specific COMBO-FISH nano-probing

Science.gov (United States)

Stuhlmüller, M.; Schwarz-Finsterle, J.; Fey, E.; Lux, J.; Bach, M.; Cremer, C.; Hinderhofer, K.; Hausmann, M.; Hildenbrand, G.

2015-10-01

Trinucleotide repeat expansions (like (CGG)n) of chromatin in the genome of cell nuclei can cause neurological disorders such as for example the Fragile-X syndrome. Until now the mechanisms are not clearly understood as to how these expansions develop during cell proliferation. Therefore in situ investigations of chromatin structures on the nanoscale are required to better understand supra-molecular mechanisms on the single cell level. By super-resolution localization microscopy (Spectral Position Determination Microscopy; SPDM) in combination with nano-probing using COMBO-FISH (COMBinatorial Oligonucleotide FISH), novel insights into the nano-architecture of the genome will become possible. The native spatial structure of trinucleotide repeat expansion genome regions was analysed and optical sequencing of repetitive units was performed within 3D-conserved nuclei using SPDM after COMBO-FISH. We analysed a (CGG)n-expansion region inside the 5' untranslated region of the FMR1 gene. The number of CGG repeats for a full mutation causing the Fragile-X syndrome was found and also verified by Southern blot. The FMR1 promotor region was similarly condensed like a centromeric region whereas the arrangement of the probes labelling the expansion region seemed to indicate a loop-like nano-structure. These results for the first time demonstrate that in situ chromatin structure measurements on the nanoscale are feasible. Due to further methodological progress it will become possible to estimate the state of trinucleotide repeat mutations in detail and to determine the associated chromatin strand structural changes on the single cell level. In general, the application of the described approach to any genome region will lead to new insights into genome nano-architecture and open new avenues for understanding mechanisms and their relevance in the development of heredity diseases.

Group normalization for genomic data.

Directory of Open Access Journals (Sweden)

Mahmoud Ghandi

Full Text Available Data normalization is a crucial preliminary step in analyzing genomic datasets. The goal of normalization is to remove global variation to make readings across different experiments comparable. In addition, most genomic loci have non-uniform sensitivity to any given assay because of variation in local sequence properties. In microarray experiments, this non-uniform sensitivity is due to different DNA hybridization and cross-hybridization efficiencies, known as the probe effect. In this paper we introduce a new scheme, called Group Normalization (GN, to remove both global and local biases in one integrated step, whereby we determine the normalized probe signal by finding a set of reference probes with similar responses. Compared to conventional normalization methods such as Quantile normalization and physically motivated probe effect models, our proposed method is general in the sense that it does not require the assumption that the underlying signal distribution be identical for the treatment and control, and is flexible enough to correct for nonlinear and higher order probe effects. The Group Normalization algorithm is computationally efficient and easy to implement. We also describe a variant of the Group Normalization algorithm, called Cross Normalization, which efficiently amplifies biologically relevant differences between any two genomic datasets.

STM-SQUID probe microscope

International Nuclear Information System (INIS)

Hayashi, Tadayuki; Tachiki, Minoru; Itozaki, Hideo

2007-01-01

We have developed a STM-SQUID probe microscope. A high T C SQUID probe microscope was combined with a scanning tunneling microscope for investigation of samples at room temperature in air. A high permeability probe needle was used as a magnetic flux guide to improve the spatial resolution. The probe with tip radius of less than 100 nm was prepared by microelectropolishing. The probe was also used as a scanning tunneling microscope tip. Topography of the sample surface could be measured by the scanning tunneling microscope with high spatial resolution prior to observation by SQUID microscopy. The SQUID probe microscope image could be observed while keeping the distance from the sample surface to the probe tip constant. We observed a topographic image and a magnetic image of Ni fine pattern and also a magnetically recorded hard disk. Furthermore we have investigated a sample vibration method of the static magnetic field emanating from a sample with the aim of achieving a higher signal-to-noise (S/N) ratio

Systematic bias in genomic classification due to contaminating non-neoplastic tissue in breast tumor samples.

Science.gov (United States)

Elloumi, Fathi; Hu, Zhiyuan; Li, Yan; Parker, Joel S; Gulley, Margaret L; Amos, Keith D; Troester, Melissa A

2011-06-30

Genomic tests are available to predict breast cancer recurrence and to guide clinical decision making. These predictors provide recurrence risk scores along with a measure of uncertainty, usually a confidence interval. The confidence interval conveys random error and not systematic bias. Standard tumor sampling methods make this problematic, as it is common to have a substantial proportion (typically 30-50%) of a tumor sample comprised of histologically benign tissue. This "normal" tissue could represent a source of non-random error or systematic bias in genomic classification. To assess the performance characteristics of genomic classification to systematic error from normal contamination, we collected 55 tumor samples and paired tumor-adjacent normal tissue. Using genomic signatures from the tumor and paired normal, we evaluated how increasing normal contamination altered recurrence risk scores for various genomic predictors. Simulations of normal tissue contamination caused misclassification of tumors in all predictors evaluated, but different breast cancer predictors showed different types of vulnerability to normal tissue bias. While two predictors had unpredictable direction of bias (either higher or lower risk of relapse resulted from normal contamination), one signature showed predictable direction of normal tissue effects. Due to this predictable direction of effect, this signature (the PAM50) was adjusted for normal tissue contamination and these corrections improved sensitivity and negative predictive value. For all three assays quality control standards and/or appropriate bias adjustment strategies can be used to improve assay reliability. Normal tissue sampled concurrently with tumor is an important source of bias in breast genomic predictors. All genomic predictors show some sensitivity to normal tissue contamination and ideal strategies for mitigating this bias vary depending upon the particular genes and computational methods used in the predictor.

Quantification of Different Eubacterium spp. in Human Fecal Samples with Species-Specific 16S rRNA-Targeted Oligonucleotide Probes

OpenAIRE

Schwiertz, Andreas; Le Blay, Gwenaelle; Blaut, Michael

2000-01-01

Species-specific 16S rRNA-targeted, Cy3 (indocarbocyanine)-labeled oligonucleotide probes were designed and validated to quantify different Eubacterium species in human fecal samples. Probes were directed at Eubacterium barkeri, E. biforme, E. contortum, E. cylindroides (two probes), E. dolichum, E. hadrum, E. lentum, E. limosum, E. moniliforme, and E. ventriosum. The specificity of the probes was tested with the type strains and a range of common intestinal bacteria. With one exception, none...

A comparison of alternative 60-mer probe designs in an in-situ synthesized oligonucleotide microarray

Directory of Open Access Journals (Sweden)

Fairbanks Benjamin D

2006-04-01

Full Text Available Abstract Background DNA microarrays have proven powerful for functional genomics studies. Several technologies exist for the generation of whole-genome arrays. It is well documented that 25mer probes directed against different regions of the same gene produce variable signal intensity values. However, the extent to which this is true for probes of greater length (60mers is not well characterized. Moreover, this information has not previously been reported for whole-genome arrays designed against bacteria, whose genomes may differ substantially in characteristics directly affecting microarray performance. Results We report here an analysis of alternative 60mer probe designs for an in-situ synthesized oligonucleotide array for the GC rich, β-proteobacterium Burkholderia cenocepacia. Probes were designed using the ArrayOligoSel3.5 software package and whole-genome microarrays synthesized by Agilent, Inc. using their in-situ, ink-jet technology platform. We first validated the quality of the microarrays as demonstrated by an average signal to noise ratio of >1000. Next, we determined that the variance of replicate probes (1178 total probes examined of identical sequence was 3.8% whereas the variance of alternative probes (558 total alternative probes examined designs was 9.5%. We determined that depending upon the definition, about 2.4% of replicate and 7.8% of alternative probes produced outlier conclusions. Finally, we determined none of the probe design subscores (GC content, internal repeat, binding energy and self annealment produced by ArrayOligoSel3.5 were predictive or probes that produced outlier signals. Conclusion Our analysis demonstrated that the use of multiple probes per target sequence is not essential for in-situ synthesized 60mer oligonucleotide arrays designed against bacteria. Although probes producing outlier signals were identified, the use of ratios results in less than 10% of such outlier conclusions. We also determined that

Development and application of a high-temperature sampling probe for burning chamber conditions in fluidized-bed combustion; Korkean laempoetilan naeytteenottosondin kehittaeminen ja soveltaminen leijukerrospolton tulipesaeolosuhteisiin

Energy Technology Data Exchange (ETDEWEB)

Larjava, K.; Paerkkae, M. [VTT Chemical Technology, Espoo (Finland); Linna, V. [VTT Energy, Jyvaeskylae (Finland). Environmental Technology

1997-10-01

Determination of heavy and alkali metals and other condensing compounds (e.g. chlorides) in combustion chamber conditions is limited by the poor suitability of traditional methods for sampling at high temperatures. IFRF has developed a high-temperature sampling probe for sampling HCN and NH{sub 3}, which has been tested for sampling of NH{sub 3} by Chalmers University of Technology in Sweden. VTT Chemical Technology and Chalmers University of Technology have in their preliminary experiments determined contents of vaporous heavy metals in the combustion chamber of a 12 MW circulating fluidized-bed boiler using this probe. According to the results, the modified probe is suitable for heavy metal determination in combustion chamber. Based on this series of experiments, modification of the probe has been started on the own financing of VTT Chemical Technology and a field measurement was performed in November 1994 to test the present version of the probe. Based on the results of that measurement, the probe has been modified further on as a part of this LIEKKI 2 project. Similar kind of a principle has been applied in the probe which has been developed by VTT Energy during 1994. The probe is built for determination of gas composition of fluidized bed in full-scale boilers. The purpose of this project is to develop and test a sampling probe for fluidized bed combustion. The main advantage of the probe is that condensation losses in sampling due to high temperature gradients can be avoided. Thus, the probe is very suitable for sampling vaporous heavy and alkali metals and other condensing species as well as burning gases and alternatively also solids at high temperatures

Probe sampling measurements and modeling of nitric oxide formation in ethane + air flames

NARCIS (Netherlands)

Dyakov, I.V.; Ruyck, de J.; Konnov, A.A.

2007-01-01

Burning velocity and probe sampling measurements of the concentrations of O2, CO2, CO and NO in the post-flame zone of ethane + air flames are reported. The heat flux method was used for stabilization of laminar, premixed, non-stretched flames on a perforated plate burner at 1 atm. Axial profiles of

Hybridization Capture Using RAD Probes (hyRAD, a New Tool for Performing Genomic Analyses on Collection Specimens.

Directory of Open Access Journals (Sweden)

Tomasz Suchan

Full Text Available In the recent years, many protocols aimed at reproducibly sequencing reduced-genome subsets in non-model organisms have been published. Among them, RAD-sequencing is one of the most widely used. It relies on digesting DNA with specific restriction enzymes and performing size selection on the resulting fragments. Despite its acknowledged utility, this method is of limited use with degraded DNA samples, such as those isolated from museum specimens, as these samples are less likely to harbor fragments long enough to comprise two restriction sites making possible ligation of the adapter sequences (in the case of double-digest RAD or performing size selection of the resulting fragments (in the case of single-digest RAD. Here, we address these limitations by presenting a novel method called hybridization RAD (hyRAD. In this approach, biotinylated RAD fragments, covering a random fraction of the genome, are used as baits for capturing homologous fragments from genomic shotgun sequencing libraries. This simple and cost-effective approach allows sequencing of orthologous loci even from highly degraded DNA samples, opening new avenues of research in the field of museum genomics. Not relying on the restriction site presence, it improves among-sample loci coverage. In a trial study, hyRAD allowed us to obtain a large set of orthologous loci from fresh and museum samples from a non-model butterfly species, with a high proportion of single nucleotide polymorphisms present in all eight analyzed specimens, including 58-year-old museum samples. The utility of the method was further validated using 49 museum and fresh samples of a Palearctic grasshopper species for which the spatial genetic structure was previously assessed using mtDNA amplicons. The application of the method is eventually discussed in a wider context. As it does not rely on the restriction site presence, it is therefore not sensitive to among-sample loci polymorphisms in the restriction sites

Four-probe measurements with a three-probe scanning tunneling microscope

International Nuclear Information System (INIS)

Salomons, Mark; Martins, Bruno V. C.; Zikovsky, Janik; Wolkow, Robert A.

2014-01-01

We present an ultrahigh vacuum (UHV) three-probe scanning tunneling microscope in which each probe is capable of atomic resolution. A UHV JEOL scanning electron microscope aids in the placement of the probes on the sample. The machine also has a field ion microscope to clean, atomically image, and shape the probe tips. The machine uses bare conductive samples and tips with a homebuilt set of pliers for heating and loading. Automated feedback controlled tip-surface contacts allow for electrical stability and reproducibility while also greatly reducing tip and surface damage due to contact formation. The ability to register inter-tip position by imaging of a single surface feature by multiple tips is demonstrated. Four-probe material characterization is achieved by deploying two tips as fixed current probes and the third tip as a movable voltage probe

Four-probe measurements with a three-probe scanning tunneling microscope

Energy Technology Data Exchange (ETDEWEB)

Salomons, Mark [National Institute for Nanotechnology, National Research Council of Canada, Edmonton, Alberta T6G 2M9 (Canada); Martins, Bruno V. C.; Zikovsky, Janik; Wolkow, Robert A., E-mail: rwolkow@ualberta.ca [National Institute for Nanotechnology, National Research Council of Canada, Edmonton, Alberta T6G 2M9 (Canada); Department of Physics, University of Alberta, Edmonton, Alberta T6G 2E1 (Canada)

2014-04-15

We present an ultrahigh vacuum (UHV) three-probe scanning tunneling microscope in which each probe is capable of atomic resolution. A UHV JEOL scanning electron microscope aids in the placement of the probes on the sample. The machine also has a field ion microscope to clean, atomically image, and shape the probe tips. The machine uses bare conductive samples and tips with a homebuilt set of pliers for heating and loading. Automated feedback controlled tip-surface contacts allow for electrical stability and reproducibility while also greatly reducing tip and surface damage due to contact formation. The ability to register inter-tip position by imaging of a single surface feature by multiple tips is demonstrated. Four-probe material characterization is achieved by deploying two tips as fixed current probes and the third tip as a movable voltage probe.

Four-probe measurements with a three-probe scanning tunneling microscope.

Science.gov (United States)

Salomons, Mark; Martins, Bruno V C; Zikovsky, Janik; Wolkow, Robert A

2014-04-01

We present an ultrahigh vacuum (UHV) three-probe scanning tunneling microscope in which each probe is capable of atomic resolution. A UHV JEOL scanning electron microscope aids in the placement of the probes on the sample. The machine also has a field ion microscope to clean, atomically image, and shape the probe tips. The machine uses bare conductive samples and tips with a homebuilt set of pliers for heating and loading. Automated feedback controlled tip-surface contacts allow for electrical stability and reproducibility while also greatly reducing tip and surface damage due to contact formation. The ability to register inter-tip position by imaging of a single surface feature by multiple tips is demonstrated. Four-probe material characterization is achieved by deploying two tips as fixed current probes and the third tip as a movable voltage probe.

Exploring the origin of the D genome of oat by fluorescence in situ hybridization.

Science.gov (United States)

Luo, Xiaomei; Zhang, Haiqin; Kang, Houyang; Fan, Xing; Wang, Yi; Sha, Lina; Zhou, Yonghong

2014-09-01

Further understanding of the origin of cultivated oat would accelerate its genetic improvement. In particular, it would be useful to clarify which diploid progenitor contributed the D genome of this allohexaploid species. In this study, we demonstrate that the landmarks produced by fluorescence in situ hybridization (FISH) of species of Avena using probes derived from Avena sativa can be used to explore the origin of the D genome. Selected sets of probes were hybridized in several sequential experiments performed on exactly the same chromosome spreads, with multiple probes of cytological preparations. Probes pITS and A3-19 showed there might be a similar distribution of pITS between the Ac and D genomes. These results indicated that the Ac genome is closely related to the D genome, and that Avena canariensis (AcAc) could be the D-genome donor of cultivated oat.

Genomic research with human samples. Points of view from scientists and research subjects about disclosure of results and risks of genomic research. Ethical and empirical approach.

Science.gov (United States)

Valle Mansilla, José Ignacio

2011-01-01

Biomedical researchers often now ask subjects to donate samples to be deposited in biobanks. This is not only of interest to researchers, patients and society as a whole can benefit from the improvements in diagnosis, treatment, and prevention that the advent of genomic medicine portends. However, there is a growing debate regarding the social and ethical implications of creating biobanks and using stored human tissue samples for genomic research. Our aim was to identify factors related to both scientists and patients' preferences regarding the sort of information to convey to subjects about the results of the study and the risks related to genomic research. The method used was a survey addressed to 204 scientists and 279 donors from the U.S. and Spain. In this sample, researchers had already published genomic epidemiology studies; and research subjects had actually volunteered to donate a human sample for genomic research. Concerning the results, patients supported more frequently than scientists their right to know individual results from future genomic research. These differences were statistically significant after adjusting by the opportunity to receive genetic research results from the research they had previously participated and their perception of risks regarding genetic information compared to other clinical data. A slight majority of researchers supported informing participants about individual genomic results only if the reliability and clinical validity of the information had been established. Men were more likely than women to believe that patients should be informed of research results even if these conditions were not met. Also among patients, almost half of them would always prefer to be informed about individual results from future genomic research. The three main factors associated to a higher support of a non-limited access to individual results were: being from the US, having previously been offered individual information and considering

On the aspiration characteristics of large-diameter, thin-walled aerosol sampling probes at yaw orientations with respect to the wind

International Nuclear Information System (INIS)

Vincent, J.H.; Mark, D.; Smith, T.A.; Stevens, D.C.; Marshall, M.

1986-01-01

Experiments were carried out in a large wind tunnel to investigate the aspiration efficiencies of thin-walled aerosol sampling probes of large diameter (up to 50 mm) at orientations with respect to the wind direction ranging from 0 to 180 degrees. Sampling conditions ranged from sub-to super-isokinetic. The experiments employed test dusts of close-graded fused alumina and were conducted under conditions of controlled freestream turbulence. For orientations up to and including 90 degrees, the results were qualitatively and quantitatively consistent with a new physical model which takes account of the fact that the sampled air not only diverges or converges (depending on the relationship between wind speed and sampling velocity) but also turns to pass through the plane of the sampling orifice. The previously published results of Durham and Lundgren (1980) and Davies and Subari (1982) for smaller probes were also in good agreement with the new model. The model breaks down, however, for orientations greater than 90 degrees due to the increasing effect of particle impaction onto the blunt leading edge of the probe body. For the probe facing directly away from the wind (180 degree orientation), aspiration efficiency is dominated almost entirely by this effect. (author)

Intra-strain polymorphisms are detected but no genomic alteration is found in cloned mice

International Nuclear Information System (INIS)

Gotoh, Koshichi; Inoue, Kimiko; Ogura, Atsuo; Oishi, Michio

2006-01-01

In-gel competitive reassociation (IGCR) is a method for differential subtraction of polymorphic (RFLP) DNA fragments between two DNA samples of interest without probes or specific sequence information. Here, we applied the IGCR procedure to two cloned mice derived from an F1 hybrid of the C57BL/6Cr and DBA/2 strains, in order to investigate the possibility of genomic alteration in the cloned mouse genomes. Each of the five of the genomic alterations we detected between the two cloned mice corresponded to the 'intra-strain' polymorphisms in the C57BL/6Cr and DBA/2 mouse strains. Our result suggests that no severe aberration of genome sequences occurs due to somatic cell nuclear transfer

DNA probe for lactobacillus delbrueckii

Energy Technology Data Exchange (ETDEWEB)

Delley, M.; Mollet, B.; Hottinger, H. (Nestle Research Centre, Lausanne (Switzerland))

1990-06-01

From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognized L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an {alpha}-{sup 32}P-labeled probe.

DNA probe for lactobacillus delbrueckii

International Nuclear Information System (INIS)

Delley, M.; Mollet, B.; Hottinger, H.

1990-01-01

From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognized L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an α- 32 P-labeled probe

Feasibility study of parallel conduction cooling of NbTi magnet and sample probe in a cryogen-free magnet system

Science.gov (United States)

Catarino, I.; Soni, V.; Barreto, J.; Martins, D.; Kar, S.

2017-02-01

The conduction cooling of both a 6 T superconducting magnet along with a sample probe in a parallel configuration is addressed in this work. A Gifford-McMahon (GM) cryocooler is directly cooling the NbTi magnet, which aims to be kept at 4 K, while a gas-gap heat switch (GGHS) manages the cooling power to be diverted to the sample probe, which may be swept from 4 K up to 300 K. A first prototype of a GGHS was customized and validated for this purpose. A sample probe assembly has been designed and assembled with the existing cryogen-free magnet system. The whole test setup and components are described and the preliminary experimental results on the integration are presented and discussed. The magnet was charged up to 3 T with a 4 K sample space and up to 1 T with a sweeping sample space temperature up to 300 K while acting on the GGHS. Despite some identified thermal insulation problems that occurred during this first test, the overall results demonstrated the feasibility of the cryogen-free parallel conduction cooling on study.

Solution-based targeted genomic enrichment for precious DNA samples

Directory of Open Access Journals (Sweden)

Shearer Aiden

2012-05-01

Full Text Available Abstract Background Solution-based targeted genomic enrichment (TGE protocols permit selective sequencing of genomic regions of interest on a massively parallel scale. These protocols could be improved by: 1 modifying or eliminating time consuming steps; 2 increasing yield to reduce input DNA and excessive PCR cycling; and 3 enhancing reproducible. Results We developed a solution-based TGE method for downstream Illumina sequencing in a non-automated workflow, adding standard Illumina barcode indexes during the post-hybridization amplification to allow for sample pooling prior to sequencing. The method utilizes Agilent SureSelect baits, primers and hybridization reagents for the capture, off-the-shelf reagents for the library preparation steps, and adaptor oligonucleotides for Illumina paired-end sequencing purchased directly from an oligonucleotide manufacturing company. Conclusions This solution-based TGE method for Illumina sequencing is optimized for small- or medium-sized laboratories and addresses the weaknesses of standard protocols by reducing the amount of input DNA required, increasing capture yield, optimizing efficiency, and improving reproducibility.

atpE gene as a new useful specific molecular target to quantify Mycobacterium in environmental samples

Science.gov (United States)

2013-01-01

Background The environment is the likely source of many pathogenic mycobacterial species but detection of mycobacteria by bacteriological tools is generally difficult and time-consuming. Consequently, several molecular targets based on the sequences of housekeeping genes, non-functional RNA and structural ribosomal RNAs have been proposed for the detection and identification of mycobacteria in clinical or environmental samples. While certain of these targets were proposed as specific for this genus, most are prone to false positive results in complex environmental samples that include related, but distinct, bacterial genera. Nowadays the increased number of sequenced genomes and the availability of software for genomic comparison provide tools to develop novel, mycobacteria-specific targets, and the associated molecular probes and primers. Consequently, we conducted an in silico search for proteins exclusive to Mycobacterium spp. genomes in order to design sensitive and specific molecular targets. Results Among the 3989 predicted proteins from M. tuberculosis H37Rv, only 11 proteins showed 80% to 100% of similarity with Mycobacterium spp. genomes, and less than 50% of similarity with genomes of closely related Corynebacterium, Nocardia and Rhodococcus genera. Based on DNA sequence alignments, we designed primer pairs and a probe that specifically detect the atpE gene of mycobacteria, as verified by quantitative real-time PCR on a collection of mycobacteria and non-mycobacterial species. The real-time PCR method we developed was successfully used to detect mycobacteria in tap water and lake samples. Conclusions The results indicate that this real-time PCR method targeting the atpE gene can serve for highly specific detection and precise quantification of Mycobacterium spp. in environmental samples. PMID:24299240

DNA Probe for Lactobacillus delbrueckii

Science.gov (United States)

Delley, Michèle; Mollet, Beat; Hottinger, Herbert

1990-01-01

From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognizes L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an α-32P-labeled DNA probe. Images PMID:16348233

Theory of NMR probe design

International Nuclear Information System (INIS)

Schnall, M.D.

1988-01-01

The NMR probe is the intrinsic part of the NMR system which allows transmission of a stimulus to a sample and the reception of a resulting signal from a sample. NMR probes are used in both imaging and spectroscopy. Optimal probe design is important to the production of adequate signal/moise. It is important for anyone using NMR techniques to understand how NMR probes work and how to optimize probe design

Radioactive probes as diagnostic tools for rice tungro viruses

International Nuclear Information System (INIS)

Azzam, O.; Arboleda, M.; Reyes. J. de los

1996-01-01

Rice tungro bacilliform (RTBV) and rice tungro spherical viruses (RTSV) are the two viral components responsible for rice tungro disease which has seriously affected the irrigated rice ecosystem in Southeast Asia for the last 30 years. RTBV has an 8 Kb double-stranded DNA circular genome, and it is primarily responsible for induction of symptoms in infected plants. RTSV has a 12 kb single-stranded RNA genome. It does not induce any apparent symptoms in the infected plant, and it is transmitted by greenleafhopper. RTBV depends upon RTSV for its own transmission. The two viruses are limited to the vascular tissue of the rice plant and are present at a low titer. Most of the detection methods used for the identification of these viruses have relied on the virus protein properties and therefore, early detection of the virus activity was not possible. We were interested in evaluating tissue printing, dot blot, and southern techniques for early detection of virus nucleic acids in rice plant using radioactive and non radioactive probes. 32 P-labeled T7 or SP6 RNA polymerase transcripts complementary to the RTBV genome and RTSV coat protein genes were used as probes of the positive stand of both viruses. For nonradioactive probes, RTBV DNA genome was labeled using the ECL detection kit (Amersham). Preliminary results show that viral nucleic acids of RTBV and RTSV could be detected using both labelling systems. Non radioactive probes were comparable in their sensitivity to the radioactive probes. Less than 100 pg of viral DNA was detected in the dot-blot assays. More data will be presented to compare the efficiency and reliability of these two techniques in detecting early virus activity in the rice plant. (author)

101-SY waste sample speed of sound/rheology testing for sonic probe program

International Nuclear Information System (INIS)

Cannon, N.S.

1994-01-01

One problem faced in the clean-up operation at Hanford is that a number of radioactive waste storage tanks are experiencing a periodic buildup and release of potentially explosive gases. The best known example is Tank 241-SY-101 (commonly referred to as 101-SY) in which hydrogen gas periodically built up within the waste to the point that increased buoyancy caused a roll-over event, in which the gas was suddenly released in potentially explosive concentrations (if an ignition source were present). The sonic probe concept is to generate acoustic vibrations in the 101-SY tank waste at nominally 100 Hz, with sufficient amplitude to cause the controlled release of hydrogen bubbles trapped in the waste. The sonic probe may provide a potentially cost-effective alternative to large mixer pumps now used for hydrogen mitigation purposes. Two important parameters needed to determine sonic probe effectiveness and design are the speed of sound and yield stress of the tank waste. Tests to determine these parameters in a 240 ml sample of 101-SY waste (obtained near the tank bottom) were performed, and the results are reported

Magnetic bead and gold nanoparticle probes based immunoassay for β-casein detection in bovine milk samples.

Science.gov (United States)

Li, Y S; Meng, X Y; Zhou, Y; Zhang, Y Y; Meng, X M; Yang, L; Hu, P; Lu, S Y; Ren, H L; Liu, Z S; Wang, X R

2015-04-15

In this work, a double-probe based immunoassay was developed for rapid and sensitive determination of β-casein in bovine milk samples. In the method, magnetic beads (MBs), employed as supports for the immobilization of anti-β-casein polyclonal antibody (PAb), were used as the capture probe. Colloidal gold nanoparticles (AuNPs), employed as a bridge for loading anti-β-casein monoclonal antibody (McAb) and horseradish peroxidase (HRP), were used as the amplification probe. The presence of β-casein causes the sandwich structures of MBs-PAb-β-casein-McAb-AuNPs through the interaction between β-casein and the anti-β-casein antibodies. The HRP, used as an enzymatic-amplified tracer, can catalytically oxidize the substrate 3,3',5,5'-tetramethylbenzidine (TMB), generating optical signals that are proportional to the quantity of β-casein. The linear range of the immunoassay was from 6.5 to 1520ngmL(-1). The limit of detection (LOD) was 4.8ngmL(-1) which was 700 times lower than that of MBs-antibody-HRP based immunoassay and 6-7 times lower than that from the microplate-antibody-HRP based assay. The recoveries of β-casein from bovine milk samples were from 95.0% to 104.3% that had a good correlation coefficient (R(2)=0.9956) with those obtained by an official standard Kjeldahl method. For higher sensitivity, simple sample pretreatment and shorter time requirement of the antigen-antibody reaction, the developed immunoassay demonstrated the viability for detection of β-casein in bovine milk samples. Copyright © 2014. Published by Elsevier B.V.

High spatial resolution Kelvin probe force microscopy with coaxial probes

International Nuclear Information System (INIS)

Brown, Keith A; Westervelt, Robert M; Satzinger, Kevin J

2012-01-01

Kelvin probe force microscopy (KPFM) is a widely used technique to measure the local contact potential difference (CPD) between an AFM probe and the sample surface via the electrostatic force. The spatial resolution of KPFM is intrinsically limited by the long range of the electrostatic interaction, which includes contributions from the macroscopic cantilever and the conical tip. Here, we present coaxial AFM probes in which the cantilever and cone are shielded by a conducting shell, confining the tip–sample electrostatic interaction to a small region near the end of the tip. We have developed a technique to measure the true CPD despite the presence of the shell electrode. We find that the behavior of these probes agrees with an electrostatic model of the force, and we observe a factor of five improvement in spatial resolution relative to unshielded probes. Our discussion centers on KPFM, but the field confinement offered by these probes may improve any variant of electrostatic force microscopy. (paper)

Frequency Response of the Sample Vibration Mode in Scanning Probe Acoustic Microscope

International Nuclear Information System (INIS)

Ya-Jun, Zhao; Qian, Cheng; Meng-Lu, Qian

2010-01-01

Based on the interaction mechanism between tip and sample in the contact mode of a scanning probe acoustic microscope (SPAM), an active mass of the sample is introduced in the mass-spring model. The tip motion and frequency response of the sample vibration mode in the SPAM are calculated by the Lagrange equation with dissipation function. For the silicon tip and glass assemblage in the SPAM the frequency response is simulated and it is in agreement with the experimental result. The living myoblast cells on the glass slide are imaged at resonance frequencies of the SPAM system, which are 20kHz, 30kHz and 120kHz. It is shown that good contrast of SPAM images could be obtained when the system is operated at the resonance frequencies of the system in high and low-frequency regions

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

DEFF Research Database (Denmark)

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

2006-01-01

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

Assessment of the LV-S2 & LV-S3 Stack Sampling Probe Locations for Compliance with ANSI/HPS N13.1-1999

Energy Technology Data Exchange (ETDEWEB)

Glissmeyer, John A.; Antonio, Ernest J.; Flaherty, Julia E.; Amidan, Brett G.

2014-09-30

This document reports on a series of tests conducted to assess the proposed air sampling locations for the Hanford Tank Waste Treatment and Immobilization Plant (WTP) Group 1-2A exhaust stacks with respect to the applicable criteria regarding the placement of an air sampling probe. The LV-C2, LV-S2, and LV-S3 exhaust stacks were tested together as a group (Test Group 1-2A). This report only covers the results of LV-S2 and LV-S3; LV-C2 will be reported on separately. Federal regulations1 require that a sampling probe be located in the exhaust stack according to the criteria established by the American National Standards Institute/Health Physics Society (ANSI/HPS) N13.1-1999, Sampling and Monitoring Releases of Airborne Radioactive Substances from the Stack and Ducts of Nuclear Facilities. 2 These criteria address the capability of the sampling probe to extract a sample that represents the effluent stream.

Sampling the genomic pool of protein tyrosine kinase genes using the polymerase chain reaction with genomic DNA.

Science.gov (United States)

Oates, A C; Wollberg, P; Achen, M G; Wilks, A F

1998-08-28

The polymerase chain reaction (PCR), with cDNA as template, has been widely used to identify members of protein families from many species. A major limitation of using cDNA in PCR is that detection of a family member is dependent on temporal and spatial patterns of gene expression. To circumvent this restriction, and in order to develop a technique that is broadly applicable we have tested the use of genomic DNA as PCR template to identify members of protein families in an expression-independent manner. This test involved amplification of DNA encoding protein tyrosine kinase (PTK) genes from the genomes of three animal species that are well known development models; namely, the mouse Mus musculus, the fruit fly Drosophila melanogaster, and the nematode worm Caenorhabditis elegans. Ten PTK genes were identified from the mouse, 13 from the fruit fly, and 13 from the nematode worm. Among these kinases were 13 members of the PTK family that had not been reported previously. Selected PTKs from this screen were shown to be expressed during development, demonstrating that the amplified fragments did not arise from pseudogenes. This approach will be useful for the identification of many novel members of gene families in organisms of agricultural, medical, developmental and evolutionary significance and for analysis of gene families from any species, or biological sample whose habitat precludes the isolation of mRNA. Furthermore, as a tool to hasten the discovery of members of gene families that are of particular interest, this method offers an opportunity to sample the genome for new members irrespective of their expression pattern.

Transcript profiling of common bean (Phaseolus vulgaris L. using the GeneChip® Soybean Genome Array: optimizing analysis by masking biased probes

Directory of Open Access Journals (Sweden)

Gronwald John W

2010-05-01

Full Text Available Abstract Background Common bean (Phaseolus vulgaris L. and soybean (Glycine max both belong to the Phaseoleae tribe and share significant coding sequence homology. This suggests that the GeneChip® Soybean Genome Array (soybean GeneChip may be used for gene expression studies using common bean. Results To evaluate the utility of the soybean GeneChip for transcript profiling of common bean, we hybridized cRNAs purified from nodule, leaf, and root of common bean and soybean in triplicate to the soybean GeneChip. Initial data analysis showed a decreased sensitivity and accuracy of measuring differential gene expression in common bean cross-species hybridization (CSH GeneChip data compared to that of soybean. We employed a method that masked putative probes targeting inter-species variable (ISV regions between common bean and soybean. A masking signal intensity threshold was selected that optimized both sensitivity and accuracy of measuring differential gene expression. After masking for ISV regions, the number of differentially-expressed genes identified in common bean was increased by 2.8-fold reflecting increased sensitivity. Quantitative RT-PCR (qRT-PCR analysis of 20 randomly selected genes and purine-ureide pathway genes demonstrated an increased accuracy of measuring differential gene expression after masking for ISV regions. We also evaluated masked probe frequency per probe set to gain insight into the sequence divergence pattern between common bean and soybean. The sequence divergence pattern analysis suggested that the genes for basic cellular functions and metabolism were highly conserved between soybean and common bean. Additionally, our results show that some classes of genes, particularly those associated with environmental adaptation, are highly divergent. Conclusions The soybean GeneChip is a suitable cross-species platform for transcript profiling in common bean when used in combination with the masking protocol described. In

Novel genomes and genome constitutions identified by GISH and 5S rDNA and knotted1 genomic sequences in the genus Setaria.

Science.gov (United States)

Zhao, Meicheng; Zhi, Hui; Doust, Andrew N; Li, Wei; Wang, Yongfang; Li, Haiquan; Jia, Guanqing; Wang, Yongqiang; Zhang, Ning; Diao, Xianmin

2013-04-11

The Setaria genus is increasingly of interest to researchers, as its two species, S. viridis and S. italica, are being developed as models for understanding C4 photosynthesis and plant functional genomics. The genome constitution of Setaria species has been studied in the diploid species S. viridis, S. adhaerans and S. grisebachii, where three genomes A, B and C were identified respectively. Two allotetraploid species, S. verticillata and S. faberi, were found to have AABB genomes, and one autotetraploid species, S. queenslandica, with an AAAA genome, has also been identified. The genomes and genome constitutions of most other species remain unknown, even though it was thought there are approximately 125 species in the genus distributed world-wide. GISH was performed to detect the genome constitutions of Eurasia species of S. glauca, S. plicata, and S. arenaria, with the known A, B and C genomes as probes. No or very poor hybridization signal was detected indicating that their genomes are different from those already described. GISH was also performed reciprocally between S. glauca, S. plicata, and S. arenaria genomes, but no hybridization signals between each other were found. The two sets of chromosomes of S. lachnea both hybridized strong signals with only the known C genome of S. grisebachii. Chromosomes of Qing 9, an accession formerly considered as S. viridis, hybridized strong signal only to B genome of S. adherans. Phylogenetic trees constructed with 5S rDNA and knotted1 markers, clearly classify the samples in this study into six clusters, matching the GISH results, and suggesting that the F genome of S. arenaria is basal in the genus. Three novel genomes in the Setaria genus were identified and designated as genome D (S. glauca), E (S. plicata) and F (S. arenaria) respectively. The genome constitution of tetraploid S. lachnea is putatively CCC'C'. Qing 9 is a B genome species indigenous to China and is hypothesized to be a newly identified species. The

Workup of Human Blood Samples for Deep Sequencing of HIV-1 Genomes

NARCIS (Netherlands)

Cornelissen, Marion; Gall, Astrid; van der Kuyl, Antoinette; Wymant, Chris; Blanquart, François; Fraser, Christophe; Berkhout, Ben

2018-01-01

We describe a detailed protocol for the manual workup of blood (plasma/serum) samples from individuals infected with the human immunodeficiency virus type 1 (HIV-1) for deep sequence analysis of the viral genome. The study optimizing the assay was performed in the context of the BEEHIVE (Bridging

Optical method for distance and displacement measurements of the probe-sample separation in a scanning near-field optical microscope

International Nuclear Information System (INIS)

Santamaria, L.; Siller, H. R.; Garcia-Ortiz, C. E.; Cortes, R.; Coello, V.

2016-01-01

In this work, we present an alternative optical method to determine the probe-sample separation distance in a scanning near-field optical microscope. The experimental method is based in a Lloyd’s mirror interferometer and offers a measurement precision deviation of ∼100 nm using digital image processing and numerical analysis. The technique can also be strategically combined with the characterization of piezoelectric actuators and stability evaluation of the optical system. It also opens the possibility for the development of an automatic approximation control system valid for probe-sample distances from 5 to 500 μm.

Optical method for distance and displacement measurements of the probe-sample separation in a scanning near-field optical microscope

Energy Technology Data Exchange (ETDEWEB)

Santamaria, L.; Siller, H. R. [Tecnológico de Monterrey, Eugenio Garza Sada 2501 Sur, Monterrey, N.L., 64849 (Mexico); Garcia-Ortiz, C. E., E-mail: cegarcia@cicese.mx [CONACYT Research Fellow – CICESE, Unidad Monterrey, Alianza Centro 504, Apodaca, NL, 66629 (Mexico); Cortes, R.; Coello, V. [CICESE, Unidad Monterrey, PIIT, Alianza Centro 504, Apodaca, NL, 66629 (Mexico)

2016-04-15

In this work, we present an alternative optical method to determine the probe-sample separation distance in a scanning near-field optical microscope. The experimental method is based in a Lloyd’s mirror interferometer and offers a measurement precision deviation of ∼100 nm using digital image processing and numerical analysis. The technique can also be strategically combined with the characterization of piezoelectric actuators and stability evaluation of the optical system. It also opens the possibility for the development of an automatic approximation control system valid for probe-sample distances from 5 to 500 μm.

Increasing Genome Sampling and Improving SNP Genotyping for Genotyping-by-Sequencing with New Combinations of Restriction Enzymes.

Science.gov (United States)

Fu, Yong-Bi; Peterson, Gregory W; Dong, Yibo

2016-04-07

Genotyping-by-sequencing (GBS) has emerged as a useful genomic approach for exploring genome-wide genetic variation. However, GBS commonly samples a genome unevenly and can generate a substantial amount of missing data. These technical features would limit the power of various GBS-based genetic and genomic analyses. Here we present software called IgCoverage for in silico evaluation of genomic coverage through GBS with an individual or pair of restriction enzymes on one sequenced genome, and report a new set of 21 restriction enzyme combinations that can be applied to enhance GBS applications. These enzyme combinations were developed through an application of IgCoverage on 22 plant, animal, and fungus species with sequenced genomes, and some of them were empirically evaluated with different runs of Illumina MiSeq sequencing in 12 plant species. The in silico analysis of 22 organisms revealed up to eight times more genome coverage for the new combinations consisted of pairing four- or five-cutter restriction enzymes than the commonly used enzyme combination PstI + MspI. The empirical evaluation of the new enzyme combination (HinfI + HpyCH4IV) in 12 plant species showed 1.7-6 times more genome coverage than PstI + MspI, and 2.3 times more genome coverage in dicots than monocots. Also, the SNP genotyping in 12 Arabidopsis and 12 rice plants revealed that HinfI + HpyCH4IV generated 7 and 1.3 times more SNPs (with 0-16.7% missing observations) than PstI + MspI, respectively. These findings demonstrate that these novel enzyme combinations can be utilized to increase genome sampling and improve SNP genotyping in various GBS applications. Copyright © 2016 Fu et al.

Distribution and evolution of repeated sequences in genomes of Triatominae (Hemiptera-Reduviidae inferred from genomic in situ hybridization.

Directory of Open Access Journals (Sweden)

Sebastian Pita

Full Text Available The subfamily Triatominae, vectors of Chagas disease, comprises 140 species characterized by a highly homogeneous chromosome number. We analyzed the chromosomal distribution and evolution of repeated sequences in Triatominae genomes by Genomic in situ Hybridization using Triatoma delpontei and Triatoma infestans genomic DNAs as probes. Hybridizations were performed on their own chromosomes and on nine species included in six genera from the two main tribes: Triatomini and Rhodniini. Genomic probes clearly generate two different hybridization patterns, dispersed or accumulated in specific regions or chromosomes. The three used probes generate the same hybridization pattern in each species. However, these patterns are species-specific. In closely related species, the probes strongly hybridized in the autosomal heterochromatic regions, resembling C-banding and DAPI patterns. However, in more distant species these co-localizations are not observed. The heterochromatic Y chromosome is constituted by highly repeated sequences, which is conserved among 10 species of Triatomini tribe suggesting be an ancestral character for this group. However, the Y chromosome in Rhodniini tribe is markedly different, supporting the early evolutionary dichotomy between both tribes. In some species, sex chromosomes and autosomes shared repeated sequences, suggesting meiotic chromatin exchanges among these heterologous chromosomes. Our GISH analyses enabled us to acquire not only reliable information about autosomal repeated sequences distribution but also an insight into sex chromosome evolution in Triatominae. Furthermore, the differentiation obtained by GISH might be a valuable marker to establish phylogenetic relationships and to test the controversial origin of the Triatominae subfamily.

Genome reorganization in Nicotiana asymmetric somatic hybrids analysed by in situ hybridization

International Nuclear Information System (INIS)

Parokonny, A.S.; Kenton, A.Y.; Gleba, Y.Y.; Bennett, M.D.

1992-01-01

In situ hybridization was used to examine genome reorganization in asymmetric somatic hybrids between Nicotiana plumbaginifolia and Nicotiana sylvestris obtained by fusion of gamma-irradiated protoplasts from one of the parents (donor) with non-irradiated protoplasts from the other (recipient). Probing with biotinylated total genomic DNA from either the donor or the recipient species unequivocally identified genetic material from both parents in 31 regenerant plants, each originating from a different nuclear hybrid colony. This method, termed genomic in situ hybridization (GISH), allowed intergenomic translocations containing chromosome segments from both species to be recognized in four regenerants. A probe homologous to the consensus sequence of the Arabidopsis thaliana telomeric repeat (5'-TTTAGGG-3')n, identified telomeres on all chromosomes, including 'mini-chromosomes' originating from the irradiated donor genome. Genomic in situ hybridization to plant chromosomes provides a rapid and reliable means of screening for recombinant genotypes in asymmetric somatic hybrids. Used in combination with other DNA probes, it also contributes to a greater understanding of the events responsible for genomic recovery and restabilization following genetic manipulation in vitro

Assessment of genomic relationship between Oryza sativa and ...

African Journals Online (AJOL)

STORAGESEVER

2010-03-01

Mar 1, 2010 ... For genomic in situ hybridization, genomic DNA from O. australiensis was used as probe for the mitotic and meiotic ... Wide hybridization is one of the plant breeding approaches ..... Disease and insect resistance in rice.

A new Langmuir probe concept for rapid sampling of space plasma electron density

International Nuclear Information System (INIS)

Jacobsen, K S; Pedersen, A; Moen, J I; Bekkeng, T A

2010-01-01

In this paper we describe a new Langmuir probe concept that was invented for the in situ investigation of HF radar backscatter irregularities, with the capability to measure absolute electron density at a resolution sufficient to resolve the finest conceivable structure in an ionospheric plasma. The instrument consists of two or more fixed-bias cylindrical Langmuir probes whose radius is small compared to the Debye length. With this configuration, it is possible to acquire absolute electron density measurements independent of electron temperature and rocket/satellite potential. The system was flown on the ICI-2 sounding rocket to investigate the plasma irregularities which cause HF backscatter. It had a sampling rate of more than 5 kHz and successfully measured structures down to the scale of one electron gyro radius. The system can easily be adapted for any ionospheric rocket or satellite, and provides high-quality measurements of electron density at any desired resolution

Chromosome-specific DNA Repeat Probes

Energy Technology Data Exchange (ETDEWEB)

Baumgartner, Adolf; Weier, Jingly Fung; Weier, Heinz-Ulrich G.

2006-03-16

In research as well as in clinical applications, fluorescence in situ hybridization (FISH) has gained increasing popularity as a highly sensitive technique to study cytogenetic changes. Today, hundreds of commercially available DNA probes serve the basic needs of the biomedical research community. Widespread applications, however, are often limited by the lack of appropriately labeled, specific nucleic acid probes. We describe two approaches for an expeditious preparation of chromosome-specific DNAs and the subsequent probe labeling with reporter molecules of choice. The described techniques allow the preparation of highly specific DNA repeat probes suitable for enumeration of chromosomes in interphase cell nuclei or tissue sections. In addition, there is no need for chromosome enrichment by flow cytometry and sorting or molecular cloning. Our PCR-based method uses either bacterial artificial chromosomes or human genomic DNA as templates with {alpha}-satellite-specific primers. Here we demonstrate the production of fluorochrome-labeled DNA repeat probes specific for human chromosomes 17 and 18 in just a few days without the need for highly specialized equipment and without the limitation to only a few fluorochrome labels.

Tracking alien chromosome in sativa background by genomic in situ hybridization

International Nuclear Information System (INIS)

Abbasi, F.M.; Iqbal, M.; Salim, M.

2004-01-01

Genomic in situ hybridization (GISH) was used to look into the genomic constitution of monosomic alien -addition line derived from O. sativa x O. brachyantha. Biotin label genomic DNA from O. brachyantha was used as probe. The probe hybridized to the brachyantha chromosome. No detectable hybridization signal was observed on sativa chromosomes. This differential painting of chromosome enables us to unequivocally discriminate brachyantha chromosome from those of sativa. Results showed the usefulness of GISH in the identification of a single alien chromosome in the sativa background. (author)

RCP: a novel probe design bias correction method for Illumina Methylation BeadChip.

Science.gov (United States)

Niu, Liang; Xu, Zongli; Taylor, Jack A

2016-09-01

The Illumina HumanMethylation450 BeadChip has been extensively utilized in epigenome-wide association studies. This array and its successor, the MethylationEPIC array, use two types of probes-Infinium I (type I) and Infinium II (type II)-in order to increase genome coverage but differences in probe chemistries result in different type I and II distributions of methylation values. Ignoring the difference in distributions between the two probe types may bias downstream analysis. Here, we developed a novel method, called Regression on Correlated Probes (RCP), which uses the existing correlation between pairs of nearby type I and II probes to adjust the beta values of all type II probes. We evaluate the effect of this adjustment on reducing probe design type bias, reducing technical variation in duplicate samples, improving accuracy of measurements against known standards, and retention of biological signal. We find that RCP is statistically significantly better than unadjusted data or adjustment with alternative methods including SWAN and BMIQ. We incorporated the method into the R package ENmix, which is freely available from the Bioconductor website (https://www.bioconductor.org/packages/release/bioc/html/ENmix.html). niulg@ucmail.uc.edu Supplementary data are available at Bioinformatics online. Published by Oxford University Press 2016. This work is written by US Government employees and is in the public domain in the US.

Use of a D17Z1 oligonucleotide probe for human DNA quantitation prior to PCR analysis of polymorphic DNA markers

Energy Technology Data Exchange (ETDEWEB)

Walsh, S.; Alavaren, M.; Varlaro, J. [Roche Molecular Systems, Alameda, CA (United States)] [and others

1994-09-01

The alpha-satellite DNA locus D17Z1 contains primate-specific sequences which are repeated several hundred times per chromosome 17. A probe that was designed to hybridize to a subset of the D17Z1 sequence can be used for very sensitive and specific quantitation of human DNA. Sample human genomic DNA is immobilized on nylon membrane using a slot blot apparatus, and then hybridized with a biotinylated D17Z1 oligonucleotide probe. The subsequent binding of streptavidin-horseradish peroxidase to the bound probe allows for either calorimetric (TMB) or chemiluminescent (ECL) detection. Signals obtained for sample DNAs are then compared to the signals obtained for a series of human DNA standards. For either detection method, forty samples can be quantitated in less than two hours, with a sensitivity of 150 pg. As little as 20 pg of DNA can be quantitated when using chemiluminescent detection with longer film exposures. PCR analysis of several VNTR and STR markers has indicated that optimal typing results are generally obtained within a relatively narrow range of input DNA quantities. Too much input DNA can lead to PCR artifacts such as preferential amplification of smaller alleles, non-specific amplification products, and exaggeration of the DNA synthesis slippage products that are seen with STR markers. Careful quantitation of human genomic DNA prior to PCR can avoid or minimize these problems and ultimately give cleaner, more unambiguous PCR results.

Development and application of a high-temperature sampling probe for burning chamber conditions of fluidized-bed combustion; Korkean laempoetilan naeytteenottosondin kehittaeminen ja soveltaminen leijukerrospolton tulipesaeolosuhteisiin

Energy Technology Data Exchange (ETDEWEB)

Larjava, K.; Paerkkae, M.; Jormanainen, P.; Roine, J.; Paakkinen, K. [VTT Chemistry, Espoo (Finland); Linna, V. [VTT Energy, Jyvaeskylae (Finland)

1996-12-01

A sampling probe for the burning chamber conditions of fluidized-bed combustion will be developed in this project. The probe will be suitable for sampling vaporous heavy and alkali metals and other condensing compounds (e.g. chlorides) as well combustion gases and alternatively also flue gas particles at high temperatures. The knowledge gained with the probe will help understanding, developing and modeling combustion processes and will thus aid the manufacturers of the boilers. (author)

CHROMATO: a new probe for well sampling and lanthanides chromatographic measurements in deep ground waters

International Nuclear Information System (INIS)

Vinson, J.M.; Rouxel, R.; Soreau, P.

1991-01-01

The important effect of natural medium on the stored radionuclides migration's alulities implies the development of all the necessary devices for the medium's chemical study. The chromato probe's target is to measure all the factors likely to be disturbed by a simple sampling. The lanthanides able to compete in all chemical migration's mechanisms with pollutants have to be considered with a special attention. The volumetric in-situ sampling of these elements is the object of this contract. 6 Refs.; 25 Photos; 15 Figs [fr

Alternative mapping of probes to genes for Affymetrix chips

DEFF Research Database (Denmark)

Gautier, Laurent; Møller, M.; Friis-Hansen, L.

2004-01-01

transcripts: the NCBI RefSeq database. We also built mappings and used them in place of the original probe to genes associations provided by the manufacturer of the arrays. Results: In a large number of cases, 36%, the probes matching a reference sequence were consistent with the grouping of probes...... by the manufacturer of the chips. For the remaining cases there were discrepancies and we show how that can affect the analysis of data. Conclusions: While the probes on Affymetrix arrays remain the same for several years, the biological knowledge concerning the genomic sequences evolves rapidly. Using up...

Ethical issues in consumer genome sequencing: Use of consumers' samples and data.

Science.gov (United States)

Niemiec, Emilia; Howard, Heidi Carmen

2016-03-01

High throughput approaches such as whole genome sequencing (WGS) and whole exome sequencing (WES) create an unprecedented amount of data providing powerful resources for clinical care and research. Recently, WGS and WES services have been made available by commercial direct-to-consumer (DTC) companies. The DTC offer of genetic testing (GT) has already brought attention to potentially problematic issues such as the adequacy of consumers' informed consent and transparency of companies' research activities. In this study, we analysed the websites of four DTC GT companies offering WGS and/or WES with regard to their policies governing storage and future use of consumers' data and samples. The results are discussed in relation to recommendations and guiding principles such as the "Statement of the European Society of Human Genetics on DTC GT for health-related purposes" (2010) and the "Framework for responsible sharing of genomic and health-related data" (Global Alliance for Genomics and Health, 2014). The analysis reveals that some companies may store and use consumers' samples or sequencing data for unspecified research and share the data with third parties. Moreover, the companies do not provide sufficient or clear information to consumers about this, which can undermine the validity of the consent process. Furthermore, while all companies state that they provide privacy safeguards for data and mention the limitations of these, information about the possibility of re-identification is lacking. Finally, although the companies that may conduct research do include information regarding proprietary claims and commercialisation of the results, it is not clear whether consumers are aware of the consequences of these policies. These results indicate that DTC GT companies still need to improve the transparency regarding handling of consumers' samples and data, including having an explicit and clear consent process for research activities.

Ethical issues in consumer genome sequencing: Use of consumers' samples and data

Directory of Open Access Journals (Sweden)

Emilia Niemiec

2016-03-01

Full Text Available High throughput approaches such as whole genome sequencing (WGS and whole exome sequencing (WES create an unprecedented amount of data providing powerful resources for clinical care and research. Recently, WGS and WES services have been made available by commercial direct-to-consumer (DTC companies. The DTC offer of genetic testing (GT has already brought attention to potentially problematic issues such as the adequacy of consumers' informed consent and transparency of companies' research activities. In this study, we analysed the websites of four DTC GT companies offering WGS and/or WES with regard to their policies governing storage and future use of consumers' data and samples. The results are discussed in relation to recommendations and guiding principles such as the “Statement of the European Society of Human Genetics on DTC GT for health-related purposes” (2010 and the “Framework for responsible sharing of genomic and health-related data” (Global Alliance for Genomics and Health, 2014. The analysis reveals that some companies may store and use consumers' samples or sequencing data for unspecified research and share the data with third parties. Moreover, the companies do not provide sufficient or clear information to consumers about this, which can undermine the validity of the consent process. Furthermore, while all companies state that they provide privacy safeguards for data and mention the limitations of these, information about the possibility of re-identification is lacking. Finally, although the companies that may conduct research do include information regarding proprietary claims and commercialisation of the results, it is not clear whether consumers are aware of the consequences of these policies. These results indicate that DTC GT companies still need to improve the transparency regarding handling of consumers' samples and data, including having an explicit and clear consent process for research activities.

Software engineering the mixed model for genome-wide association studies on large samples

Science.gov (United States)

Mixed models improve the ability to detect phenotype-genotype associations in the presence of population stratification and multiple levels of relatedness in genome-wide association studies (GWAS), but for large data sets the resource consumption becomes impractical. At the same time, the sample siz...

Genomic regions associated with bovine milk fatty acids in both summer and winter milk samples

NARCIS (Netherlands)

Bouwman, A.C.; Visker, M.H.P.W.; Arendonk, van J.A.M.; Bovenhuis, H.

2012-01-01

Background - In this study we perform a genome-wide association study (GWAS) for bovine milk fatty acids from summer milk samples. This study replicates a previous study where we performed a GWAS for bovine milk fatty acids based on winter milk samples from the same population. Fatty acids from

Inferring Population Size History from Large Samples of Genome-Wide Molecular Data - An Approximate Bayesian Computation Approach.

Directory of Open Access Journals (Sweden)

Simon Boitard

2016-03-01

Full Text Available Inferring the ancestral dynamics of effective population size is a long-standing question in population genetics, which can now be tackled much more accurately thanks to the massive genomic data available in many species. Several promising methods that take advantage of whole-genome sequences have been recently developed in this context. However, they can only be applied to rather small samples, which limits their ability to estimate recent population size history. Besides, they can be very sensitive to sequencing or phasing errors. Here we introduce a new approximate Bayesian computation approach named PopSizeABC that allows estimating the evolution of the effective population size through time, using a large sample of complete genomes. This sample is summarized using the folded allele frequency spectrum and the average zygotic linkage disequilibrium at different bins of physical distance, two classes of statistics that are widely used in population genetics and can be easily computed from unphased and unpolarized SNP data. Our approach provides accurate estimations of past population sizes, from the very first generations before present back to the expected time to the most recent common ancestor of the sample, as shown by simulations under a wide range of demographic scenarios. When applied to samples of 15 or 25 complete genomes in four cattle breeds (Angus, Fleckvieh, Holstein and Jersey, PopSizeABC revealed a series of population declines, related to historical events such as domestication or modern breed creation. We further highlight that our approach is robust to sequencing errors, provided summary statistics are computed from SNPs with common alleles.

High Quality Genomic Copy Number Data from Archival Formalin-Fixed Paraffin-Embedded Leiomyosarcoma: Optimisation of Universal Linkage System Labelling

Science.gov (United States)

Salawu, Abdulazeez; Ul-Hassan, Aliya; Hammond, David; Fernando, Malee; Reed, Malcolm; Sisley, Karen

2012-01-01

Most soft tissue sarcomas are characterized by genetic instability and frequent genomic copy number aberrations that are not subtype-specific. Oligonucleotide microarray-based Comparative Genomic Hybridisation (array CGH) is an important technique used to map genome-wide copy number aberrations, but the traditional requirement for high-quality DNA typically obtained from fresh tissue has limited its use in sarcomas. Although large archives of Formalin-fixed Paraffin-embedded (FFPE) tumour samples are available for research, the degradative effects of formalin on DNA from these tissues has made labelling and analysis by array CGH technically challenging. The Universal Linkage System (ULS) may be used for a one-step chemical labelling of such degraded DNA. We have optimised the ULS labelling protocol to perform aCGH on archived FFPE leiomyosarcoma tissues using the 180k Agilent platform. Preservation age of samples ranged from a few months to seventeen years and the DNA showed a wide range of degradation (when visualised on agarose gels). Consistently high DNA labelling efficiency and low microarray probe-to-probe variation (as measured by the derivative log ratio spread) was seen. Comparison of paired fresh and FFPE samples from identical tumours showed good correlation of CNAs detected. Furthermore, the ability to macro-dissect FFPE samples permitted the detection of CNAs that were masked in fresh tissue. Aberrations were visually confirmed using Fluorescence in situ Hybridisation. These results suggest that archival FFPE tissue, with its relative abundance and attendant clinical data may be used for effective mapping for genomic copy number aberrations in such rare tumours as leiomyosarcoma and potentially unravel clues to tumour origins, progression and ultimately, targeted treatment. PMID:23209738

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

Directory of Open Access Journals (Sweden)

Minsoung Rhee

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

Assessment of the National Research Universal Reactor Proposed New Stack Sampling Probe Location for Compliance with ANSI/HPS N13.1-1999

Energy Technology Data Exchange (ETDEWEB)

Glissmeyer, John A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Antonio, Ernest J. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Flaherty, Julia E. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

2016-02-29

This document reports on a series of tests conducted to assess the proposed air sampling location for the National Research Universal reactor (NRU) complex exhaust stack, located in Chalk River, Ontario, Canada, with respect to the applicable criteria regarding the placement of an air sampling probe. Due to the age of the equipment in the existing monitoring system, and the increasing difficulty in acquiring replacement parts to maintain this equipment, a more up-to-date system is planned to replace the current effluent monitoring system, and a new monitoring location has been proposed. The new sampling probe should be located within the exhaust stack according to the criteria established by the American National Standards Institute/Health Physics Society (ANSI/HPS) N13.1-1999, Sampling and Monitoring Releases of Airborne Radioactive Substances from the Stack and Ducts of Nuclear Facilities. These criteria address the capability of the sampling probe to extract a sample that represents the effluent stream. The internal Pacific Northwest National Laboratory (PNNL) project for this task was 65167, Atomic Energy Canada Ltd. Chalk River Effluent Duct Flow Qualification. The testing described in this document was guided by the Test Plan: Testing of the NRU Stack Air Sampling Position (TP-STMON-032).

Direct protein quantification in complex sample solutions by surface-engineered nanorod probes

KAUST Repository

Schrittwieser, Stefan

2017-06-30

Detecting biomarkers from complex sample solutions is the key objective of molecular diagnostics. Being able to do so in a simple approach that does not require laborious sample preparation, sophisticated equipment and trained staff is vital for point-of-care applications. Here, we report on the specific detection of the breast cancer biomarker sHER2 directly from serum and saliva samples by a nanorod-based homogeneous biosensing approach, which is easy to operate as it only requires mixing of the samples with the nanorod probes. By careful nanorod surface engineering and homogeneous assay design, we demonstrate that the formation of a protein corona around the nanoparticles does not limit the applicability of our detection method, but on the contrary enables us to conduct in-situ reference measurements, thus further strengthening the point-of-care applicability of our method. Making use of sandwich assays on top of the nanorods, we obtain a limit of detection of 110 pM and 470 pM in 10-fold diluted spiked saliva and serum samples, respectively. In conclusion, our results open up numerous applications in direct protein biomarker quantification, specifically in point-of-care settings where resources are limited and ease-of-use is of essence.

Direct protein quantification in complex sample solutions by surface-engineered nanorod probes

KAUST Repository

Schrittwieser, Stefan; Pelaz, Beatriz; Parak, Wolfgang J.; Lentijo Mozo, Sergio; Soulantica, Katerina; Dieckhoff, Jan; Ludwig, Frank; Schotter, Joerg

2017-01-01

Detecting biomarkers from complex sample solutions is the key objective of molecular diagnostics. Being able to do so in a simple approach that does not require laborious sample preparation, sophisticated equipment and trained staff is vital for point-of-care applications. Here, we report on the specific detection of the breast cancer biomarker sHER2 directly from serum and saliva samples by a nanorod-based homogeneous biosensing approach, which is easy to operate as it only requires mixing of the samples with the nanorod probes. By careful nanorod surface engineering and homogeneous assay design, we demonstrate that the formation of a protein corona around the nanoparticles does not limit the applicability of our detection method, but on the contrary enables us to conduct in-situ reference measurements, thus further strengthening the point-of-care applicability of our method. Making use of sandwich assays on top of the nanorods, we obtain a limit of detection of 110 pM and 470 pM in 10-fold diluted spiked saliva and serum samples, respectively. In conclusion, our results open up numerous applications in direct protein biomarker quantification, specifically in point-of-care settings where resources are limited and ease-of-use is of essence.

Aneris: Devlopment of an intelligent oceanographic probe with high resolution autonomous sampling and col·lecting capabilities

OpenAIRE

Cano, V.; Pujol, N.; Piera Fernández, Jaume; Torrecilla Ribalta, Elena; Aymerich, I.F.

2010-01-01

ANERIS is a multidisciplinary project focused on the design and development of a innovative sampling and collecting sonde. The probe design pretend to minimize actual sea water collecting devices limitations by providing autonomy and intelligence to the system. Peer Reviewed

Development and operation of an integrated sampling probe and gas analyzer for turbulent mixing studies in complex supersonic flows

Science.gov (United States)

Wiswall, John D.

For many aerospace applications, mixing enhancement between co-flowing streams has been identified as a critical and enabling technology. Due to short fuel residence times in scramjet combustors, combustion is limited by the molecular mixing of hydrogen (fuel) and air. Determining the mixedness of fuel and air in these complex supersonic flowfields is critical to the advancement of novel injection schemes currently being developed at UTA in collaboration with NASA Langley and intended to be used on a future two-stage to orbit (~Mach 16) hypersonic air-breathing vehicle for space access. Expanding on previous work, an instrument has been designed, fabricated, and tested in order to measure mean concentrations of injected helium (a passive scalar used instead of hazardous hydrogen) and to quantitatively characterize the nature of the high-frequency concentration fluctuations encountered in the compressible, turbulent, and high-speed (up to Mach 3.5) complex flows associated with the new supersonic injection schemes. This important high-frequency data is not yet attainable when employing other techniques such as Laser Induced Fluorescence, Filtered Rayleigh Scattering or mass spectroscopy in the same complex supersonic flows. The probe operates by exploiting the difference between the thermodynamic properties of two species through independent massflow measurements and calibration. The probe samples isokinetically from the flowfield's area of interest and the helium concentration may be uniquely determined by hot-film anemometry and internally measured stagnation conditions. The final design has a diameter of 0.25" and is only 2.22" long. The overall accuracy of the probe is 3% in molar fraction of helium. The frequency response of mean concentration measurements is estimated at 103 Hz, while high-frequency hot-film measurements were conducted at 60 kHz. Additionally, the work presents an analysis of the probe's internal mixing effects and the effects of the spatial

Characterization of noncoding regulatory DNA in the human genome.

Science.gov (United States)

Elkon, Ran; Agami, Reuven

2017-08-08

Genetic variants associated with common diseases are usually located in noncoding parts of the human genome. Delineation of the full repertoire of functional noncoding elements, together with efficient methods for probing their biological roles, is therefore of crucial importance. Over the past decade, DNA accessibility and various epigenetic modifications have been associated with regulatory functions. Mapping these features across the genome has enabled researchers to begin to document the full complement of putative regulatory elements. High-throughput reporter assays to probe the functions of regulatory regions have also been developed but these methods separate putative regulatory elements from the chromosome so that any effects of chromatin context and long-range regulatory interactions are lost. Definitive assignment of function(s) to putative cis-regulatory elements requires perturbation of these elements. Genome-editing technologies are now transforming our ability to perturb regulatory elements across entire genomes. Interpretation of high-throughput genetic screens that incorporate genome editors might enable the construction of an unbiased map of functional noncoding elements in the human genome.

Gene probes : principles and protocols [Methods in molecular biology, v. 179

National Research Council Canada - National Science Library

Rapley, Ralph; Aquino de Muro, Marilena

2002-01-01

... of labeled DNA has allowed genes to be mapped to single chromosomes and in many cases to a single chromosome band, promoting significant advance in human genome mapping. Gene Probes: Principles and Protocols presents the principles for gene probe design, labeling, detection, target format, and hybridization conditions together with detailed protocols, accom...

Detection of alien genetic introgressions in bread wheat using dot-blot genomic hybridisation.

Science.gov (United States)

Rey, María-Dolores; Prieto, Pilar

2017-01-01

Simple, reliable methods for the identification of alien genetic introgressions are required in plant breeding programmes. The use of genomic dot-blot hybridisation allows the detection of small Hordeum chilense genomic introgressions in the descendants of genetic crosses between wheat and H. chilense addition or substitution lines in wheat when molecular markers are difficult to use. Based on genomic in situ hybridisation, DNA samples from wheat lines carrying putatively H. chilense introgressions were immobilised on a membrane, blocked with wheat genomic DNA and hybridised with biotin-labelled H. chilense genomic DNA as a probe. This dot-blot screening reduced the number of plants necessary to be analysed by molecular markers or in situ hybridisation, saving time and money. The technique was sensitive enough to detect a minimum of 5 ng of total genomic DNA immobilised on the membrane or about 1/420 dilution of H. chilense genomic DNA in the wheat background. The robustness of the technique was verified by in situ hybridisation. In addition, the detection of other wheat relative species such as Hordeum vulgare , Secale cereale and Agropyron cristatum in the wheat background was also reported .

Preventing probe induced topography correlated artifacts in Kelvin Probe Force Microscopy

NARCIS (Netherlands)

Polak, L.; Wijngaarden, Rinke J.

2016-01-01

Kelvin Probe Force Microscopy (KPFM) on samples with rough surface topography can be hindered by topography correlated artifacts. We show that, with the proper experimental configuration and using homogeneously metal coated probes, we are able to obtain amplitude modulation (AM) KPFM results on a

Ultraspecific probes for high throughput HLA typing

Directory of Open Access Journals (Sweden)

Eggers Rick

2009-02-01

Full Text Available Abstract Background The variations within an individual's HLA (Human Leukocyte Antigen genes have been linked to many immunological events, e.g. susceptibility to disease, response to vaccines, and the success of blood, tissue, and organ transplants. Although the microarray format has the potential to achieve high-resolution typing, this has yet to be attained due to inefficiencies of current probe design strategies. Results We present a novel three-step approach for the design of high-throughput microarray assays for HLA typing. This approach first selects sequences containing the SNPs present in all alleles of the locus of interest and next calculates the number of base changes necessary to convert a candidate probe sequences to the closest subsequence within the set of sequences that are likely to be present in the sample including the remainder of the human genome in order to identify those candidate probes which are "ultraspecific" for the allele of interest. Due to the high specificity of these sequences, it is possible that preliminary steps such as PCR amplification are no longer necessary. Lastly, the minimum number of these ultraspecific probes is selected such that the highest resolution typing can be achieved for the minimal cost of production. As an example, an array was designed and in silico results were obtained for typing of the HLA-B locus. Conclusion The assay presented here provides a higher resolution than has previously been developed and includes more alleles than previously considered. Based upon the in silico and preliminary experimental results, we believe that the proposed approach can be readily applied to any highly polymorphic gene system.

A DATABASE FOR TRACKING TOXICOGENOMIC SAMPLES AND PROCEDURES WITH GENOMIC, PROTEOMIC AND METABONOMIC COMPONENTS

Science.gov (United States)

A Database for Tracking Toxicogenomic Samples and Procedures with Genomic, Proteomic and Metabonomic Components Wenjun Bao1, Jennifer Fostel2, Michael D. Waters2, B. Alex Merrick2, Drew Ekman3, Mitchell Kostich4, Judith Schmid1, David Dix1Office of Research and Developmen...

A BAC-based physical map of the Drosophila buzzatii genome

Energy Technology Data Exchange (ETDEWEB)

Gonzalez, Josefa; Nefedov, Michael; Bosdet, Ian; Casals, Ferran; Calvete, Oriol; Delprat, Alejandra; Shin, Heesun; Chiu, Readman; Mathewson, Carrie; Wye, Natasja; Hoskins, Roger A.; Schein, JacquelineE.; de Jong, Pieter; Ruiz, Alfredo

2005-03-18

Large-insert genomic libraries facilitate cloning of large genomic regions, allow the construction of clone-based physical maps and provide useful resources for sequencing entire genomes. Drosophilabuzzatii is a representative species of the repleta group in the Drosophila subgenus, which is being widely used as a model in studies of genome evolution, ecological adaptation and speciation. We constructed a Bacterial Artificial Chromosome (BAC) genomic library of D. buzzatii using the shuttle vector pTARBAC2.1. The library comprises 18,353 clones with an average insert size of 152 kb and a {approx}18X expected representation of the D. buzzatii euchromatic genome. We screened the entire library with six euchromatic gene probes and estimated the actual genome representation to be {approx}23X. In addition, we fingerprinted by restriction digestion and agarose gel electrophoresis a sample of 9,555 clones, and assembled them using Finger Printed Contigs (FPC) software and manual editing into 345 contigs (mean of 26 clones per contig) and 670singletons. Finally, we anchored 181 large contigs (containing 7,788clones) to the D. buzzatii salivary gland polytene chromosomes by in situ hybridization of 427 representative clones. The BAC library and a database with all the information regarding the high coverage BAC-based physical map described in this paper are available to the research community.

Depth Probing Soft X-ray Microprobe (DPSXRM) for High Resolution Probing of Earth's Microstructural Samples

Science.gov (United States)

Dikedi, P. N.

2015-12-01

The Cambrian explosion; occurrence of landslides in very dry weather conditions; rockslides; dead, shriveled-up and crumbled leaves possessing fossil records with the semblance of well preserved, flat leaves; abundance of trilobite tracks in lower and higher rock layers; and sailing stones are enigmas demanding demystifications. These enigmas could be elucidated when data on soil structure, texture and strength are provided by some device with submicrometre accuracy; for these and other reasons, the design of a Depth Probing Soft X-ray Microprobe (DPSXRM), is being proposed; it is expected to deliver soft X-rays, at spatial resolution, ϛ≥600nm and to probe at the depth of 0.5m in 17s. The microprobe is portable compared to a synchrotron radiation facility (Diamond Light Source has land size of 43,300m2); spatial resolution,ϛ , of the DPSXRM surpasses those of the X-ray Fluorescence microanalysis (10µm), electron microprobe (1-3µm) and ion microprobe (5->30µm); the DPSXRM has allowance for multiple targets. Vanadium and Manganese membranes are proposed owing to respective 4.952KeV VKα1 and 5.899KeV MnKα1 X-rays emitted, which best suits micro-probing of Earth's microstructural samples. Compound systems like the Kirk-Patrick and Baez and Wolter optics, aspheric mirrors like elliptical and parabolic optics, small apertures and Abbe sine condition are employed to reduce or remove astigmatism, obliquity, comatic and spherical aberrations—leading to good image quality. Results show that 5.899KeV MnKα1 and 4.952KeV VKα1 soft X-rays will travel a distance of 2.75mm to form circular patches of radii 2.2mm and 2.95mm respectively. Zone plate with nth zone radius of 1.5mm must be positioned 1.5mm and 2mm from the electron gun if circular patches must be formed from 4.952KeV VKα1 and 5.899KeV MnKα1 soft X-rays respectively. The focal lengths of 0.25μm≤ƒ≤1.50μm and 0.04μm≤ƒ≤0.2μm covered by 4.952KeV VKα1 and 5.899KeV Mn Kα1 soft X-Rays, will

GenomeRNAi: a database for cell-based RNAi phenotypes.

Science.gov (United States)

Horn, Thomas; Arziman, Zeynep; Berger, Juerg; Boutros, Michael

2007-01-01

RNA interference (RNAi) has emerged as a powerful tool to generate loss-of-function phenotypes in a variety of organisms. Combined with the sequence information of almost completely annotated genomes, RNAi technologies have opened new avenues to conduct systematic genetic screens for every annotated gene in the genome. As increasing large datasets of RNAi-induced phenotypes become available, an important challenge remains the systematic integration and annotation of functional information. Genome-wide RNAi screens have been performed both in Caenorhabditis elegans and Drosophila for a variety of phenotypes and several RNAi libraries have become available to assess phenotypes for almost every gene in the genome. These screens were performed using different types of assays from visible phenotypes to focused transcriptional readouts and provide a rich data source for functional annotation across different species. The GenomeRNAi database provides access to published RNAi phenotypes obtained from cell-based screens and maps them to their genomic locus, including possible non-specific regions. The database also gives access to sequence information of RNAi probes used in various screens. It can be searched by phenotype, by gene, by RNAi probe or by sequence and is accessible at http://rnai.dkfz.de.

Design of an Enterobacteriaceae Pan-genome Microarray Chip

DEFF Research Database (Denmark)

Lukjancenko, Oksana; Ussery, David

2010-01-01

-density microarray chip has been designed, using 116 Enterobacteriaceae genome sequences, taking into account the enteric pan-genome. Probes for the microarray were checked in silico and performance of the chip, based on experimental strains from four different genera, demonstrate a relatively high ability...... to distinguish those strains on genus, species, and pathotype/serovar levels. Additionally, the microarray performed well when investigating which genes were found in a given strain of interest. The Enterobacteriaceae pan-genome microarray, based on 116 genomes, provides a valuable tool for determination...

EXONSAMPLER: a computer program for genome-wide and candidate gene exon sampling for targeted next-generation sequencing.

Science.gov (United States)

Cosart, Ted; Beja-Pereira, Albano; Luikart, Gordon

2014-11-01

The computer program EXONSAMPLER automates the sampling of thousands of exon sequences from publicly available reference genome sequences and gene annotation databases. It was designed to provide exon sequences for the efficient, next-generation gene sequencing method called exon capture. The exon sequences can be sampled by a list of gene name abbreviations (e.g. IFNG, TLR1), or by sampling exons from genes spaced evenly across chromosomes. It provides a list of genomic coordinates (a bed file), as well as a set of sequences in fasta format. User-adjustable parameters for collecting exon sequences include a minimum and maximum acceptable exon length, maximum number of exonic base pairs (bp) to sample per gene, and maximum total bp for the entire collection. It allows for partial sampling of very large exons. It can preferentially sample upstream (5 prime) exons, downstream (3 prime) exons, both external exons, or all internal exons. It is written in the Python programming language using its free libraries. We describe the use of EXONSAMPLER to collect exon sequences from the domestic cow (Bos taurus) genome for the design of an exon-capture microarray to sequence exons from related species, including the zebu cow and wild bison. We collected ~10% of the exome (~3 million bp), including 155 candidate genes, and ~16,000 exons evenly spaced genomewide. We prioritized the collection of 5 prime exons to facilitate discovery and genotyping of SNPs near upstream gene regulatory DNA sequences, which control gene expression and are often under natural selection. © 2014 John Wiley & Sons Ltd.

Genomic Characterization for Parasitic Weeds of the Genus Striga by Sample Sequence Analysis

Directory of Open Access Journals (Sweden)

Matt C. Estep

2012-03-01

Full Text Available Generation of ∼2200 Sanger sequence reads or ∼10,000 454 reads for seven Lour. DNA samples (five species allowed identification of the highly repetitive DNA content in these genomes. The 14 most abundant repeats in these species were identified and partially assembled. Annotation indicated that they represent nine long terminal repeat (LTR retrotransposon families, three tandem satellite repeats, one long interspersed element (LINE retroelement, and one DNA transposon. All of these repeats are most closely related to repetitive elements in other closely related plants and are not products of horizontal transfer from their host species. These repeats were differentially abundant in each species, with the LTR retrotransposons and satellite repeats most responsible for variation in genome size. Each species had some repetitive elements that were more abundant and some less abundant than the other species examined, indicating that no single element or any unilateral growth or decrease trend in genome behavior was responsible for variation in genome size and composition. Genome sizes were determined by flow sorting, and the values of 615 Mb [ (L. Kuntze], 1330 Mb [ (Willd. Vatke], 1425 Mb [ (Delile Benth.] and 2460 Mb ( Benth. suggest a ploidy series, a prediction supported by repetitive DNA sequence analysis. Phylogenetic analysis using six chloroplast loci indicated the ancestral relationships of the five most agriculturally important species, with the unexpected result that the one parasite of dicotyledonous plants ( was found to be more closely related to some of the grass parasites than many of the grass parasites are to each other.

Multi-point probe for testing electrical properties and a method of producing a multi-point probe

DEFF Research Database (Denmark)

2011-01-01

A multi-point probe for testing electrical properties of a number of specific locations of a test sample comprises a supporting body defining a first surface, a first multitude of conductive probe arms (101-101'''), each of the probe arms defining a proximal end and a distal end. The probe arms...... of contact with the supporting body, and a maximum thickness perpendicular to its perpendicular bisector and its line of contact with the supporting body. Each of the probe arms has a specific area or point of contact (111-111''') at its distal end for contacting a specific location among the number...... of specific locations of the test sample. At least one of the probe arms has an extension defining a pointing distal end providing its specific area or point of contact located offset relative to its perpendicular bisector....

MALDI MS peptide mapping performance by in-gel digestion on a probe with prestructured sample supports

DEFF Research Database (Denmark)

Klenø, Tina Guldberg; Andreasen, Christian Maaløv; Kjeldal, Helle Ørsted

2004-01-01

Matrix-assisted laser desorption/ionization (tandem) mass spectrometry (MALDI MS) is widely used in protein chemistry and proteomics research for the identification and characterization of proteins isolated by polyacrylamide gel electrophoresis. In an effort to minimize sample handling and increa......-probe digestion protocol combined with MALDI tandem mass spectrometry provides a robust platform for proteomics research, including protein identification and determination of posttranslational modifications....

DNA polymorphisms in the Sahiwal breed of Zebu cattle revealed by synthetic oligonucleotide probes

International Nuclear Information System (INIS)

Shashikanth; Yadav, B.R.

2005-01-01

Genomic DNA of 15 randomly selected unrelated animals and from two sire families (11 animals) of the Sahiwal breed of Zebu cattle were investigated. Four oligonucleotide probes - (GTG) 5 , (TCC) 5 , (GT) 8 and (GT) 12 - were used on genomic DNA digested with restriction enzymes AluI, HinfI, MboI, EcoRI and HaeIII in different combinations. All four probes produced multiloci fingerprints with differing levels of polymorphisms. Total bands and shared bands in the fingerprints of each individual were in the range of 2.5 to 23.0 KB. Band number ranged from 9 to 17, with 0.48 average band sharing. Probes (GT) 8 , (GT) 12 and (TCC) 5 produced fingerprinting patterns of medium to low polymorphism, whereas probe (GTG) 5 produced highly polymorphic patterns. Probe (GTG) 5 in combination with the HaeIII enzyme was highly polymorphic with a heterozygosity level of 0.85, followed by (GT) 8 , (TCC) 5 and (GT) 12 with heterozygosity levels of 0.70, 0.65 and 0.30, respectively. Probe GTG 5 or its complementary sequence CAC 5 produced highly polymorphic fingerprints, indicating that the probe can be used for analysing population structure, parentage verification and identifying loci controlling quantitative traits and fertility status. (author)

ProSeeK: a web server for MLPA probe design.

Science.gov (United States)

Pantano, Lorena; Armengol, Lluís; Villatoro, Sergi; Estivill, Xavier

2008-11-28

The technological evolution of platforms for detecting genome-wide copy number imbalances has allowed the discovery of an unexpected amount of human sequence that is variable in copy number among individuals. This type of human variation can make an important contribution to human diversity and disease susceptibility. Multiplex Ligation-dependent Probe Amplification (MLPA) is a targeted method to assess copy number differences for up to 40 genomic loci in one single experiment. Although specific MLPA assays can be ordered from MRC-Holland (the proprietary company of the MLPA technology), custom designs are also developed in many laboratories worldwide. After our own experience, an important drawback of custom MLPA assays is the time spent during the design of the specific oligonucleotides that are used as probes. Due to the large number of probes included in a single assay, a number of restrictions need to be met in order to maximize specificity and to increase success likelihood. We have developed a web tool for facilitating and optimising custom probe design for MLPA experiments. The algorithm only requires the target sequence in FASTA format and a set of parameters, that are provided by the user according to each specific MLPA assay, to identify the best probes inside the given region. To our knowledge, this is the first available tool for optimizing custom probe design of MLPA assays. The ease-of-use and speed of the algorithm dramatically reduces the turn around time of probe design. ProSeeK will become a useful tool for all laboratories that are currently using MLPA in their research projects for CNV studies.

ProSeeK: A web server for MLPA probe design

Directory of Open Access Journals (Sweden)

Villatoro Sergi

2008-11-01

Full Text Available Abstract Background The technological evolution of platforms for detecting genome-wide copy number imbalances has allowed the discovery of an unexpected amount of human sequence that is variable in copy number among individuals. This type of human variation can make an important contribution to human diversity and disease susceptibility. Multiplex Ligation-dependent Probe Amplification (MLPA is a targeted method to assess copy number differences for up to 40 genomic loci in one single experiment. Although specific MLPA assays can be ordered from MRC-Holland (the proprietary company of the MLPA technology, custom designs are also developed in many laboratories worldwide. After our own experience, an important drawback of custom MLPA assays is the time spent during the design of the specific oligonucleotides that are used as probes. Due to the large number of probes included in a single assay, a number of restrictions need to be met in order to maximize specificity and to increase success likelihood. Results We have developed a web tool for facilitating and optimising custom probe design for MLPA experiments. The algorithm only requires the target sequence in FASTA format and a set of parameters, that are provided by the user according to each specific MLPA assay, to identify the best probes inside the given region. Conclusion To our knowledge, this is the first available tool for optimizing custom probe design of MLPA assays. The ease-of-use and speed of the algorithm dramatically reduces the turn around time of probe design. ProSeeK will become a useful tool for all laboratories that are currently using MLPA in their research projects for CNV studies.

ReseqChip: Automated integration of multiple local context probe data from the MitoChip array in mitochondrial DNA sequence assembly

Directory of Open Access Journals (Sweden)

Spang Rainer

2009-12-01

Full Text Available Abstract Background The Affymetrix MitoChip v2.0 is an oligonucleotide tiling array for the resequencing of the human mitochondrial (mt genome. For each of 16,569 nucleotide positions of the mt genome it holds two sets of four 25-mer probes each that match the heavy and the light strand of a reference mt genome and vary only at their central position to interrogate all four possible alleles. In addition, the MitoChip v2.0 carries alternative local context probes to account for known mtDNA variants. These probes have been neglected in most studies due to the lack of software for their automated analysis. Results We provide ReseqChip, a free software that automates the process of resequencing mtDNA using multiple local context probes on the MitoChip v2.0. ReseqChip significantly improves base call rate and sequence accuracy. ReseqChip is available at http://code.open-bio.org/svnweb/index.cgi/bioperl/browse/bioperl-live/trunk/Bio/Microarray/Tools/. Conclusions ReseqChip allows for the automated consolidation of base calls from alternative local mt genome context probes. It thereby improves the accuracy of resequencing, while reducing the number of non-called bases.

Next-generation sampling: Pairing genomics with herbarium specimens provides species-level signal in Solidago (Asteraceae).

Science.gov (United States)

Beck, James B; Semple, John C

2015-06-01

The ability to conduct species delimitation and phylogeny reconstruction with genomic data sets obtained exclusively from herbarium specimens would rapidly enhance our knowledge of large, taxonomically contentious plant genera. In this study, the utility of genotyping by sequencing is assessed in the notoriously difficult genus Solidago (Asteraceae) by attempting to obtain an informative single-nucleotide polymorphism data set from a set of specimens collected between 1970 and 2010. Reduced representation libraries were prepared and Illumina-sequenced from 95 Solidago herbarium specimen DNAs, and resulting reads were processed with the nonreference Universal Network-Enabled Analysis Kit (UNEAK) pipeline. Multidimensional clustering was used to assess the correspondence between genetic groups and morphologically defined species. Library construction and sequencing were successful in 93 of 95 samples. The UNEAK pipeline identified 8470 single-nucleotide polymorphisms, and a filtered data set was analyzed for each of three Solidago subsections. Although results varied, clustering identified genomic groups that often corresponded to currently recognized species or groups of closely related species. These results suggest that genotyping by sequencing is broadly applicable to DNAs obtained from herbarium specimens. The data obtained and their biological signal suggest that pairing genomics with large-scale herbarium sampling is a promising strategy in species-rich plant groups.

PRIMEGENSw3: a web-based tool for high-throughput primer and probe design.

Science.gov (United States)

Kushwaha, Garima; Srivastava, Gyan Prakash; Xu, Dong

2015-01-01

Highly specific and efficient primer and probe design has been a major hurdle in many high-throughput techniques. Successful implementation of any PCR or probe hybridization technique depends on the quality of primers and probes used in terms of their specificity and cross-hybridization. Here we describe PRIMEGENSw3, a set of web-based utilities for high-throughput primer and probe design. These utilities allow users to select genomic regions and to design primer/probe for selected regions in an interactive, user-friendly, and automatic fashion. The system runs the PRIMEGENS algorithm in the back-end on the high-performance server with the stored genomic database or user-provided custom database for cross-hybridization check. Cross-hybridization is checked not only using BLAST but also by checking mismatch positions and energy calculation of potential hybridization hits. The results can be visualized online and also can be downloaded. The average success rate of primer design using PRIMEGENSw3 is ~90 %. The web server also supports primer design for methylated sequences, which is used in epigenetic studies. Stand-alone version of the software is also available for download at the website.

Using nanopore sequencing to get complete genomes from complex samples

DEFF Research Database (Denmark)

Kirkegaard, Rasmus Hansen; Karst, Søren Michael; Nielsen, Per Halkjær

The advantages of “next generation sequencing” has come at the cost of genome finishing. The dominant sequencing technology provides short reads of 150-300 bp, which has made genome assembly very difficult as the reads do not span important repeat regions. Genomes have thus been added...... to the databases as fragmented assemblies and not as finished contigs that resemble the chromosomes in which the DNA is organised within the cells. This is especially troublesome for genomes derived from complex metagenome sequencing. Databases with incomplete genomes can lead to false conclusions about...... the absence of genes and functional predictions of the organisms. Furthermore, it is common that repetitive elements and marker genes such as the 16S rRNA gene are missing completely from these genome bins. Using nanopore long reads, we demonstrate that it is possible to span these regions and make complete...

A generic approach for the design of whole-genome oligoarrays, validated for genomotyping, deletion mapping and gene expression analysis on Staphylococcus aureus

Directory of Open Access Journals (Sweden)

Renzoni Adriana

2005-06-01

Full Text Available Abstract Background DNA microarray technology is widely used to determine the expression levels of thousands of genes in a single experiment, for a broad range of organisms. Optimal design of immobilized nucleic acids has a direct impact on the reliability of microarray results. However, despite small genome size and complexity, prokaryotic organisms are not frequently studied to validate selected bioinformatics approaches. Relying on parameters shown to affect the hybridization of nucleic acids, we designed freely available software and validated experimentally its performance on the bacterial pathogen Staphylococcus aureus. Results We describe an efficient procedure for selecting 40–60 mer oligonucleotide probes combining optimal thermodynamic properties with high target specificity, suitable for genomic studies of microbial species. The algorithm for filtering probes from extensive oligonucleotides libraries fitting standard thermodynamic criteria includes positional information of predicted target-probe binding regions. This algorithm efficiently selected probes recognizing homologous gene targets across three different sequenced genomes of Staphylococcus aureus. BLAST analysis of the final selection of 5,427 probes yielded >97%, 93%, and 81% of Staphylococcus aureus genome coverage in strains N315, Mu50, and COL, respectively. A manufactured oligoarray including a subset of control Escherichia coli probes was validated for applications in the fields of comparative genomics and molecular epidemiology, mapping of deletion mutations and transcription profiling. Conclusion This generic chip-design process merging sequence information from several related genomes improves genome coverage even in conserved regions.

Rapid whole genome sequencing for the detection and characterization of microorganisms directly from clinical samples

DEFF Research Database (Denmark)

Hasman, Henrik; Saputra, Dhany; Sicheritz-Pontén, Thomas

2014-01-01

Whole genome sequencing (WGS) is becoming available as a routine tool for clinical microbiology. If applied directly on clinical samples this could further reduce diagnostic time and thereby improve control and treatment. A major bottle-neck is the availability of fast and reliable bioinformatics...

Hyb-Seq: combining target enrichment and genome skimming for plant phylogenomics

Science.gov (United States)

Kevin Weitemier; Shannon C.K. Straub; Richard C. Cronn; Mark Fishbein; Roswitha Schmickl; Angela McDonnell; Aaron. Liston

2014-01-01

• Premise of the study: Hyb-Seq, the combination of target enrichment and genome skimming, allows simultaneous data collection for low-copy nuclear genes and high-copy genomic targets for plant systematics and evolution studies. • Methods and Results: Genome and transcriptome assemblies for milkweed ( Asclepias syriaca ) were used to design enrichment probes for 3385...

Genome Sequences of Industrially Relevant Saccharomyces cerevisiae Strain M3707, Isolated from a Sample of Distillers Yeast and Four Haploid Derivatives

Energy Technology Data Exchange (ETDEWEB)

Brown, Steven D.; Klingeman, Dawn M.; Johnson, Courtney M.; Clum, Alicia; Aerts, Andrea; Salamov, Asaf; Sharma, Aditi; Zane, Matthew; Barry, Kerrie; Grigoriev, Igor V.; Davison, Brian H.; Lynd, Lee R.; Gilna, Paul; Hau, Heidi; Hogsett, David A.; Froehlich, Allan C.

2013-04-19

Saccharomyces cerevisiae strain M3707 was isolated from a sample of commercial distillers yeast, and its genome sequence together with the genome sequences for the four derived haploid strains M3836, M3837, M3838, and M3839 has been determined. Yeasts have potential for consolidated bioprocessing (CBP) for biofuel production, and access to these genome sequences will facilitate their development.

Genomic Characterization of DArT Markers Based on High-Density Linkage Analysis and Physical Mapping to the Eucalyptus Genome

Science.gov (United States)

Petroli, César D.; Sansaloni, Carolina P.; Carling, Jason; Steane, Dorothy A.; Vaillancourt, René E.; Myburg, Alexander A.; da Silva, Orzenil Bonfim; Pappas, Georgios Joannis; Kilian, Andrzej; Grattapaglia, Dario

2012-01-01

Diversity Arrays Technology (DArT) provides a robust, high throughput, cost-effective method to query thousands of sequence polymorphisms in a single assay. Despite the extensive use of this genotyping platform for numerous plant species, little is known regarding the sequence attributes and genome-wide distribution of DArT markers. We investigated the genomic properties of the 7,680 DArT marker probes of a Eucalyptus array, by sequencing them, constructing a high density linkage map and carrying out detailed physical mapping analyses to the Eucalyptus grandis reference genome. A consensus linkage map with 2,274 DArT markers anchored to 210 microsatellites and a framework map, with improved support for ordering, displayed extensive collinearity with the genome sequence. Only 1.4 Mbp of the 75 Mbp of still unplaced scaffold sequence was captured by 45 linkage mapped but physically unaligned markers to the 11 main Eucalyptus pseudochromosomes, providing compelling evidence for the quality and completeness of the current Eucalyptus genome assembly. A highly significant correspondence was found between the locations of DArT markers and predicted gene models, while most of the 89 DArT probes unaligned to the genome correspond to sequences likely absent in E. grandis, consistent with the pan-genomic feature of this multi-Eucalyptus species DArT array. These comprehensive linkage-to-physical mapping analyses provide novel data regarding the genomic attributes of DArT markers in plant genomes in general and for Eucalyptus in particular. DArT markers preferentially target the gene space and display a largely homogeneous distribution across the genome, thereby providing superb coverage for mapping and genome-wide applications in breeding and diversity studies. Data reported on these ubiquitous properties of DArT markers will be particularly valuable to researchers working on less-studied crop species who already count on DArT genotyping arrays but for which no reference

Genomic characterization of DArT markers based on high-density linkage analysis and physical mapping to the Eucalyptus genome.

Directory of Open Access Journals (Sweden)

César D Petroli

Full Text Available Diversity Arrays Technology (DArT provides a robust, high throughput, cost-effective method to query thousands of sequence polymorphisms in a single assay. Despite the extensive use of this genotyping platform for numerous plant species, little is known regarding the sequence attributes and genome-wide distribution of DArT markers. We investigated the genomic properties of the 7,680 DArT marker probes of a Eucalyptus array, by sequencing them, constructing a high density linkage map and carrying out detailed physical mapping analyses to the Eucalyptus grandis reference genome. A consensus linkage map with 2,274 DArT markers anchored to 210 microsatellites and a framework map, with improved support for ordering, displayed extensive collinearity with the genome sequence. Only 1.4 Mbp of the 75 Mbp of still unplaced scaffold sequence was captured by 45 linkage mapped but physically unaligned markers to the 11 main Eucalyptus pseudochromosomes, providing compelling evidence for the quality and completeness of the current Eucalyptus genome assembly. A highly significant correspondence was found between the locations of DArT markers and predicted gene models, while most of the 89 DArT probes unaligned to the genome correspond to sequences likely absent in E. grandis, consistent with the pan-genomic feature of this multi-Eucalyptus species DArT array. These comprehensive linkage-to-physical mapping analyses provide novel data regarding the genomic attributes of DArT markers in plant genomes in general and for Eucalyptus in particular. DArT markers preferentially target the gene space and display a largely homogeneous distribution across the genome, thereby providing superb coverage for mapping and genome-wide applications in breeding and diversity studies. Data reported on these ubiquitous properties of DArT markers will be particularly valuable to researchers working on less-studied crop species who already count on DArT genotyping arrays but for

Improved reproducibility in genome-wide DNA methylation analysis for PAXgene® fixed samples compared to restored FFPE DNA

DEFF Research Database (Denmark)

Andersen, Gitte Brinch; Hager, Henrik; Hansen, Lise Lotte

2014-01-01

Chip. Quantitative DNA methylation analysis demonstrated that the methylation profile in PAXgene-fixed tissues showed, in comparison with restored FFPE samples, a higher concordance with the profile detected in frozen samples. We demonstrate, for the first time, that DNA from PAXgene conserved tissue performs better......Formalin fixation has been the standard method for conservation of clinical specimens for decades. However, a major drawback is the high degradation of nucleic acids, which complicates its use in genome-wide analyses. Unbiased identification of biomarkers, however, requires genome-wide studies......, precluding the use of the valuable archives of specimens with long-term follow-up data. Therefore, restoration protocols for DNA from formalin-fixed and paraffin-embedded (FFPE) samples have been developed, although they are cost-intensive and time-consuming. An alternative to FFPE and snap...

Quality of the neutron probe calibration curve

International Nuclear Information System (INIS)

Libardi, Paulo Leonel; Moraes, Sergio Oliveira

1997-01-01

An experiment of neutron probe calibration has been performed, involving various volume size samples and collected at various distances from the access tubes. The experiment aimed to give some answers to questions such as suitable sample physical volume, always use of the same volume and sample distance from the neutron probe access tube

Characterizing restriction enzyme-associated loci in historic ragweed (Ambrosia artemisiifolia) voucher specimens using custom-designed RNA probes

DEFF Research Database (Denmark)

Sanchez Barreiro, Fatima; Garrett Vieira, Filipe Jorge; Martin, Michael David

2017-01-01

Population genetic studies of non-model organisms frequently employ reduced representation library (RRL) methodologies, many of which rely on protocols in which genomic DNA is digested by one or more restriction enzymes. However, because high molecular weight DNA is recommended for these protocols......, samples with degraded DNA are generally unsuitable for RRL methods. Given that ancient and historic specimens can provide key temporal perspectives to evolutionary questions, we explored how custom-designed RNA probes could enrich for RRL loci (Restriction Enzyme-Associated Loci baits, or REALbaits...

Identification and Characterization of Epstein-Barr Virus Genomes in Lung Carcinoma Biopsy Samples by Next-Generation Sequencing Technology.

Science.gov (United States)

Wang, Shanshan; Xiong, Hongchao; Yan, Shi; Wu, Nan; Lu, Zheming

2016-05-18

Epstein-Barr virus (EBV) has been detected in the tumor cells of several cancers, including some cases of lung carcinoma (LC). However, the genomic characteristics and diversity of EBV strains associated with LC are poorly understood. In this study, we sequenced the EBV genomes isolated from four primary LC tumor biopsy samples, designated LC1 to LC4. Comparative analysis demonstrated that LC strains were more closely related to GD1 strain. Compared to GD1 reference genome, a total of 520 variations in all, including 498 substitutions, 12 insertions, and 10 deletions were found. Latent genes were found to harbor the most numbers of nonsynonymous mutations. Phylogenetic analysis showed that all LC strains were closely related to Asian EBV strains, whereas different from African/American strains. LC2 genome was distinct from the other three LC genomes, suggesting at least two parental lineages of EBV among the LC genomes may exist. All LC strains could be classified as China 1 and V-val subtype according to the amino acid sequence of LMP1 and EBNA1, respectively. In conclusion, our results showed the genomic diversity among EBV genomes isolated from LC, which might facilitate to uncover the previously unknown variations of pathogenic significance.

Karyotypic relationships among Equus grevyi, Equus burchelli and domestic horse defined using horse chromosome arm-specific probes.

Science.gov (United States)

Musilova, P; Kubickova, S; Zrnova, E; Horin, P; Vahala, J; Rubes, J

2007-01-01

Using laser microdissection we prepared a set of horse chromosome arm-specific probes. Most of the probes were generated from horse chromosomes, some of them were derived from Equus zebra hartmannae. The set of probes were hybridized onto E. grevyi chromosomes in order to establish a genome-wide chromosomal correspondence between this zebra and horse. The use of arm-specific probes provided us with more information on the mutual arrangement of the genomes than we could obtain by means of whole-chromosome paints generated by flow sorting, even if we used reciprocal painting with probe sets from both species. By comparison of our results and results of comparative mapping in E. burchelli, we also established the chromosomal correspondence between E. grevyi and E. burchelli, providing evidence for a very close karyotypic relationship between these two zebra species. Establishment of the comparative map for E. grevyi contributes to the knowledge of the karyotypic phylogeny in the Equidae family.

Lab-scale development of a high temperature aerosol particle sampling probe system for field measurements in thermochemical conversion of biomass

Energy Technology Data Exchange (ETDEWEB)

Lindskog, M.; Malik, A.; Pagels, J.; Sanati, M. [Lund Univ., Lund (Sweden). Div. of Ergonomics and Aerosol Technology

2010-07-01

Thermochemical conversion of biomass requires both combustion in an oxygen rich environment and gasification in an oxygen deficient environment. Therefore, the mass concentration of fly ash from combustion processes is dominated by inorganic compounds, and the particulate matter obtained from gasification is dominated by carbonaceous compounds. The fine fly ash particles can initiate corrosion and fouling and also increases emissions of fine particulates to the atmosphere. This study involved the design of a laboratory scale setup consisting of a high temperature sampling probe and an aerosol generation system to study the formation of fine particle from biomass gasification processes. An aerosol model system using potassium chloride (KCl) as the ash compound and Di Octyl Sebacate oil (DOS) as the volatile organic part was used to test the high temperature sampling probe. Tests conducted at 200 degrees C showed good reproducibility of the aerosol generator. The tests also demonstrated suitable dilution ratios which enabled the denuder to absorb all of the gaseous organic compounds in the set up, thus enabling measurement of only the particle phase. Condensable organic concentrations of 1-68 mg/m{sup 3} were easily handled by the high temperature sampling probe system, indicating that the denuder worked well. Additional tests will be performed using an Aerosol Mass Spectrometer (AMST) to verify that the denuder can capture all of the gaseous organic compounds also when condensed onto agglomerated soot particles. 6 refs., 1 tab., 9 figs.

Simplified sample preparation method for protein identification by matrix-assisted laser desorption/ionization mass spectrometry: in-gel digestion on the probe surface

DEFF Research Database (Denmark)

Stensballe, A; Jensen, Ole Nørregaard

2001-01-01

/ionization-time of flight mass spectrometry (MALDI-TOF-MS) is used as the first protein screening method in many laboratories because of its inherent simplicity, mass accuracy, sensitivity and relatively high sample throughput. We present a simplified sample preparation method for MALDI-MS that enables in-gel digestion...... for protein identification similar to that obtained by the traditional protocols for in-gel digestion and MALDI peptide mass mapping of human proteins, i.e. approximately 60%. The overall performance of the novel on-probe digestion method is comparable with that of the standard in-gel sample preparation...... protocol while being less labour intensive and more cost-effective due to minimal consumption of reagents, enzymes and consumables. Preliminary data obtained on a MALDI quadrupole-TOF tandem mass spectrometer demonstrated the utility of the on-probe digestion protocol for peptide mass mapping and peptide...

Focussed ion beam thin sample microanalysis using a field emission gun electron probe microanalyser

Science.gov (United States)

Kubo, Y.

2018-01-01

Field emission gun electron probe microanalysis (FEG-EPMA) in conjunction with wavelength-dispersive X-ray spectrometry using a low acceleration voltage (V acc) allows elemental analysis with sub-micrometre lateral spatial resolution (SR). However, this degree of SR does not necessarily meet the requirements associated with increasingly miniaturised devices. Another challenge related to performing FEG-EPMA with a low V acc is that the accuracy of quantitative analyses is adversely affected, primarily because low energy X-ray lines such as the L- and M-lines must be employed and due to the potential of line interference. One promising means of obtaining high SR with FEG-EPMA is to use thin samples together with high V acc values. This mini-review covers the basic principles of thin-sample FEG-EPMA and describes an application of this technique to the analysis of optical fibres. Outstanding issues related to this technique that must be addressed are also discussed, which include the potential for electron beam damage during analysis of insulating materials and the development of methods to use thin samples for quantitative analysis.

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

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Amanda eMartino

2012-01-01

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

Moth sex chromatin probed by comparative genomic hybridization (CGH)

Czech Academy of Sciences Publication Activity Database

Sahara, K.; Marec, František; Eickhoff, U.; Traut, W.

2003-01-01

Roč. 46, - (2003), s. 339-342 ISSN 0831-2796 R&D Projects: GA AV ČR IAA6007307 Institutional research plan: CEZ:AV0Z5007907 Keywords : Lepidoptera * comparative genomic hybridization Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.861, year: 2003

DNA Probe for Lactobacillus delbrueckii

OpenAIRE

Delley, Michèle; Mollet, Beat; Hottinger, Herbert

1990-01-01

From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognizes L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an α-32P-l...

Genome-wide identification of significant aberrations in cancer genome.

Science.gov (United States)

Yuan, Xiguo; Yu, Guoqiang; Hou, Xuchu; Shih, Ie-Ming; Clarke, Robert; Zhang, Junying; Hoffman, Eric P; Wang, Roger R; Zhang, Zhen; Wang, Yue

2012-07-27

Somatic Copy Number Alterations (CNAs) in human genomes are present in almost all human cancers. Systematic efforts to characterize such structural variants must effectively distinguish significant consensus events from random background aberrations. Here we introduce Significant Aberration in Cancer (SAIC), a new method for characterizing and assessing the statistical significance of recurrent CNA units. Three main features of SAIC include: (1) exploiting the intrinsic correlation among consecutive probes to assign a score to each CNA unit instead of single probes; (2) performing permutations on CNA units that preserve correlations inherent in the copy number data; and (3) iteratively detecting Significant Copy Number Aberrations (SCAs) and estimating an unbiased null distribution by applying an SCA-exclusive permutation scheme. We test and compare the performance of SAIC against four peer methods (GISTIC, STAC, KC-SMART, CMDS) on a large number of simulation datasets. Experimental results show that SAIC outperforms peer methods in terms of larger area under the Receiver Operating Characteristics curve and increased detection power. We then apply SAIC to analyze structural genomic aberrations acquired in four real cancer genome-wide copy number data sets (ovarian cancer, metastatic prostate cancer, lung adenocarcinoma, glioblastoma). When compared with previously reported results, SAIC successfully identifies most SCAs known to be of biological significance and associated with oncogenes (e.g., KRAS, CCNE1, and MYC) or tumor suppressor genes (e.g., CDKN2A/B). Furthermore, SAIC identifies a number of novel SCAs in these copy number data that encompass tumor related genes and may warrant further studies. Supported by a well-grounded theoretical framework, SAIC has been developed and used to identify SCAs in various cancer copy number data sets, providing useful information to study the landscape of cancer genomes. Open-source and platform-independent SAIC software is

Increased detectability of somatic changes in the DNA from human tumours after probing with "synthetic" and "genome-derived" hypervariable multilocus probes

DEFF Research Database (Denmark)

Lagoda, P J; Seitz, G; Epplen, J T

1989-01-01

intensities were observed. Together the probes 33.15 and (CAC)5/(GTG)5 detected deviating fingerprint patterns in 63% of the colorectal carcinomas investigated. In mammary and stomach carcinomas, only 1/11 and 2/11 tumours, respectively, showed differences with either of the three probes, 33.15, (GACA)4...

A probe-based quantitative PCR assay for detecting Tetracapsuloides bryosalmonae in fish tissue and environmental DNA water samples

Science.gov (United States)

Hutchins, Patrick; Sepulveda, Adam; Martin, Renee; Hopper, Lacey

2017-01-01

A probe-based quantitative real-time PCR assay was developed to detect Tetracapsuloides bryosalmonae, which causes proliferative kidney disease in salmonid fish, in kidney tissue and environmental DNA (eDNA) water samples. The limits of detection and quantification were 7 and 100 DNA copies for calibration standards and T. bryosalmonae was reliably detected down to 100 copies in tissue and eDNA samples. The assay presented here is a highly sensitive and quantitative tool for detecting T. bryosalmonae with potential applications for tissue diagnostics and environmental detection.

The Japanese Society of Pathology Guidelines on the handling of pathological tissue samples for genomic research: Standard operating procedures based on empirical analyses.

Science.gov (United States)

Kanai, Yae; Nishihara, Hiroshi; Miyagi, Yohei; Tsuruyama, Tatsuhiro; Taguchi, Kenichi; Katoh, Hiroto; Takeuchi, Tomoyo; Gotoh, Masahiro; Kuramoto, Junko; Arai, Eri; Ojima, Hidenori; Shibuya, Ayako; Yoshida, Teruhiko; Akahane, Toshiaki; Kasajima, Rika; Morita, Kei-Ichi; Inazawa, Johji; Sasaki, Takeshi; Fukayama, Masashi; Oda, Yoshinao

2018-02-01

Genome research using appropriately collected pathological tissue samples is expected to yield breakthroughs in the development of biomarkers and identification of therapeutic targets for diseases such as cancers. In this connection, the Japanese Society of Pathology (JSP) has developed "The JSP Guidelines on the Handling of Pathological Tissue Samples for Genomic Research" based on an abundance of data from empirical analyses of tissue samples collected and stored under various conditions. Tissue samples should be collected from appropriate sites within surgically resected specimens, without disturbing the features on which pathological diagnosis is based, while avoiding bleeding or necrotic foci. They should be collected as soon as possible after resection: at the latest within about 3 h of storage at 4°C. Preferably, snap-frozen samples should be stored in liquid nitrogen (about -180°C) until use. When intending to use genomic DNA extracted from formalin-fixed paraffin-embedded tissue, 10% neutral buffered formalin should be used. Insufficient fixation and overfixation must both be avoided. We hope that pathologists, clinicians, clinical laboratory technicians and biobank operators will come to master the handling of pathological tissue samples based on the standard operating procedures in these Guidelines to yield results that will assist in the realization of genomic medicine. © 2018 The Authors. Pathology International published by Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.

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.

High sensitivity high-resolution full range relaxometry using a fast mechanical sample shuttling device and a cryo-probe

Energy Technology Data Exchange (ETDEWEB)

Chou, Ching-Yu [Université Paris-Saclay, NIMBE, CEA, CNRS (France); Chu, Minglee [Academia Sinica, Institute of Physics (China); Chang, Chi-Fon [Academia Sinica, Genomics Research Center (China); Yu, Tsunai; Huang, Tai-huang, E-mail: bmthh@gate.sinica.edu.tw [Academia Sinica, Institute of Biomedical Science (China); Sakellariou, Dimitris, E-mail: dimitrios.sakellariou@cea.fr [Université Paris-Saclay, NIMBE, CEA, CNRS (France)

2016-11-15

Field-dependent NMR studies of bio-molecular systems using a sample shuttling hardware operating on a high-field NMR apparatus have provided valuable structural and dynamic information. We have recently published a design of a compact sample transportation device, called “field-cycler”, which was installed in a commercial spectrometer and which provided highly precise positioning and stability during high speed shuttling. In this communication, we demonstrate the first use of a sample shuttling device on a commercial high field standard bore NMR spectrometer, equipped with a commercial triple resonance cryogenically cooled NMR probe. The performance and robustness of the hardware operating in 1D and 2D field cycling experiments, as well as the impact of the sample shuttling time on the signal intensity are discussed.

Evaluation of SNP Data from the Malus Infinium Array Identifies Challenges for Genetic Analysis of Complex Genomes of Polyploid Origin.

Directory of Open Access Journals (Sweden)

Michela Troggio

Full Text Available High throughput arrays for the simultaneous genotyping of thousands of single-nucleotide polymorphisms (SNPs have made the rapid genetic characterisation of plant genomes and the development of saturated linkage maps a realistic prospect for many plant species of agronomic importance. However, the correct calling of SNP genotypes in divergent polyploid genomes using array technology can be problematic due to paralogy, and to divergence in probe sequences causing changes in probe binding efficiencies. An Illumina Infinium II whole-genome genotyping array was recently developed for the cultivated apple and used to develop a molecular linkage map for an apple rootstock progeny (M432, but a large proportion of segregating SNPs were not mapped in the progeny, due to unexpected genotype clustering patterns. To investigate the causes of this unexpected clustering we performed BLAST analysis of all probe sequences against the 'Golden Delicious' genome sequence and discovered evidence for paralogous annealing sites and probe sequence divergence for a high proportion of probes contained on the array. Following visual re-evaluation of the genotyping data generated for 8,788 SNPs for the M432 progeny using the array, we manually re-scored genotypes at 818 loci and mapped a further 797 markers to the M432 linkage map. The newly mapped markers included the majority of those that could not be mapped previously, as well as loci that were previously scored as monomorphic, but which segregated due to divergence leading to heterozygosity in probe annealing sites. An evaluation of the 8,788 probes in a diverse collection of Malus germplasm showed that more than half the probes returned genotype clustering patterns that were difficult or impossible to interpret reliably, highlighting implications for the use of the array in genome-wide association studies.

BarleyBase—an expression profiling database for plant genomics

Science.gov (United States)

Shen, Lishuang; Gong, Jian; Caldo, Rico A.; Nettleton, Dan; Cook, Dianne; Wise, Roger P.; Dickerson, Julie A.

2005-01-01

BarleyBase (BB) (www.barleybase.org) is an online database for plant microarrays with integrated tools for data visualization and statistical analysis. BB houses raw and normalized expression data from the two publicly available Affymetrix genome arrays, Barley1 and Arabidopsis ATH1 with plans to include the new Affymetrix 61K wheat, maize, soybean and rice arrays, as they become available. BB contains a broad set of query and display options at all data levels, ranging from experiments to individual hybridizations to probe sets down to individual probes. Users can perform cross-experiment queries on probe sets based on observed expression profiles and/or based on known biological information. Probe set queries are integrated with visualization and analysis tools such as the R statistical toolbox, data filters and a large variety of plot types. Controlled vocabularies for gene and plant ontologies, as well as interconnecting links to physical or genetic map and other genomic data in PlantGDB, Gramene and GrainGenes, allow users to perform EST alignments and gene function prediction using Barley1 exemplar sequences, thus, enhancing cross-species comparison. PMID:15608273

Four-point probe measurements using current probes with voltage feedback to measure electric potentials

Science.gov (United States)

Lüpke, Felix; Cuma, David; Korte, Stefan; Cherepanov, Vasily; Voigtländer, Bert

2018-02-01

We present a four-point probe resistance measurement technique which uses four equivalent current measuring units, resulting in minimal hardware requirements and corresponding sources of noise. Local sample potentials are measured by a software feedback loop which adjusts the corresponding tip voltage such that no current flows to the sample. The resulting tip voltage is then equivalent to the sample potential at the tip position. We implement this measurement method into a multi-tip scanning tunneling microscope setup such that potentials can also be measured in tunneling contact, allowing in principle truly non-invasive four-probe measurements. The resulting measurement capabilities are demonstrated for \

PROBING GENOME MAINTENANCE FUNCTIONS OF HUMAN RECQ1

Directory of Open Access Journals (Sweden)

Furqan Sami

2013-03-01

Full Text Available The RecQ helicases are a highly conserved family of DNA-unwinding enzymes that play key roles in protecting the genome stability in all kingdoms of life.'Human RecQ homologs include RECQ1, BLM, WRN, RECQ4, and RECQ5β.'Although the individual RecQ-related diseases are characterized by a variety of clinical features encompassing growth defects (Bloom Syndrome and Rothmund Thomson Syndrome to premature aging (Werner Syndrome, all these patients have a high risk of cancer predisposition.'Here, we present an overview of recent progress towards elucidating functions of RECQ1 helicase, the most abundant but poorly characterized RecQ homolog in humans.'Consistent with a conserved role in genome stability maintenance, deficiency of RECQ1 results in elevated frequency of spontaneous sister chromatid exchanges, chromosomal instability, increased DNA damage and greater sensitivity to certain genotoxic stress.'Delineating what aspects of RECQ1 catalytic functions contribute to the observed cellular phenotypes, and how this is regulated is critical to establish its biological functions in DNA metabolism.'Recent studies have identified functional specialization of RECQ1 in DNA repair; however, identification of fundamental similarities will be just as critical in developing a unifying theme for RecQ actions, allowing the functions revealed from studying one homolog to be extrapolated and generalized to other RecQ homologs.

Whole genome DNA copy number changes identified by high density oligonucleotide arrays

Directory of Open Access Journals (Sweden)

Huang Jing

2004-05-01

Full Text Available Abstract Changes in DNA copy number are one of the hallmarks of the genetic instability common to most human cancers. Previous micro-array-based methods have been used to identify chromosomal gains and losses; however, they are unable to genotype alleles at the level of single nucleotide polymorphisms (SNPs. Here we describe a novel algorithm that uses a recently developed high-density oligonucleotide array-based SNP genotyping method, whole genome sampling analysis (WGSA, to identify genome-wide chromosomal gains and losses at high resolution. WGSA simultaneously genotypes over 10,000 SNPs by allele-specific hybridisation to perfect match (PM and mismatch (MM probes synthesised on a single array. The copy number algorithm jointly uses PM intensity and discrimination ratios between paired PM and MM intensity values to identify and estimate genetic copy number changes. Values from an experimental sample are compared with SNP-specific distributions derived from a reference set containing over 100 normal individuals to gain statistical power. Genomic regions with statistically significant copy number changes can be identified using both single point analysis and contiguous point analysis of SNP intensities. We identified multiple regions of amplification and deletion using a panel of human breast cancer cell lines. We verified these results using an independent method based on quantitative polymerase chain reaction and found that our approach is both sensitive and specific and can tolerate samples which contain a mixture of both tumour and normal DNA. In addition, by using known allele frequencies from the reference set, statistically significant genomic intervals can be identified containing contiguous stretches of homozygous markers, potentially allowing the detection of regions undergoing loss of heterozygosity (LOH without the need for a matched normal control sample. The coupling of LOH analysis, via SNP genotyping, with copy number

Genome-wide haplotype analysis of cis expression quantitative trait loci in monocytes.

Directory of Open Access Journals (Sweden)

Sophie Garnier

Full Text Available In order to assess whether gene expression variability could be influenced by several SNPs acting in cis, either through additive or more complex haplotype effects, a systematic genome-wide search for cis haplotype expression quantitative trait loci (eQTL was conducted in a sample of 758 individuals, part of the Cardiogenics Transcriptomic Study, for which genome-wide monocyte expression and GWAS data were available. 19,805 RNA probes were assessed for cis haplotypic regulation through investigation of ~2,1 × 10(9 haplotypic combinations. 2,650 probes demonstrated haplotypic p-values >10(4-fold smaller than the best single SNP p-value. Replication of significant haplotype effects were tested for 412 probes for which SNPs (or proxies that defined the detected haplotypes were available in the Gutenberg Health Study composed of 1,374 individuals. At the Bonferroni correction level of 1.2 × 10(-4 (~0.05/412, 193 haplotypic signals replicated. 1000 G imputation was then conducted, and 105 haplotypic signals still remained more informative than imputed SNPs. In-depth analysis of these 105 cis eQTL revealed that at 76 loci genetic associations were compatible with additive effects of several SNPs, while for the 29 remaining regions data could be compatible with a more complex haplotypic pattern. As 24 of the 105 cis eQTL have previously been reported to be disease-associated loci, this work highlights the need for conducting haplotype-based and 1000 G imputed cis eQTL analysis before commencing functional studies at disease-associated loci.

Deep whole-genome sequencing of 90 Han Chinese genomes.

Science.gov (United States)

Lan, Tianming; Lin, Haoxiang; Zhu, Wenjuan; Laurent, Tellier Christian Asker Melchior; Yang, Mengcheng; Liu, Xin; Wang, Jun; Wang, Jian; Yang, Huanming; Xu, Xun; Guo, Xiaosen

2017-09-01

Next-generation sequencing provides a high-resolution insight into human genetic information. However, the focus of previous studies has primarily been on low-coverage data due to the high cost of sequencing. Although the 1000 Genomes Project and the Haplotype Reference Consortium have both provided powerful reference panels for imputation, low-frequency and novel variants remain difficult to discover and call with accuracy on the basis of low-coverage data. Deep sequencing provides an optimal solution for the problem of these low-frequency and novel variants. Although whole-exome sequencing is also a viable choice for exome regions, it cannot account for noncoding regions, sometimes resulting in the absence of important, causal variants. For Han Chinese populations, the majority of variants have been discovered based upon low-coverage data from the 1000 Genomes Project. However, high-coverage, whole-genome sequencing data are limited for any population, and a large amount of low-frequency, population-specific variants remain uncharacterized. We have performed whole-genome sequencing at a high depth (∼×80) of 90 unrelated individuals of Chinese ancestry, collected from the 1000 Genomes Project samples, including 45 Northern Han Chinese and 45 Southern Han Chinese samples. Eighty-three of these 90 have been sequenced by the 1000 Genomes Project. We have identified 12 568 804 single nucleotide polymorphisms, 2 074 210 short InDels, and 26 142 structural variations from these 90 samples. Compared to the Han Chinese data from the 1000 Genomes Project, we have found 7 000 629 novel variants with low frequency (defined as minor allele frequency genome. Compared to the 1000 Genomes Project, these Han Chinese deep sequencing data enhance the characterization of a large number of low-frequency, novel variants. This will be a valuable resource for promoting Chinese genetics research and medical development. Additionally, it will provide a valuable supplement to the 1000

Innovative SPM Probes for Energy-Storage Science: MWCNT-Nanopipettes to Nanobattery Probes

Science.gov (United States)

Larson, Jonathan; Talin, Alec; Pearse, Alexander; Kozen, Alexander; Reutt-Robey, Janice

As energy-storage materials and designs continue to advance, new tools are needed to direct and explore ion insertion/de-insertion at well-defined battery materials interfaces. Scanned probe tips, assembled from actual energy-storage materials, permit SPM measures of local cathode-anode (tip-sample) interactions, including ion transfer. We present examples of ``cathode'' MWCNT-terminated STM probe tips interacting with Li(s)/Si(111) anode substrates. The MWCNT tip functions as both SPM probe and Li-nanopipette,[1] for controlled transport and manipulation of Li. Local field conditions for lithium ionization and transfer are determined and compared to electrostatic models. Additional lithium metallic and oxide tips have been prepared by thin film deposition on conventional W tips, the latter of which effectively functions as a nanobattery. We demonstrate use of these novel probe materials in the local lithiation of low-index Si anode interfaces, probing local barriers for lithium insertion. Prospects and limitations of these novel SPM probes will be discussed. U.S. Department of Energy Award Number DESC0001160.

A new theoretical probe for the magnetic force microscope

Energy Technology Data Exchange (ETDEWEB)

Windmill, J.F.C. E-mail: jwindmill@plymouth.ac.uk; Clegg, W.W.; Jenkins, D.F.L.; Davey, P.J

2001-05-01

The magnetic force microscope (MFM) is established as a valuable tool for the analysis of magnetic structures. The standard design of MFM incorporates a silicon tip coated with a magnetic material. However, these tips are subject to several inherent problems, e.g. changing characteristics over time due to damage or magnetic hysteresis. A new theoretical electromagnetic MFM probe is introduced here. Although electromagnetic MFM has been discussed before by Zhou et al. (J. Vac. Sci. Technol. A 17 (1999) 2233), the design presented here is a different approach. Two different probe iterations and their magnetic field intensity distribution are modelled. The probe imaging capability is compared using the reciprocity principle (Wright and Hill, Appl. Phys. Lett. 68 (1996) 1726) to image the simulated force interaction between a sample and the probe fields. Thus, images of a sample's magnetic distribution are produced by the convolution of the different probe gradient field distributions and the sample magnetisation. Both perpendicular and longitudinal magnetisation patterns were simulated with the different probe iterations. This clearly showed the improvement of the second probe iteration, particularly for longitudinal patterns. The practical use of the new probe is also discussed, and future work outlined.

Mismatch oligonucleotides in human and yeast: guidelines for probe design on tiling microarrays

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Jee Justin

2008-12-01

Full Text Available Abstract Background Mismatched oligonucleotides are widely used on microarrays to differentiate specific from nonspecific hybridization. While many experiments rely on such oligos, the hybridization behavior of various degrees of mismatch (MM structure has not been extensively studied. Here, we present the results of two large-scale microarray experiments on S. cerevisiae and H. sapiens genomic DNA, to explore MM oligonucleotide behavior with real sample mixtures under tiling-array conditions. Results We examined all possible nucleotide substitutions at the central position of 36-nucleotide probes, and found that nonspecific binding by MM oligos depends upon the individual nucleotide substitutions they incorporate: C→A, C→G and T→A (yielding purine-purine mispairs are most disruptive, whereas A→X were least disruptive. We also quantify a marked GC skew effect: substitutions raising probe GC content exhibit higher intensity (and vice versa. This skew is small in highly-expressed regions (± 0.5% of total intensity range and large (± 2% or more elsewhere. Multiple mismatches per oligo are largely additive in effect: each MM added in a distributed fashion causes an additional 21% intensity drop relative to PM, three-fold more disruptive than adding adjacent mispairs (7% drop per MM. Conclusion We investigate several parameters for oligonucleotide design, including the effects of each central nucleotide substitution on array signal intensity and of multiple MM per oligo. To avoid GC skew, individual substitutions should not alter probe GC content. RNA sample mixture complexity may increase the amount of nonspecific hybridization, magnify GC skew and boost the intensity of MM oligos at all levels.

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

Science.gov (United States)

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

2011-01-01

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

Probing the genome-scale metabolic landscape of Bordetella pertussis, the causative agent of whooping cough.

Science.gov (United States)

Branco Dos Santos, Filipe; Olivier, Brett G; Boele, Joost; Smessaert, Vincent; De Rop, Philippe; Krumpochova, Petra; Klau, Gunnar W; Giera, Martin; Dehottay, Philippe; Teusink, Bas; Goffin, Philippe

2017-08-25

Whooping cough is a highly-contagious respiratory disease caused by Bordetella pertussi s. Despite vaccination, its incidence has been rising alarmingly, and yet, the physiology of B. pertussis remains poorly understood. We combined genome-scale metabolic reconstruction, a novel optimization algorithm and experimental data to probe the full metabolic potential of this pathogen, using strain Tohama I as a reference. Experimental validation showed that B. pertussis secretes a significant proportion of nitrogen as arginine and purine nucleosides, which may contribute to modulation of the host response. We also found that B. pertussis can be unexpectedly versatile, being able to metabolize many compounds while displaying minimal nutrient requirements. It can grow without cysteine - using inorganic sulfur sources such as thiosulfate - and it can grow on organic acids such as citrate or lactate as sole carbon sources, providing in vivo demonstration that its TCA cycle is functional. Although the metabolic reconstruction of eight additional strains indicates that the structural genes underlying this metabolic flexibility are widespread, experimental validation suggests a role of strain-specific regulatory mechanisms in shaping metabolic capabilities. Among five alternative strains tested, three were shown to grow on substrate combinations requiring a functional TCA cycle, but only one could use thiosulfate. Finally, the metabolic model was used to rationally design growth media with over two-fold improvements in pertussis toxin production. This study thus provides novel insights into B. pertussis physiology, and highlights the potential, but also limitations of models solely based on metabolic gene content. IMPORTANCE The metabolic capabilities of Bordetella pertussis - the causative agent of whooping cough - were investigated from a systems-level perspective. We constructed a comprehensive genome-scale metabolic model for B. pertussis , and challenged its predictions

Sandwich hybridization probes for the detection of Pseudo-nitzschia (Bacillariophyceae) species: An update to existing probes and a description of new probes.

Science.gov (United States)

Bowers, Holly A; Marin, Roman; Birch, James M; Scholin, Christopher A

2017-12-01

New sandwich hybridization assay (SHA) probes for detecting Pseudo-nitzschia species (P. arenysensis, P. fraudulenta, P. hasleana, P. pungens) are presented, along with updated cross-reactivity information on historical probes (SHA and FISH; fluorescence in situ hybridization) targeting P. australis and P. multiseries. Pseudo-nitzschia species are a cosmopolitan group of diatoms that produce varying levels of domoic acid (DA), a neurotoxin that can accumulate in finfish and shellfish and transfer throughout the food web. Consumption of infected food sources can lead to illness in humans (amnesic shellfish poisoning; ASP) and marine wildlife (domoic acid poisoning; DAP). The threat of human illness, along with economic loss from fishery closures has resulted in the implementation of monitoring protocols and intensive ecological studies. SHA probes have been instrumental in some of these efforts, as the technique performs well in complex heterogeneous sample matrices and has been adapted to benchtop and deployable (Environmental Sample Processor) platforms. The expanded probe set will enhance future efforts towards understanding spatial, temporal and successional patterns in species during bloom and non-bloom periods. Copyright © 2017 Elsevier B.V. All rights reserved.

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

DEFF Research Database (Denmark)

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

2013-01-01

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

Modulated microwave microscopy and probes used therewith

Science.gov (United States)

Lai, Keji; Kelly, Michael; Shen, Zhi-Xun

2012-09-11

A microwave microscope including a probe tip electrode vertically positionable over a sample and projecting downwardly from the end of a cantilever. A transmission line connecting the tip electrode to the electronic control system extends along the cantilever and is separated from a ground plane at the bottom of the cantilever by a dielectric layer. The probe tip may be vertically tapped near or at the sample surface at a low frequency and the microwave signal reflected from the tip/sample interaction is demodulated at the low frequency. Alternatively, a low-frequency electrical signal is also a non-linear electrical element associated with the probe tip to non-linearly interact with the applied microwave signal and the reflected non-linear microwave signal is detected at the low frequency. The non-linear element may be semiconductor junction formed near the apex of the probe tip or be an FET formed at the base of a semiconducting tip.

Differences between the genomes of lymphoblastoid cell lines and blood-derived samples

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Joesch-Cohen LM

2017-02-01

Full Text Available Lena M Joesch-Cohen, Gustavo Glusman Institute for Systems Biology, Seattle, WA, USA Abstract: Lymphoblastoid cell lines (LCLs represent a convenient research tool for expanding the amount of biologic material available from an individual. LCLs are commonly used as reference materials, most notably from the Genome in a Bottle Consortium. However, the question remains how faithfully LCL-derived genome assemblies represent the germline genome of the donor individual as compared to the genome assemblies derived from peripheral blood mononuclear cells. We present an in-depth comparison of a large collection of LCL- and peripheral blood mononuclear cell-derived genomes in terms of distributions of coverage and copy number alterations. We found significant differences in the depth of coverage and copy number calls, which may be driven by differential replication timing. Importantly, these copy number changes preferentially affect regions closer to genes and with higher GC content. This suggests that genomic studies based on LCLs may display locus-specific biases, and that conclusions based on analysis of depth of coverage and copy number variation may require further scrutiny. Keywords: genomics, whole-genome sequencing, viral transformation, copy number changes, bioinformatics

Three-way flexible cantilever probes for static contact

DEFF Research Database (Denmark)

Wang, Fei; Petersen, Dirch Hjorth; Jensen, Helle Vendelbo

2011-01-01

In micro four-point probe measurements, three-way flexible L-shaped cantilever probes show significant advantages over conventional straight cantilever probes. The L-shaped cantilever allows static contact to the sample surface which reduces the frictional wear of the cantilever tips. We analyze...

Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes.

Science.gov (United States)

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

2011-05-01

New DNA amplification methods are continuously developed for sensitive detection and quantification of specific DNA target sequences for, e.g. clinical, environmental or food applications. These new applications often require the use of long DNA oligonucleotides as probes for target sequences hybridization. Depending on the molecular technique, the length of DNA probes ranges from 40 to 450 nucleotides, solid-phase chemical synthesis being the strategy generally used for their production. However, the fidelity of chemical synthesis of DNA decreases for larger DNA probes. Defects in the oligonucleotide sequence result in the loss of hybridization efficiency, affecting the sensitivity and selectivity of the amplification method. In this work, an enzymatic procedure has been developed as an alternative to solid-phase chemical synthesis for the production of long oligonucleotides. The enzymatic procedure for probe production was based on ligation of short DNA sequences. Long DNA probes were obtained from smaller oligonucleotides together with a short sequence that acts as bridge stabilizing the molecular complex for DNA ligation. The ligation reactions were monitored by capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) using a bare fused-silica capillary. The capillary gel electrophoresis-LIF method demonstrated to be very useful and informative for the characterization of the ligation reaction, providing important information about the nature of some impurities, as well as for the fine optimization of the ligation conditions (i.e. ligation cycles, oligonucleotide and enzyme concentration). As a result, the yield and quality of the ligation product were highly improved. The in-lab prepared DNA probes were used in a novel multiplex ligation-dependent genome amplification (MLGA) method for the detection of genetically modified maize in samples. The great possibilities of the whole approach were demonstrated by the specific and sensitive

33S NMR cryogenic probe for taurine detection

Science.gov (United States)

Hobo, Fumio; Takahashi, Masato; Maeda, Hideaki

2009-03-01

With the goal of a S33 nuclear magnetic resonance (NMR) probe applicable to in vivo NMR on taurine-biological samples, we have developed the S33 NMR cryogenic probe, which is applicable to taurine solutions. The NMR sensitivity gain relative to a conventional broadband probe is as large as 3.5. This work suggests that improvements in the preamplifier could allow NMR measurements on 100 μM taurine solutions, which is the level of sensitivity necessary for biological samples.

Microarray MAPH: accurate array-based detection of relative copy number in genomic DNA

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Chan Alan

2006-06-01

Full Text Available Abstract Background Current methods for measurement of copy number do not combine all the desirable qualities of convenience, throughput, economy, accuracy and resolution. In this study, to improve the throughput associated with Multiplex Amplifiable Probe Hybridisation (MAPH we aimed to develop a modification based on the 3-Dimensional, Flow-Through Microarray Platform from PamGene International. In this new method, electrophoretic analysis of amplified products is replaced with photometric analysis of a probed oligonucleotide array. Copy number analysis of hybridised probes is based on a dual-label approach by comparing the intensity of Cy3-labelled MAPH probes amplified from test samples co-hybridised with similarly amplified Cy5-labelled reference MAPH probes. The key feature of using a hybridisation-based end point with MAPH is that discrimination of amplified probes is based on sequence and not fragment length. Results In this study we showed that microarray MAPH measurement of PMP22 gene dosage correlates well with PMP22 gene dosage determined by capillary MAPH and that copy number was accurately reported in analyses of DNA from 38 individuals, 12 of which were known to have Charcot-Marie-Tooth disease type 1A (CMT1A. Conclusion Measurement of microarray-based endpoints for MAPH appears to be of comparable accuracy to electrophoretic methods, and holds the prospect of fully exploiting the potential multiplicity of MAPH. The technology has the potential to simplify copy number assays for genes with a large number of exons, or of expanded sets of probes from dispersed genomic locations.

Microarray MAPH: accurate array-based detection of relative copy number in genomic DNA.

Science.gov (United States)

Gibbons, Brian; Datta, Parikkhit; Wu, Ying; Chan, Alan; Al Armour, John

2006-06-30

Current methods for measurement of copy number do not combine all the desirable qualities of convenience, throughput, economy, accuracy and resolution. In this study, to improve the throughput associated with Multiplex Amplifiable Probe Hybridisation (MAPH) we aimed to develop a modification based on the 3-Dimensional, Flow-Through Microarray Platform from PamGene International. In this new method, electrophoretic analysis of amplified products is replaced with photometric analysis of a probed oligonucleotide array. Copy number analysis of hybridised probes is based on a dual-label approach by comparing the intensity of Cy3-labelled MAPH probes amplified from test samples co-hybridised with similarly amplified Cy5-labelled reference MAPH probes. The key feature of using a hybridisation-based end point with MAPH is that discrimination of amplified probes is based on sequence and not fragment length. In this study we showed that microarray MAPH measurement of PMP22 gene dosage correlates well with PMP22 gene dosage determined by capillary MAPH and that copy number was accurately reported in analyses of DNA from 38 individuals, 12 of which were known to have Charcot-Marie-Tooth disease type 1A (CMT1A). Measurement of microarray-based endpoints for MAPH appears to be of comparable accuracy to electrophoretic methods, and holds the prospect of fully exploiting the potential multiplicity of MAPH. The technology has the potential to simplify copy number assays for genes with a large number of exons, or of expanded sets of probes from dispersed genomic locations.

Development of Prevotella intermedia-specific PCR primers based on the nucleotide sequences of a DNA probe Pig27.

Science.gov (United States)

Kim, Min Jung; Hwang, Kyung Hwan; Lee, Young-Seok; Park, Jae-Yoon; Kook, Joong-Ki

2011-03-01

The aim of this study was to develop Prevotella intermedia-specific PCR primers based on the P. intermedia-specific DNA probe. The P. intermedia-specific DNA probe was screened by inverted dot blot hybridization and confirmed by Southern blot hybridization. The nucleotide sequences of the species-specific DNA probes were determined using a chain termination method. Southern blot analysis showed that the DNA probe, Pig27, detected only the genomic DNA of P. intermedia strains. PCR showed that the PCR primers, Pin-F1/Pin-R1, had species-specificity for P. intermedia. The detection limits of the PCR primer sets were 0.4pg of the purified genomic DNA of P. intermedia ATCC 49046. These results suggest that the PCR primers, Pin-F1/Pin-R1, could be useful in the detection of P. intermedia as well as in the development of a PCR kit in epidemiological studies related to periodontal diseases. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

Genomic heterogeneity among human and nonhuman strains of hepatitis A virus

International Nuclear Information System (INIS)

Lemon, S.M.; Chao, S.F.; Jansen, R.W.; Binn, L.N.; LeDuc, J.W.

1987-01-01

Cloned cDNA probes derived from the P1 and P2 regions of the genome of HM175 virus, a reference strain of human hepatitis A virus (HAV), failed to hybridize under standard stringency criteria with RNA from PA21 and PA33 viruses, two epizootiologically related HAV strains recovered from naturally infected New World owl monkeys. Hybridization of these probes to PA21 RNA was only evident under reduced stringency conditions. However, cDNA representing the 5' nontranslated region of the MH175 genome hybridized equally to HM175 and PA21 RNA under standard stringency conditions, while a probe derived from the 3', 1400 bases of the genome yielded a reduced hybridization signal with PA21 RNA. In contrast, no differences could be discerned between HM175 virus and three other HAV strains of human origin (GR8, LV374, and MS1) in any region of the genome, unless increased stringency conditions were used. These results suggest that PA21 and PA33 are unique among HAV isolates and may represent a virus native to the owl monkey. Despite extremely poor homology within the P1 region, which encodes capsid polypeptides, monoclonal antibody analysis confirmed that the immunodominant neutralization epitopes of HAV were highly conserved between HM175 and PA21 viruses. These data provide molecular evidence for the existence of HAV strains unique to nonhuman species and indicate that strict conservation of antigenic function may accompany substantial genetic divergence in HAV

A statistical method for predicting splice variants between two groups of samples using GeneChip® expression array data

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Olson James M

2006-04-01

Full Text Available Abstract Background Alternative splicing of pre-messenger RNA results in RNA variants with combinations of selected exons. It is one of the essential biological functions and regulatory components in higher eukaryotic cells. Some of these variants are detectable with the Affymetrix GeneChip® that uses multiple oligonucleotide probes (i.e. probe set, since the target sequences for the multiple probes are adjacent within each gene. Hybridization intensity from a probe correlates with abundance of the corresponding transcript. Although the multiple-probe feature in the current GeneChip® was designed to assess expression values of individual genes, it also measures transcriptional abundance for a sub-region of a gene sequence. This additional capacity motivated us to develop a method to predict alternative splicing, taking advance of extensive repositories of GeneChip® gene expression array data. Results We developed a two-step approach to predict alternative splicing from GeneChip® data. First, we clustered the probes from a probe set into pseudo-exons based on similarity of probe intensities and physical adjacency. A pseudo-exon is defined as a sequence in the gene within which multiple probes have comparable probe intensity values. Second, for each pseudo-exon, we assessed the statistical significance of the difference in probe intensity between two groups of samples. Differentially expressed pseudo-exons are predicted to be alternatively spliced. We applied our method to empirical data generated from GeneChip® Hu6800 arrays, which include 7129 probe sets and twenty probes per probe set. The dataset consists of sixty-nine medulloblastoma (27 metastatic and 42 non-metastatic samples and four cerebellum samples as normal controls. We predicted that 577 genes would be alternatively spliced when we compared normal cerebellum samples to medulloblastomas, and predicted that thirteen genes would be alternatively spliced when we compared metastatic

COMBO-FISH Enables High Precision Localization Microscopy as a Prerequisite for Nanostructure Analysis of Genome Loci

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Rainer Kaufmann

2010-10-01

Full Text Available With the completeness of genome databases, it has become possible to develop a novel FISH (Fluorescence in Situ Hybridization technique called COMBO-FISH (COMBinatorial Oligo FISH. In contrast to other FISH techniques, COMBO-FISH makes use of a bioinformatics approach for probe set design. By means of computer genome database searching, several oligonucleotide stretches of typical lengths of 15–30 nucleotides are selected in such a way that all uniquely colocalize at the given genome target. The probes applied here were Peptide Nucleic Acids (PNAs—synthetic DNA analogues with a neutral backbone—which were synthesized under high purity conditions. For a probe repetitively highlighted in centromere 9, PNAs labeled with different dyes were tested, among which Alexa 488Ò showed reversible photobleaching (blinking between dark and bright state a prerequisite for the application of SPDM (Spectral Precision Distance/Position Determination Microscopy a novel technique of high resolution fluorescence localization microscopy. Although COMBO-FISH labeled cell nuclei under SPDM conditions sometimes revealed fluorescent background, the specific locus was clearly discriminated by the signal intensity and the resulting localization accuracy in the range of 10–20 nm for a detected oligonucleotide stretch. The results indicate that COMBO-FISH probes with blinking dyes are well suited for SPDM, which will open new perspectives on molecular nanostructural analysis of the genome.

Genome-wide identification of significant aberrations in cancer genome

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Yuan Xiguo

2012-07-01

Full Text Available Abstract Background Somatic Copy Number Alterations (CNAs in human genomes are present in almost all human cancers. Systematic efforts to characterize such structural variants must effectively distinguish significant consensus events from random background aberrations. Here we introduce Significant Aberration in Cancer (SAIC, a new method for characterizing and assessing the statistical significance of recurrent CNA units. Three main features of SAIC include: (1 exploiting the intrinsic correlation among consecutive probes to assign a score to each CNA unit instead of single probes; (2 performing permutations on CNA units that preserve correlations inherent in the copy number data; and (3 iteratively detecting Significant Copy Number Aberrations (SCAs and estimating an unbiased null distribution by applying an SCA-exclusive permutation scheme. Results We test and compare the performance of SAIC against four peer methods (GISTIC, STAC, KC-SMART, CMDS on a large number of simulation datasets. Experimental results show that SAIC outperforms peer methods in terms of larger area under the Receiver Operating Characteristics curve and increased detection power. We then apply SAIC to analyze structural genomic aberrations acquired in four real cancer genome-wide copy number data sets (ovarian cancer, metastatic prostate cancer, lung adenocarcinoma, glioblastoma. When compared with previously reported results, SAIC successfully identifies most SCAs known to be of biological significance and associated with oncogenes (e.g., KRAS, CCNE1, and MYC or tumor suppressor genes (e.g., CDKN2A/B. Furthermore, SAIC identifies a number of novel SCAs in these copy number data that encompass tumor related genes and may warrant further studies. Conclusions Supported by a well-grounded theoretical framework, SAIC has been developed and used to identify SCAs in various cancer copy number data sets, providing useful information to study the landscape of cancer genomes

Detecting the effects of toxic agents on spermatogenesis using DNA probes

International Nuclear Information System (INIS)

Hecht, N.B.

1987-01-01

Advances in the molecular biology of spermatogenesis suggest that DNA probes can be used to monitor the effects of toxic agents in male germ cells of mammals. Molecular hybridization analyses with DNA probes can provide a reproducible methodology capable of detecting changes ranging from massive deletions to single base pair substitutions in the genome of exposed individuals. A constantly increasing number of DNA probes that can be used to detect such alterations in human sperm DNA exist for both ubiquitously expressed proteins and for genes solely expressed in the testis. In this chapter, the currently available testicular stage-specific and/or cell type-specific DNA probes and the techniques by which they can be utilized in reproductive toxicology studies are discussed. The advantages, limitations, and future technological advances of this novel biological marker system for the human male reproductive system are also considered

Hall probe magnetometer for SSC magnet cables

International Nuclear Information System (INIS)

Cross, R.W.; Goldfarb, R.B.

1991-01-01

The authors of this paper constructed a Hall probe magnetometer to measure the magnetization hysteresis loops of Superconducting Super Collider magnet cables. The instrument uses two Hall-effect field sensors to measure the applied field H and the magnetic induction B. Magnetization M is calculated from the difference of the two quantities. The Hall probes are centered coaxially in the bore of a superconducting solenoid with the B probe against the sample's broad surface. An alternative probe arrangement, in which M is measured directly, aligns the sample probe parallel to the field. The authors measured M as a function of H and field cycle rate both with and without a dc transport current. Flux creep as a function of current was measured from the dependence of ac loss on the cycling rate and from the decay of magnetization with time. Transport currents up to 20% of the critical current have minimal effect on magnetization and flux creep

Reproducibility of subgingival bacterial samples from patients with peri-implant mucositis

DEFF Research Database (Denmark)

Hallström, Hadar; Persson, G Rutger; Strömberg, Ulf

2015-01-01

collected with paper points and analyzed using the checkerboard DNA-DNA hybridization technique. Whole genomic probes of 74 preselected bacterial species were used. Based on the bacterial scores, Cohen's kappa coefficient was used to calculate the inter-annotator agreement for categorical data......OBJECTIVE: The aim of the present study was to investigate the reproducibility of bacterial enumeration from subsequent subgingival samples collected from patients with peri-implant mucositis. MATERIAL AND METHODS: Duplicate microbial samples from 222 unique implant sites in 45 adult subjects were....... The percentage agreement was considered as "good" when the two samples showed the same score or differed by 1 to the power of 10. RESULTS: Moderate to fair kappa values were displayed for all bacterial species in the test panel (range 0.21-0.58). There were no significant differences between Gram...

Optimised padlock probe ligation and microarray detection of multiple (non-authorised GMOs in a single reaction

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Schoen Cor D

2008-12-01

Full Text Available Abstract Background To maintain EU GMO regulations, producers of new GM crop varieties need to supply an event-specific method for the new variety. As a result methods are nowadays available for EU-authorised genetically modified organisms (GMOs, but only to a limited extent for EU-non-authorised GMOs (NAGs. In the last decade the diversity of genetically modified (GM ingredients in food and feed has increased significantly. As a result of this increase GMO laboratories currently need to apply many different methods to establish to potential presence of NAGs in raw materials and complex derived products. Results In this paper we present an innovative method for detecting (approved GMOs as well as the potential presence of NAGs in complex DNA samples containing different crop species. An optimised protocol has been developed for padlock probe ligation in combination with microarray detection (PPLMD that can easily be scaled up. Linear padlock probes targeted against GMO-events, -elements and -species have been developed that can hybridise to their genomic target DNA and are visualised using microarray hybridisation. In a tenplex PPLMD experiment, different genomic targets in Roundup-Ready soya, MON1445 cotton and Bt176 maize were detected down to at least 1%. In single experiments, the targets were detected down to 0.1%, i.e. comparable to standard qPCR. Conclusion Compared to currently available methods this is a significant step forward towards multiplex detection in complex raw materials and derived products. It is shown that the PPLMD approach is suitable for large-scale detection of GMOs in real-life samples and provides the possibility to detect and/or identify NAGs that would otherwise remain undetected.

Optimised padlock probe ligation and microarray detection of multiple (non-authorised) GMOs in a single reaction.

Science.gov (United States)

Prins, Theo W; van Dijk, Jeroen P; Beenen, Henriek G; Van Hoef, Am Angeline; Voorhuijzen, Marleen M; Schoen, Cor D; Aarts, Henk J M; Kok, Esther J

2008-12-04

To maintain EU GMO regulations, producers of new GM crop varieties need to supply an event-specific method for the new variety. As a result methods are nowadays available for EU-authorised genetically modified organisms (GMOs), but only to a limited extent for EU-non-authorised GMOs (NAGs). In the last decade the diversity of genetically modified (GM) ingredients in food and feed has increased significantly. As a result of this increase GMO laboratories currently need to apply many different methods to establish to potential presence of NAGs in raw materials and complex derived products. In this paper we present an innovative method for detecting (approved) GMOs as well as the potential presence of NAGs in complex DNA samples containing different crop species. An optimised protocol has been developed for padlock probe ligation in combination with microarray detection (PPLMD) that can easily be scaled up. Linear padlock probes targeted against GMO-events, -elements and -species have been developed that can hybridise to their genomic target DNA and are visualised using microarray hybridisation.In a tenplex PPLMD experiment, different genomic targets in Roundup-Ready soya, MON1445 cotton and Bt176 maize were detected down to at least 1%. In single experiments, the targets were detected down to 0.1%, i.e. comparable to standard qPCR. Compared to currently available methods this is a significant step forward towards multiplex detection in complex raw materials and derived products. It is shown that the PPLMD approach is suitable for large-scale detection of GMOs in real-life samples and provides the possibility to detect and/or identify NAGs that would otherwise remain undetected.

The effects of sampling on the efficiency and accuracy of k-mer indexes: Theoretical and empirical comparisons using the human genome.

Science.gov (United States)

Almutairy, Meznah; Torng, Eric

2017-01-01

One of the most common ways to search a sequence database for sequences that are similar to a query sequence is to use a k-mer index such as BLAST. A big problem with k-mer indexes is the space required to store the lists of all occurrences of all k-mers in the database. One method for reducing the space needed, and also query time, is sampling where only some k-mer occurrences are stored. Most previous work uses hard sampling, in which enough k-mer occurrences are retained so that all similar sequences are guaranteed to be found. In contrast, we study soft sampling, which further reduces the number of stored k-mer occurrences at a cost of decreasing query accuracy. We focus on finding highly similar local alignments (HSLA) over nucleotide sequences, an operation that is fundamental to biological applications such as cDNA sequence mapping. For our comparison, we use the NCBI BLAST tool with the human genome and human ESTs. When identifying HSLAs, we find that soft sampling significantly reduces both index size and query time with relatively small losses in query accuracy. For the human genome and HSLAs of length at least 100 bp, soft sampling reduces index size 4-10 times more than hard sampling and processes queries 2.3-6.8 times faster, while still achieving retention rates of at least 96.6%. When we apply soft sampling to the problem of mapping ESTs against the genome, we map more than 98% of ESTs perfectly while reducing the index size by a factor of 4 and query time by 23.3%. These results demonstrate that soft sampling is a simple but effective strategy for performing efficient searches for HSLAs. We also provide a new model for sampling with BLAST that predicts empirical retention rates with reasonable accuracy by modeling two key problem factors.

Improved controlled atmosphere high temperature scanning probe microscope

DEFF Research Database (Denmark)

Hansen, Karin Vels; Wu, Yuehua; Jacobsen, Torben

2013-01-01

fuel cells and electrolyzer cells. Here, we report on advanced improvements of our original controlled atmosphere high temperature scanning probe microscope, CAHT-SPM. The new microscope can employ a broad range of the scanning probe techniques including tapping mode, scanning tunneling microscopy......, scanning tunneling spectroscopy, conductive atomic force microscopy, and Kelvin probe force microscopy. The temperature of the sample can be as high as 850 °C. Both reducing and oxidizing gases such as oxygen, hydrogen, and nitrogen can be added in the sample chamber and the oxygen partial pressure (pO2...

Molluscan Evolutionary Genomics

Energy Technology Data Exchange (ETDEWEB)

Simison, W. Brian; Boore, Jeffrey L.

2005-12-01

In the last 20 years there have been dramatic advances in techniques of high-throughput DNA sequencing, most recently accelerated by the Human Genome Project, a program that has determined the three billion base pair code on which we are based. Now this tremendous capability is being directed at other genome targets that are being sampled across the broad range of life. This opens up opportunities as never before for evolutionary and organismal biologists to address questions of both processes and patterns of organismal change. We stand at the dawn of a new 'modern synthesis' period, paralleling that of the early 20th century when the fledgling field of genetics first identified the underlying basis for Darwin's theory. We must now unite the efforts of systematists, paleontologists, mathematicians, computer programmers, molecular biologists, developmental biologists, and others in the pursuit of discovering what genomics can teach us about the diversity of life. Genome-level sampling for mollusks to date has mostly been limited to mitochondrial genomes and it is likely that these will continue to provide the best targets for broad phylogenetic sampling in the near future. However, we are just beginning to see an inroad into complete nuclear genome sequencing, with several mollusks and other eutrochozoans having been selected for work about to begin. Here, we provide an overview of the state of molluscan mitochondrial genomics, highlight a few of the discoveries from this research, outline the promise of broadening this dataset, describe upcoming projects to sequence whole mollusk nuclear genomes, and challenge the community to prepare for making the best use of these data.

Full information acquisition in scanning probe microscopy and spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Jesse, Stephen; Belianinov, Alex; Kalinin, Sergei V.; Somnath, Suhas

2017-04-04

Apparatus and methods are described for scanning probe microscopy and spectroscopy based on acquisition of full probe response. The full probe response contains valuable information about the probe-sample interaction that is lost in traditional scanning probe microscopy and spectroscopy methods. The full probe response is analyzed post data acquisition using fast Fourier transform and adaptive filtering, as well as multivariate analysis. The full response data is further compressed to retain only statistically significant components before being permanently stored.

DNA probes for distinguishing Psychodopygus wellcomei from Psychodopygus complexus (Diptera: Psychodidae

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P. D. Ready

1991-03-01

Full Text Available Genomic DNA fragments from males of Psychodopygus wellcomei were isolated and shown to be useful as sensitive diagnostic probles for positively separting individuals of this species from those of Ps. complexus. These two members of the Ps. squamiventris series are found sympatrically in foci of cutaneous leishmaniasis in the hill forests of southern Pará State. Of the two species, only Ps. welcomei is thought to be an important vector of Leishmania braziliensis sensu stricto, buth this is based on circumstantial evidence because of the difficulties of identifying female sandflies wothin the series. The diagnostic probes were isolated from a library of Ps. wellcomei built by ligationg short fragments of Sau 3A-resistricted, genomic DNA into the plasmid vector PUC 18. Differential screening of 1316 library clones with total genomic DNA of Ps. Wellcomei and Ps. complexus identified 5 recombinants, with cross-hybridizing inserts of repetitive DNA, that showed strong specificity for Ps. wellcomei. As little as 0.4% of the DNA extracted from an individual sandfly (=ca. 0.5 namograms was specifically detected. The diagnostic probes were used to identify as Ps. wellcomei a wild-caught female sandfly found infected with L. braziliensis s.s., providing only the second positive association between these two species.

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

Science.gov (United States)

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

2011-01-01

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

A hidden Markov model approach for determining expression from genomic tiling micro arrays

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Krogh Anders

2006-05-01

Full Text Available Abstract Background Genomic tiling micro arrays have great potential for identifying previously undiscovered coding as well as non-coding transcription. To-date, however, analyses of these data have been performed in an ad hoc fashion. Results We present a probabilistic procedure, ExpressHMM, that adaptively models tiling data prior to predicting expression on genomic sequence. A hidden Markov model (HMM is used to model the distributions of tiling array probe scores in expressed and non-expressed regions. The HMM is trained on sets of probes mapped to regions of annotated expression and non-expression. Subsequently, prediction of transcribed fragments is made on tiled genomic sequence. The prediction is accompanied by an expression probability curve for visual inspection of the supporting evidence. We test ExpressHMM on data from the Cheng et al. (2005 tiling array experiments on ten Human chromosomes 1. Results can be downloaded and viewed from our web site 2. Conclusion The value of adaptive modelling of fluorescence scores prior to categorisation into expressed and non-expressed probes is demonstrated. Our results indicate that our adaptive approach is superior to the previous analysis in terms of nucleotide sensitivity and transfrag specificity.

Sparse representation and Bayesian detection of genome copy number alterations from microarray data.

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Pique-Regi, Roger; Monso-Varona, Jordi; Ortega, Antonio; Seeger, Robert C; Triche, Timothy J; Asgharzadeh, Shahab

2008-02-01

Genomic instability in cancer leads to abnormal genome copy number alterations (CNA) that are associated with the development and behavior of tumors. Advances in microarray technology have allowed for greater resolution in detection of DNA copy number changes (amplifications or deletions) across the genome. However, the increase in number of measured signals and accompanying noise from the array probes present a challenge in accurate and fast identification of breakpoints that define CNA. This article proposes a novel detection technique that exploits the use of piece wise constant (PWC) vectors to represent genome copy number and sparse Bayesian learning (SBL) to detect CNA breakpoints. First, a compact linear algebra representation for the genome copy number is developed from normalized probe intensities. Second, SBL is applied and optimized to infer locations where copy number changes occur. Third, a backward elimination (BE) procedure is used to rank the inferred breakpoints; and a cut-off point can be efficiently adjusted in this procedure to control for the false discovery rate (FDR). The performance of our algorithm is evaluated using simulated and real genome datasets and compared to other existing techniques. Our approach achieves the highest accuracy and lowest FDR while improving computational speed by several orders of magnitude. The proposed algorithm has been developed into a free standing software application (GADA, Genome Alteration Detection Algorithm). http://biron.usc.edu/~piquereg/GADA

Copper nanocluster coupling europium as an off-to-on fluorescence probe for the determination of phosphate ion in water samples.

Science.gov (United States)

Cao, Haiyan; Chen, Zhaohui; Huang, Yuming

2015-10-01

This paper reports an "off-to-on" fluorescence (FL) probe for sensitively and selectively detecting phosphate ions (Pi's). Fabrication of the probe was based on the competition between Pi's and tannic acid-stabilized copper nanoclusters (TA-Cu NCs) for Eu(3+) binding. The addition of Eu(3+) ions to TA-Cu NCs triggered the aggregation of TA-Cu NCs, which quenched the FL of TA-Cu NCs. After Pi addition, the aggregated TA-Cu NCs solubilized into the aqueous solution to facilitate the Pi-triggered dispersion of TA-Cu NCs. This phenomenon was due to the stronger binding ability between Pi's and Eu(3+) than that between TA and Eu(3+), leading to FL recovery of Cu NCs. The degree of redispersion of TA-Cu NCs was directly related to Pi concentration. Thus, Pi concentration can be quantitatively determined by the change in FL of the TA-Cu NCs dispersion. Under the optimized conditions, the change in FL presented a linear relationship with Pi concentration from 0.07 μmol L(-1) to 80 μmol L(-1). The limit of detection for Pi was 9.6×10(-3) μmol L(-1) at a signal-to-noise ratio of 3. For Pi determination in real samples, only 1 mL water sample was needed. The proposed probe was highly sensitive, free from the interference of other common species in aqueous media, and particularly useful for the fast and simple diagnosis of water-eutrophication extent. Copyright © 2015 Elsevier B.V. All rights reserved.

Development and Antarctic Testing of a Maneuverable Probe for Clean In-Situ Analysis and Sampling of Subsurface Ice and Subglacial Aquatic Ecosystems

Science.gov (United States)

Francke, G.; Dachwald, B.; Kowalski, J.; Digel, I.; Tulaczyk, S. M.; Mikucki, J.; Feldmann, M.; Espe, C.; Schöngarth, S.; Hiecker, S.; Blandfort, D.; Schüller, K.; Plescher, E.

2016-12-01

There is significant interest in sampling subglacial environments for geochemical and microbiological studies, but those environments are difficult to access. Such environments exist not only on Earth but are also expected beneath the icy crusts of some outer solar system bodies, like the Jovian moon Europa and the Saturnian moon Enceladus. Existing ice drilling technologies make it cumbersome to maintain microbiologically clean access for sample acquisition and environmental stewardship of potentially fragile subglacial aquatic ecosystems. The "IceMole" is a maneuverable subsurface ice probe for clean in-situ analysis and sampling of glacial ice and subglacial materials. The design is based on combining melting and mechanical propulsion, using an ice screw at the tip of the melting head to maintain firm contact between the melting head and the ice. It can change melting direction by differential heating of the melting head and optional side wall heaters. The first two prototypes were successfully tested between 2010 and 2012 on glaciers in Switzerland and Iceland, where they demonstrated downward, horizontal and upward melting, as well as curve driving and dirt layer penetration. Hence, the IceMole allows maneuvers which may be necessary for obstacle avoidance or target selection. Maneuverability, however, necessitates a sophisticated on-board navigation system capable of autonomous operations. Therefore, between 2012 and 2014, a more advanced probe was developed as part of the "Enceladus Explorer" (EnEx) project. The EnEx-IceMole offers systems for relative positioning based on in-ice attitude determination, acoustic positioning, ultrasonic obstacle and target detection, which is all integrated through a high-level sensor fusion. In December 2014, it was used for clean access into a unique subglacial aquatic environment at Blood Falls, Antarctica, where a subglacial brine sample was successfully obtained after about 17 meters of oblique melting. Particular

Immunoglobulin heavy-chain fluorescence in situ hybridization-chromogenic in situ hybridization DNA probe split signal in the clonality assessment of lymphoproliferative processes on cytological samples.

Science.gov (United States)

Zeppa, Pio; Sosa Fernandez, Laura Virginia; Cozzolino, Immacolata; Ronga, Valentina; Genesio, Rita; Salatiello, Maria; Picardi, Marco; Malapelle, Umberto; Troncone, Giancarlo; Vigliar, Elena

2012-12-25

The human immunoglobulin heavy-chain (IGH) locus at chromosome 14q32 is frequently involved in different translocations of non-Hodgkin lymphoma (NHL), and the detection of any breakage involving the IGH locus should identify a B-cell NHL. The split-signal IGH fluorescence in situ hybridization-chromogenic in situ hybridization (FISH-CISH) DNA probe is a mixture of 2 fluorochrome-labeled DNAs: a green one that binds the telomeric segment and a red one that binds the centromeric segment, both on the IGH breakpoint. In the current study, the authors tested the capability of the IGH FISH-CISH DNA probe to detect IGH translocations and diagnose B-cell lymphoproliferative processes on cytological samples. Fifty cytological specimens from cases of lymphoproliferative processes were tested using the split-signal IGH FISH-CISH DNA probe and the results were compared with light-chain assessment by flow cytometry (FC), IGH status was tested by polymerase chain reaction (PCR), and clinicohistological data. The signal score produced comparable results on FISH and CISH analysis and detected 29 positive, 15 negative, and 6 inadequate cases; there were 29 true-positive cases (66%), 9 true-negative cases (20%), 6 false-negative cases (14%), and no false-positive cases (0%). Comparing the sensitivity of the IGH FISH-CISH DNA split probe with FC and PCR, the highest sensitivity was obtained by FC, followed by FISH-CISH and PCR. The split-signal IGH FISH-CISH DNA probe is effective in detecting any translocation involving the IGH locus. This probe can be used on different samples from different B-cell lymphoproliferative processes, although it is not useful for classifying specific entities. Cancer (Cancer Cytopathol) 2012;. © 2012 American Cancer Society. Copyright © 2012 American Cancer Society.

Visualization of RNA structure models within the Integrative Genomics Viewer.

Science.gov (United States)

Busan, Steven; Weeks, Kevin M

2017-07-01

Analyses of the interrelationships between RNA structure and function are increasingly important components of genomic studies. The SHAPE-MaP strategy enables accurate RNA structure probing and realistic structure modeling of kilobase-length noncoding RNAs and mRNAs. Existing tools for visualizing RNA structure models are not suitable for efficient analysis of long, structurally heterogeneous RNAs. In addition, structure models are often advantageously interpreted in the context of other experimental data and gene annotation information, for which few tools currently exist. We have developed a module within the widely used and well supported open-source Integrative Genomics Viewer (IGV) that allows visualization of SHAPE and other chemical probing data, including raw reactivities, data-driven structural entropies, and data-constrained base-pair secondary structure models, in context with linear genomic data tracks. We illustrate the usefulness of visualizing RNA structure in the IGV by exploring structure models for a large viral RNA genome, comparing bacterial mRNA structure in cells with its structure under cell- and protein-free conditions, and comparing a noncoding RNA structure modeled using SHAPE data with a base-pairing model inferred through sequence covariation analysis. © 2017 Busan and Weeks; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Piezoelectric Sensor for Determination of Genetically Modified Soybean Roundup Readyâ in Samples not Amplified by PCR

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Hanna Radecka

2007-08-01

Full Text Available The chemically modified piezoelectrodes were utilized to develop relativelycheap and easy to use biosensor for determination of genetically modified Roundup Readysoybean (RR soybean. The biosensor relies on the immobilization onto goldpiezoelectrodes of the 21-mer single stranded oligonucleotide (probes related to5-enolpyruvylshikimate-3-phosphate synthase (EPSPS gene, which is an active componentof an insert integrated into RR soybean genome. The hybridization reaction between theprobe and the target complementary sequence in solution was monitored. The system wasoptimized using synthetic oligonucleotides, which were applied for EPSPS gene detectionin DNA samples extracted from animal feed containing 30% RR soybean amplified by thePCR and nonamplified by PCR. The detection limit for genomic DNA was in the range of4.7·105 numbers of genom copies contained EPSPS gene in the QCM cell. The propertiessuch as sensitivity and selectivity of piezoelectric senor presented here indicated that it could be applied for the direct determination of genetically modified RR soybean in the samples non-amplified by PCR.

Genomic Variants Revealed by Invariably Missing Genotypes in Nelore Cattle.

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Joaquim Manoel da Silva

Full Text Available High density genotyping panels have been used in a wide range of applications. From population genetics to genome-wide association studies, this technology still offers the lowest cost and the most consistent solution for generating SNP data. However, in spite of the application, part of the generated data is always discarded from final datasets based on quality control criteria used to remove unreliable markers. Some discarded data consists of markers that failed to generate genotypes, labeled as missing genotypes. A subset of missing genotypes that occur in the whole population under study may be caused by technical issues but can also be explained by the presence of genomic variations that are in the vicinity of the assayed SNP and that prevent genotyping probes from annealing. The latter case may contain relevant information because these missing genotypes might be used to identify population-specific genomic variants. In order to assess which case is more prevalent, we used Illumina HD Bovine chip genotypes from 1,709 Nelore (Bos indicus samples. We found 3,200 missing genotypes among the whole population. NGS re-sequencing data from 8 sires were used to verify the presence of genomic variations within their flanking regions in 81.56% of these missing genotypes. Furthermore, we discovered 3,300 novel SNPs/Indels, 31% of which are located in genes that may affect traits of importance for the genetic improvement of cattle production.

A Thousand Fly Genomes: An Expanded Drosophila Genome Nexus.

Science.gov (United States)

Lack, Justin B; Lange, Jeremy D; Tang, Alison D; Corbett-Detig, Russell B; Pool, John E

2016-12-01

The Drosophila Genome Nexus is a population genomic resource that provides D. melanogaster genomes from multiple sources. To facilitate comparisons across data sets, genomes are aligned using a common reference alignment pipeline which involves two rounds of mapping. Regions of residual heterozygosity, identity-by-descent, and recent population admixture are annotated to enable data filtering based on the user's needs. Here, we present a significant expansion of the Drosophila Genome Nexus, which brings the current data object to a total of 1,121 wild-derived genomes. New additions include 305 previously unpublished genomes from inbred lines representing six population samples in Egypt, Ethiopia, France, and South Africa, along with another 193 genomes added from recently-published data sets. We also provide an aligned D. simulans genome to facilitate divergence comparisons. This improved resource will broaden the range of population genomic questions that can addressed from multi-population allele frequencies and haplotypes in this model species. The larger set of genomes will also enhance the discovery of functionally relevant natural variation that exists within and between populations. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Intrauterine photoacoustic and ultrasound imaging probe

Science.gov (United States)

Miranda, Christopher; Barkley, Joel; Smith, Barbara S.

2018-04-01

Intrauterine photoacoustic and ultrasound imaging are probe-based imaging modalities with translational potential for use in detecting endometrial diseases. This deep-tissue imaging probe design allows for the retrofitting of commercially available endometrial sampling curettes. The imaging probe presented here has a 2.92-mm diameter and approximate length of 26 cm, which allows for entry into the human endometrial cavity, making it possible to use photoacoustic imaging and high-resolution ultrasound to characterize the uterus. We demonstrate the imaging probes' ability to provide structural information of an excised pig uterus using ultrasound imaging and detect photoacoustic signals at a radial depth of 1 cm.

Why nano-oxidation with carbon nanotube probes is so stable: II. Bending behaviour of CNT probes during nano-oxidation

International Nuclear Information System (INIS)

Kuramochi, H; Tokizaki, T; Ando, K; Yokoyama, H; Dagata, J A

2007-01-01

Part I demonstrated that nano-oxidation in the dynamic-force mode was enhanced by the use of conductive carbon nanotube (CNT) probes. Fabrication of oxide nanostructures using CNT probes benefited not only from the smaller tip apex compared to conventional probes but from improved operational stability over a wide range of exposure conditions primarily due to the hydrophobic nature of the CNT. Here we investigate the bending response of CNT probes to electrostatic and meniscus forces during nano-oxidation. We conclude that bending of the CNT introduces an additional cushion in the combined cantilever-probe deflection system, thus improving overall stability of the tip-sample junction during nano-oxidation

Direct and quantitative broadband absorptance spectroscopy with multilayer cantilever probes

Science.gov (United States)

Hsu, Wei-Chun; Tong, Jonathan Kien-Kwok; Liao, Bolin; Chen, Gang

2015-04-21

A system for measuring the absorption spectrum of a sample is provided that includes a broadband light source that produces broadband light defined within a range of an absorptance spectrum. An interferometer modulates the intensity of the broadband light source for a range of modulation frequencies. A bi-layer cantilever probe arm is thermally connected to a sample arm having at most two layers of materials. The broadband light modulated by the interferometer is directed towards the sample and absorbed by the sample and converted into heat, which causes a temperature rise and bending of the bi-layer cantilever probe arm. A detector mechanism measures and records the deflection of the probe arm so as to obtain the absorptance spectrum of the sample.

A DNA-based pattern classifier with in vitro learning and associative recall for genomic characterization and biosensing without explicit sequence knowledge.

Science.gov (United States)

Lee, Ju Seok; Chen, Junghuei; Deaton, Russell; Kim, Jin-Woo

2014-01-01

Genetic material extracted from in situ microbial communities has high promise as an indicator of biological system status. However, the challenge is to access genomic information from all organisms at the population or community scale to monitor the biosystem's state. Hence, there is a need for a better diagnostic tool that provides a holistic view of a biosystem's genomic status. Here, we introduce an in vitro methodology for genomic pattern classification of biological samples that taps large amounts of genetic information from all genes present and uses that information to detect changes in genomic patterns and classify them. We developed a biosensing protocol, termed Biological Memory, that has in vitro computational capabilities to "learn" and "store" genomic sequence information directly from genomic samples without knowledge of their explicit sequences, and that discovers differences in vitro between previously unknown inputs and learned memory molecules. The Memory protocol was designed and optimized based upon (1) common in vitro recombinant DNA operations using 20-base random probes, including polymerization, nuclease digestion, and magnetic bead separation, to capture a snapshot of the genomic state of a biological sample as a DNA memory and (2) the thermal stability of DNA duplexes between new input and the memory to detect similarities and differences. For efficient read out, a microarray was used as an output method. When the microarray-based Memory protocol was implemented to test its capability and sensitivity using genomic DNA from two model bacterial strains, i.e., Escherichia coli K12 and Bacillus subtilis, results indicate that the Memory protocol can "learn" input DNA, "recall" similar DNA, differentiate between dissimilar DNA, and detect relatively small concentration differences in samples. This study demonstrated not only the in vitro information processing capabilities of DNA, but also its promise as a genomic pattern classifier that could

Continuous-flow liquid microjunction surface sampling probe connected on-line with high-performance liquid chromatography/mass spectrometry for spatially resolved analysis of small molecules and proteins.

Science.gov (United States)

Van Berkel, Gary J; Kertesz, Vilmos

2013-06-30

A continuous-flow liquid microjunction surface sampling probe extracts soluble material from surfaces for direct ionization and detection by mass spectrometry. Demonstrated here is the on-line coupling of such a probe with high-performance liquid chromatography/mass spectrometry (HPLC/MS) enabling extraction, separation and detection of small molecules and proteins from surfaces in a spatially resolved (~0.5 mm diameter spots) manner. A continuous-flow liquid microjunction surface sampling probe was connected to a six-port, two-position valve for extract collection and injection to an HPLC column. A QTRAP® 5500 hybrid triple quadrupole linear ion trap equipped with a Turbo V™ ion source operated in positive electrospray ionization (ESI) mode was used for all experiments. The system operation was tested with the extraction, separation and detection of propranolol and associated metabolites from drug dosed tissues, caffeine from a coffee bean, cocaine from paper currency, and proteins from dried sheep blood spots on paper. Confirmed in the tissue were the parent drug and two different hydroxypropranolol glucuronides. The mass spectrometric response for these compounds from different locations in the liver showed an increase with increasing extraction time (5, 20 and 40 s). For on-line separation and detection/identification of extracted proteins from dried sheep blood spots, two major protein peaks dominated the chromatogram and could be correlated with the expected masses for the hemoglobin α and β chains. Spatially resolved sampling, separation, and detection of small molecules and proteins from surfaces can be accomplished using a continuous-flow liquid microjunction surface sampling probe coupled on-line with HPLC/MS detection. Published in 2013. This article is a U.S. Government work and is in the public domain in the USA.

Development and Characterization of Complex DNA Fingerprinting Probes for the Infectious Yeast Candida dubliniensis

Science.gov (United States)

Joly, Sophie; Pujol, Claude; Rysz, Michal; Vargas, Kaaren; Soll, David R.

1999-01-01

Using a strategy to clone large genomic sequences containing repetitive elements from the infectious yeast Candida dubliniensis, the three unrelated sequences Cd1, Cd24, and Cd25, with respective molecular sizes of 15,500, 10,000, and 16,000 bp, were cloned and analyzed for their efficacy as DNA fingerprinting probes. Each generated a complex Southern blot hybridization pattern with endonuclease-digested genomic DNA. Cd1 generated an extremely variable pattern that contained all of the bands of the pattern generated by the repeat element RPS of Candida albicans. We demonstrated that Cd1 does not contain RPS but does contain a repeat element associated with RPS throughout the C. dubliniensis genome. The Cd1 pattern was the least stable over time both in vitro and in vivo and for that reason proved most effective in assessing microevolution. Cd24, which did not exhibit microevolution in vitro, was highly variable in vivo, suggesting in vivo-dependent microevolution. Cd25 was deemed the best probe for broad epidemiological studies, since it was the most stable over time, was the only truly C. dubliniensis-specific probe of the three, generated the most complex pattern, was distributed throughout all C. dubliniensis chromosomes, and separated a worldwide collection of 57 C. dubliniensis isolates into two distinct groups. The presence of a species-specific repetitive element in Cd25 adds weight to the already substantial evidence that C. dubliniensis represents a bona fide species. PMID:10074523

Modeling Atom Probe Tomography: A review

Energy Technology Data Exchange (ETDEWEB)

Vurpillot, F., E-mail: francois.vurpillot@univ-rouen.fr [Groupe de Physique des Matériaux, UMR CNRS 6634, Université de Rouen, Saint Etienne du Rouvray 76801 (France); Oberdorfer, C. [Institut für Materialwissenschaft, Lehrstuhl für Materialphysik, Universität Stuttgart, Heisenbergstr. 3, 70569 Stuttgart (Germany)

2015-12-15

Improving both the precision and the accuracy of Atom Probe Tomography reconstruction requires a correct understanding of the imaging process. In this aim, numerical modeling approaches have been developed for 15 years. The injected ingredients of these modeling tools are related to the basic physic of the field evaporation mechanism. The interplay between the sample nature and structure of the analyzed sample and the reconstructed image artefacts have pushed to gradually improve and make the model more and more sophisticated. This paper reviews the evolution of the modeling approach in Atom Probe Tomography and presents some future potential directions in order to improve the method. - Highlights: • The basics of field evaporation. • The main aspects of Atom Probe Tomography modeling. • The intrinsic limitations of the current method and future potential directions to improve the understanding of tip to image ion projection.

Persistence of microbial contamination on transvaginal ultrasound probes despite low-level disinfection procedure.

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Fatima M'Zali

Full Text Available AIM OF THE STUDY: In many countries, Low Level Disinfection (LLD of covered transvaginal ultrasound probes is recommended between patients' examinations. The aim of this study was to evaluate the antimicrobial efficacy of LLD under routine conditions on a range of microorganisms. MATERIALS AND METHODS: Samples were taken over a six month period in a private French Radiology Center. 300 specimens derived from endovaginal ultrasound probes were analyzed after disinfection of the probe with wipes impregnated with a quaternary ammonium compound and chlorhexidine. Human papillomavirus (HPV was sought in the first set of s100 samples, Chlamydia trachomatis and mycoplasmas were searched in the second set of 100 samples, bacteria and fungi in the third 100 set samples. HPV, C. trachomatis and mycoplasmas were detected by PCR amplification. PCR positive samples were subjected to a nuclease treatment before an additional PCR assay to assess the likely viable microorganisms. Bacteria and fungi were investigated by conventional methods. RESULTS: A substantial persistence of microorganisms was observed on the disinfected probes: HPV DNA was found on 13% of the samples and 7% in nuclease-resistant form. C. trachomatis DNA was detected on 20% of the probes by primary PCR but only 2% after nuclease treatment, while mycoplasma DNA was amplified in 8% and 4%, respectively. Commensal and/or environmental bacterial flora was present on 86% of the probes, occasionally in mixed culture, and at various levels (10->3000 CFU/probe; Staphylococcus aureus was cultured from 4% of the probes (10-560 CFU/probe. No fungi were isolated. CONCLUSION: Our findings raise concerns about the efficacy of impregnated towels as a sole mean for disinfection of ultrasound probes. Although the ultrasound probes are used with disposable covers, our results highlight the potential risk of cross contamination between patients during ultrasound examination and emphasize the need for reviewing

Complete genome sequences of two strains of Treponema pallidum subsp. pertenue from Ghana, Africa: Identical genome sequences in samples isolated more than 7 years apart.

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Michal Strouhal

2017-09-01

Full Text Available Treponema pallidum subsp. pertenue (TPE is the causative agent of yaws, a multi-stage disease, endemic in tropical regions of Africa, Asia, Oceania, and South America. To date, four TPE strains have been completely sequenced including three TPE strains of human origin (Samoa D, CDC-2, and Gauthier and one TPE strain (Fribourg-Blanc isolated from a baboon. All TPE strains are highly similar to T. pallidum subsp. pallidum (TPA strains. The mutation rate in syphilis and related treponemes has not been experimentally determined yet.Complete genomes of two TPE strains, CDC 2575 and Ghana-051, that infected patients in Ghana and were isolated in 1980 and 1988, respectively, were sequenced and analyzed. Both strains had identical consensus genome nucleotide sequences raising the question whether TPE CDC 2575 and Ghana-051 represent two different strains. Several lines of evidence support the fact that both strains represent independent samples including regions showing intrastrain heterogeneity (13 and 5 intrastrain heterogeneous sites in TPE Ghana-051 and TPE CDC 2575, respectively. Four of these heterogeneous sites were found in both genomes but the frequency of alternative alleles differed. The identical consensus genome sequences were used to estimate the upper limit of the yaws treponeme evolution rate, which was 4.1 x 10-10 nucleotide changes per site per generation.The estimated upper limit for the mutation rate of TPE was slightly lower than the mutation rate of E. coli, which was determined during a long-term experiment. Given the known diversity between TPA and TPE genomes and the assumption that both TPA and TPE have a similar mutation rate, the most recent common ancestor of syphilis and yaws treponemes appears to be more than ten thousand years old and likely even older.

The whole genome sequences and experimentally phased haplotypes of over 100 personal genomes.

Science.gov (United States)

Mao, Qing; Ciotlos, Serban; Zhang, Rebecca Yu; Ball, Madeleine P; Chin, Robert; Carnevali, Paolo; Barua, Nina; Nguyen, Staci; Agarwal, Misha R; Clegg, Tom; Connelly, Abram; Vandewege, Ward; Zaranek, Alexander Wait; Estep, Preston W; Church, George M; Drmanac, Radoje; Peters, Brock A

2016-10-11

Since the completion of the Human Genome Project in 2003, it is estimated that more than 200,000 individual whole human genomes have been sequenced. A stunning accomplishment in such a short period of time. However, most of these were sequenced without experimental haplotype data and are therefore missing an important aspect of genome biology. In addition, much of the genomic data is not available to the public and lacks phenotypic information. As part of the Personal Genome Project, blood samples from 184 participants were collected and processed using Complete Genomics' Long Fragment Read technology. Here, we present the experimental whole genome haplotyping and sequencing of these samples to an average read coverage depth of 100X. This is approximately three-fold higher than the read coverage applied to most whole human genome assemblies and ensures the highest quality results. Currently, 114 genomes from this dataset are freely available in the GigaDB repository and are associated with rich phenotypic data; the remaining 70 should be added in the near future as they are approved through the PGP data release process. For reproducibility analyses, 20 genomes were sequenced at least twice using independent LFR barcoded libraries. Seven genomes were also sequenced using Complete Genomics' standard non-barcoded library process. In addition, we report 2.6 million high-quality, rare variants not previously identified in the Single Nucleotide Polymorphisms database or the 1000 Genomes Project Phase 3 data. These genomes represent a unique source of haplotype and phenotype data for the scientific community and should help to expand our understanding of human genome evolution and function.

Scanning vector Hall probe microscopy

International Nuclear Information System (INIS)

Cambel, V.; Gregusova, D.; Fedor, J.; Kudela, R.; Bending, S.J.

2004-01-01

We have developed a scanning vector Hall probe microscope for mapping magnetic field vector over magnetic samples. The microscope is based on a micromachined Hall sensor and the cryostat with scanning system. The vector Hall sensor active area is ∼5x5 μm 2 . It is realized by patterning three Hall probes on the tilted faces of GaAs pyramids. Data from these 'tilted' Hall probes are used to reconstruct the full magnetic field vector. The scanning area of the microscope is 5x5 mm 2 , space resolution 2.5 μm, field resolution ∼1 μT Hz -1/2 at temperatures 10-300 K

A new type of compensated neutron logging probe

International Nuclear Information System (INIS)

Ji Changsong; Shang Xiulan; Dai Zhude; Huang Heyi; Zhang Jianguo; Zu Shihuan; Zhao Jianqiang

1988-01-01

A new type of compensated neutron logging probe with glass scintillators has been designed. High sensitivity, long plateau and stability are the features of this probe which differs from the probes with 3 He or LiI(Eu) detector. From the results of field application the measured porosity is in good correspondence with the one obtained by rock core sampling method

Intrauterine photoacoustic and ultrasound imaging probe.

Science.gov (United States)

Miranda, Christopher; Barkley, Joel; Smith, Barbara

2018-04-01

Intrauterine photoacoustic and ultrasound imaging are probe-based imaging modalities with translational potential for use in detecting endometrial diseases. This deep-tissue imaging probe design allows for the retrofitting of commercially available endometrial sampling curettes. The imaging probe presented here has a 2.92-mm diameter and approximate length of 26 cm, which allows for entry into the human endometrial cavity, making it possible to use photoacoustic imaging and high-resolution ultrasound to characterize the uterus. We demonstrate the imaging probes' ability to provide structural information of an excised pig uterus using ultrasound imaging and detect photoacoustic signals at a radial depth of 1 cm. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

The application of micro-coil NMR probe technology to metabolomics of urine and serum

International Nuclear Information System (INIS)

Grimes, John H.; O’Connell, Thomas M.

2011-01-01

Increasing the sensitivity and throughput of NMR-based metabolomics is critical for the continued growth of this field. In this paper the application of micro-coil NMR probe technology was evaluated for this purpose. The most commonly used biofluids in metabolomics are urine and serum. In this study we examine different sample limited conditions and compare the detection sensitivity of the micro-coil with a standard 5 mm NMR probe. Sample concentration is evaluated as a means to leverage the greatly improved mass sensitivity of the micro-coil probes. With very small sample volumes, the sensitivity of the micro-coil probe does indeed provide a significant advantage over the standard probe. Concentrating the samples does improve the signal detection, but the benefits do not follow the expected linear increase and are both matrix and metabolite specific. Absolute quantitation will be affected by concentration, but an analysis of relative concentrations is still possible. The choice of the micro-coil probe over a standard tube based probe will depend upon a number of factors including number of samples and initial volume but this study demonstrates the feasibility of high-throughput metabolomics with the micro-probe platform.

The Drosophila genome nexus: a population genomic resource of 623 Drosophila melanogaster genomes, including 197 from a single ancestral range population.

Science.gov (United States)

Lack, Justin B; Cardeno, Charis M; Crepeau, Marc W; Taylor, William; Corbett-Detig, Russell B; Stevens, Kristian A; Langley, Charles H; Pool, John E

2015-04-01

Hundreds of wild-derived Drosophila melanogaster genomes have been published, but rigorous comparisons across data sets are precluded by differences in alignment methodology. The most common approach to reference-based genome assembly is a single round of alignment followed by quality filtering and variant detection. We evaluated variations and extensions of this approach and settled on an assembly strategy that utilizes two alignment programs and incorporates both substitutions and short indels to construct an updated reference for a second round of mapping prior to final variant detection. Utilizing this approach, we reassembled published D. melanogaster population genomic data sets and added unpublished genomes from several sub-Saharan populations. Most notably, we present aligned data from phase 3 of the Drosophila Population Genomics Project (DPGP3), which provides 197 genomes from a single ancestral range population of D. melanogaster (from Zambia). The large sample size, high genetic diversity, and potentially simpler demographic history of the DPGP3 sample will make this a highly valuable resource for fundamental population genetic research. The complete set of assemblies described here, termed the Drosophila Genome Nexus, presently comprises 623 consistently aligned genomes and is publicly available in multiple formats with supporting documentation and bioinformatic tools. This resource will greatly facilitate population genomic analysis in this model species by reducing the methodological differences between data sets. Copyright © 2015 by the Genetics Society of America.

The use of small diameter probing equipment for contaminated site investigation

International Nuclear Information System (INIS)

Christy, T.M.; Spradlin, S.C.

1992-01-01

The past decade has witnessed a dramatic increase in the number of contaminated sites being investigated in the United States. This increase in subsurface investigation has spurred a corresponding increase in the development of subsurface sampling tools and methods. The past five years, in particular, have seen the development of small diameter (1 double-prime to 1.4 double-prime O.D.) percussion driven probing tools which can be used for the recovery of soil vapor, soil core and groundwater samples. This development has placed heretofore unavailable tools at the disposal of site investigators. Mechanized, vehicle mounted soil probe systems apply both static force and hydraulically powered percussion hammers for tool placement. Static down forces up to 3,000 lbs combined with percussion hammers of eight (8) horsepower continuous output are typical on equipment available to the field investigator. Using these energies, probing tools have been used for sampling a variety of media at depths exceeding 70 feet. Advantages of probing equipment which have contributed to its increasing usage in recent years include: ease of mobilization, absence of borehole cuttings, minimization of surface disturbance, and speed of sample collection. This paper focuses on the field application of hydraulic probing equipment including: the suitability of probing operations with respect to various Boil types and lithologies to probing operations; sampler types and recovery quantities for various media, and innovative probing applications presently being tested

DNA Probes Show Genetic Variation in Cyanobacterial Symbionts of the Azolla Fern and a Closer Relationship to Free-Living Nostoc Strains than to Free-Living Anabaena Strains

Science.gov (United States)

Plazinski, Jacek; Zheng, Qi; Taylor, Rona; Croft, Lynn; Rolfe, Barry G.; Gunning, Brian E. S.

1990-01-01

Twenty-two isolates of Anabaena azollae derived from seven Azolla species from various geographic and ecological sources were characterized by DNA-DNA hybridization. Cloned DNA fragments derived from the genomic sequences of three different A. azollae isolates were used to detect restriction fragment length polymorphism among all symbiotic anabaenas. DNA clones were radiolabeled and hybridized against southern blot transfers of genomic DNAs of different isolates of A. azollae digested with restriction endonucleases. Eight DNA probes were selected to identify the Anabaena strains tested. Two were strain specific and hybridized only to A. azollae strains isolated from Azolla microphylla or Azolla caroliniana. One DNA probe was section specific (hybridized only to anabaenas isolated from Azolla ferns representing the section Euazolla), and five other probes gave finer discrimination among anabaenas representing various ecotypes of Azolla species. These cloned genomic DNA probes identified 11 different genotypes of A. azollae isolates. These included three endosymbiotic genotypes within Azolla filiculoides species and two genotypes within both A. caroliniana and Azolla pinnata endosymbionts. Although we were not able to discriminate among anabaenas extracted from different ecotypes of Azolla nilotica, Azolla mexicina, Azolla rubra and Azolla microphylla species, each of the endosymbionts was easily identified as a unique genotype. When total DNA isolated from free-living Anabaena sp. strain PCC7120 was screened, none of the genomic DNA probes gave detectable positive hybridization. Total DNA of Nostoc cycas PCC7422 hybridized with six of eight genomic DNA fragments. These data imply that the dominant symbiotic organism in association with Azolla spp. is more closely related to Nostoc spp. than to free-living Anabaena spp. Images PMID:16348182

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

Science.gov (United States)

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

2016-03-20

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

Detection of DNA fingerprints of cultivated rice by hybridization with a human minisatellite DNA probe

International Nuclear Information System (INIS)

Dallas, J.F.

1988-01-01

A human minisatellite DNA probe detects several restriction fragment length polymorphisms in cultivars of Asian and African rice. Certain fragments appear to be inherited in a Mendelian fashion and may represent unlinked loci. The hybridization patterns appear to be cultivar-specific and largely unchanged after the regeneration of plants from tissue culture. The results suggest that these regions of the rice genome may be used to generate cultivar-specific DNA fingerprints. The demonstration of similarity between a human minisatellite sequence and polymorphic regions in the rice genome suggests that such regions also occur in the genomes of many other plant species

Direct Analysis of Amphetamine Stimulants in a Whole Urine Sample by Atmospheric Solids Analysis Probe Tandem Mass Spectrometry

Science.gov (United States)

Crevelin, Eduardo J.; Salami, Fernanda H.; Alves, Marcela N. R.; De Martinis, Bruno S.; Crotti, Antônio E. M.; Moraes, Luiz A. B.

2016-05-01

Amphetamine-type stimulants (ATS) are among illicit stimulant drugs that are most often used worldwide. A major challenge is to develop a fast and efficient methodology involving minimal sample preparation to analyze ATS in biological fluids. In this study, a urine pool solution containing amphetamine, methamphetamine, ephedrine, sibutramine, and fenfluramine at concentrations ranging from 0.5 pg/mL to 100 ng/mL was prepared and analyzed by atmospheric solids analysis probe tandem mass spectrometry (ASAP-MS/MS) and multiple reaction monitoring (MRM). A urine sample and saliva collected from a volunteer contributor (V1) were also analyzed. The limit of detection of the tested compounds ranged between 0.002 and 0.4 ng/mL in urine samples; the signal-to-noise ratio was 5. These results demonstrated that the ASAP-MS/MS methodology is applicable for the fast detection of ATS in urine samples with great sensitivity and specificity, without the need for cleanup, preconcentration, or chromatographic separation. Thus ASAP-MS/MS could potentially be used in clinical and forensic toxicology applications.

SBH and the integration of complementary approaches in the mapping, sequencing, and understanding of complex genomes

Energy Technology Data Exchange (ETDEWEB)

Drmanac, R.; Drmanac, S.; Labat, I.; Vicentic, A.; Gemmell, A.; Stavropoulos, N.; Jarvis, J.

1992-01-01

A variant of sequencing by hybridization (SBH) is being developed with a potential to inexpensively determine up to 100 million base pairs per year. The method comprises (1) arraying short clones in 864-well plates; (2) growth of the M13 clones or PCR of the inserts; (3) automated spotting of DNAs by corresponding pin-arrays; (4) hybridization of dotted samples with 200-3000 [sup 32]P- or [sup 33]P-labeled 6- to 8-mer probes; and (5) scoring hybridization signals using storage phosphor plates. Some 200 7- to 8-mers can provide an inventory of the genes if CDNA clones are hybridized, or can define the order of 2-kb genomic clones, creating physical and structural maps with 100-bp resolution; the distribution of G+C, LINEs, SINEs, and gene families would be revealed. cDNAs that represent new genes and genomic clones in regions of interest selected by SBH can be sequenced by a gel method. Uniformly distributed clones from the previous step will be hybridized with 2000--3000 6- to 8-mers. As a result, approximately 50--60% of the genomic regions containing members of large repetitive and gene families and those families represented in GenBank would be completely sequenced. In the less redundant regions, every base pair is expected to be read with 3-4 probes, but the complete sequence can not be reconstructed. Such partial sequences allow the inference of similarity and the recognition of coding, regulatory, and repetitive sequences, as well as study of the evolutionary processes all the way up to the species delineation.

SBH and the integration of complementary approaches in the mapping, sequencing, and understanding of complex genomes

Energy Technology Data Exchange (ETDEWEB)

Drmanac, R.; Drmanac, S.; Labat, I.; Vicentic, A.; Gemmell, A.; Stavropoulos, N.; Jarvis, J.

1992-12-01

A variant of sequencing by hybridization (SBH) is being developed with a potential to inexpensively determine up to 100 million base pairs per year. The method comprises (1) arraying short clones in 864-well plates; (2) growth of the M13 clones or PCR of the inserts; (3) automated spotting of DNAs by corresponding pin-arrays; (4) hybridization of dotted samples with 200-3000 {sup 32}P- or {sup 33}P-labeled 6- to 8-mer probes; and (5) scoring hybridization signals using storage phosphor plates. Some 200 7- to 8-mers can provide an inventory of the genes if CDNA clones are hybridized, or can define the order of 2-kb genomic clones, creating physical and structural maps with 100-bp resolution; the distribution of G+C, LINEs, SINEs, and gene families would be revealed. cDNAs that represent new genes and genomic clones in regions of interest selected by SBH can be sequenced by a gel method. Uniformly distributed clones from the previous step will be hybridized with 2000--3000 6- to 8-mers. As a result, approximately 50--60% of the genomic regions containing members of large repetitive and gene families and those families represented in GenBank would be completely sequenced. In the less redundant regions, every base pair is expected to be read with 3-4 probes, but the complete sequence can not be reconstructed. Such partial sequences allow the inference of similarity and the recognition of coding, regulatory, and repetitive sequences, as well as study of the evolutionary processes all the way up to the species delineation.

SBH and the integration of complementary approaches in the mapping, sequencing, and understanding of complex genomes

International Nuclear Information System (INIS)

Drmanac, R.; Drmanac, S.; Labat, I.; Vicentic, A.; Gemmell, A.; Stavropoulos, N.; Jarvis, J.

1992-01-01

A variant of sequencing by hybridization (SBH) is being developed with a potential to inexpensively determine up to 100 million base pairs per year. The method comprises (1) arraying short clones in 864-well plates; (2) growth of the M13 clones or PCR of the inserts; (3) automated spotting of DNAs by corresponding pin-arrays; (4) hybridization of dotted samples with 200-3000 32 P- or 33 P-labeled 6- to 8-mer probes; and (5) scoring hybridization signals using storage phosphor plates. Some 200 7- to 8-mers can provide an inventory of the genes if CDNA clones are hybridized, or can define the order of 2-kb genomic clones, creating physical and structural maps with 100-bp resolution; the distribution of G+C, LINEs, SINEs, and gene families would be revealed. cDNAs that represent new genes and genomic clones in regions of interest selected by SBH can be sequenced by a gel method. Uniformly distributed clones from the previous step will be hybridized with 2000--3000 6- to 8-mers. As a result, approximately 50--60% of the genomic regions containing members of large repetitive and gene families and those families represented in GenBank would be completely sequenced. In the less redundant regions, every base pair is expected to be read with 3-4 probes, but the complete sequence can not be reconstructed. Such partial sequences allow the inference of similarity and the recognition of coding, regulatory, and repetitive sequences, as well as study of the evolutionary processes all the way up to the species delineation

A Method to Correlate mRNA Expression Datasets Obtained from Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Tissue Samples: A Matter of Thresholds.

Directory of Open Access Journals (Sweden)

Dana A M Mustafa

Full Text Available Gene expression profiling of tumors is a successful tool for the discovery of new cancer biomarkers and potential targets for the development of new therapeutic strategies. Reliable profiling is preferably performed on fresh frozen (FF tissues in which the quality of nucleic acids is better preserved than in formalin-fixed paraffin-embedded (FFPE material. However, since snap-freezing of biopsy materials is often not part of daily routine in pathology laboratories, one may have to rely on archival FFPE material. Procedures to retrieve the RNAs from FFPE materials have been developed and therefore, datasets obtained from FFPE and FF materials need to be made compatible to ensure reliable comparisons are possible.To develop an efficient method to compare gene expression profiles obtained from FFPE and FF samples using the same platform.Twenty-six FFPE-FF sample pairs of the same tumors representing various cancer types, and two FFPE-FF sample pairs of breast cancer cell lines, were included. Total RNA was extracted and gene expression profiling was carried out using Illumina's Whole-Genome cDNA-mediated Annealing, Selection, extension and Ligation (WG-DASL V3 arrays, enabling the simultaneous detection of 24,526 mRNA transcripts. A sample exclusion criterion was created based on the expression of 11 stably expressed reference genes. Pearson correlation at the probe level was calculated for paired FFPE-FF, and three cut-off values were chosen. Spearman correlation coefficients between the matched FFPE and FF samples were calculated for three probe lists with varying levels of significance and compared to the correlation based on all measured probes. Unsupervised hierarchical cluster analysis was performed to verify performance of the included probe lists to compare matched FPPE-FF samples.Twenty-seven FFPE-FF pairs passed the sample exclusion criterion. From the profiles of 27 FFPE and FF matched samples, the best correlating probes were identified

Genome U-Plot: a whole genome visualization.

Science.gov (United States)

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

2018-05-15

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

Genomic profiling of oral squamous cell carcinoma by array-based comparative genomic hybridization.

Directory of Open Access Journals (Sweden)

Shunichi Yoshioka

Full Text Available We designed a study to investigate genetic relationships between primary tumors of oral squamous cell carcinoma (OSCC and their lymph node metastases, and to identify genomic copy number aberrations (CNAs related to lymph node metastasis. For this purpose, we collected a total of 42 tumor samples from 25 patients and analyzed their genomic profiles by array-based comparative genomic hybridization. We then compared the genetic profiles of metastatic primary tumors (MPTs with their paired lymph node metastases (LNMs, and also those of LNMs with non-metastatic primary tumors (NMPTs. Firstly, we found that although there were some distinctive differences in the patterns of genomic profiles between MPTs and their paired LNMs, the paired samples shared similar genomic aberration patterns in each case. Unsupervised hierarchical clustering analysis grouped together 12 of the 15 MPT-LNM pairs. Furthermore, similarity scores between paired samples were significantly higher than those between non-paired samples. These results suggested that MPTs and their paired LNMs are composed predominantly of genetically clonal tumor cells, while minor populations with different CNAs may also exist in metastatic OSCCs. Secondly, to identify CNAs related to lymph node metastasis, we compared CNAs between grouped samples of MPTs and LNMs, but were unable to find any CNAs that were more common in LNMs. Finally, we hypothesized that subpopulations carrying metastasis-related CNAs might be present in both the MPT and LNM. Accordingly, we compared CNAs between NMPTs and LNMs, and found that gains of 7p, 8q and 17q were more common in the latter than in the former, suggesting that these CNAs may be involved in lymph node metastasis of OSCC. In conclusion, our data suggest that in OSCCs showing metastasis, the primary and metastatic tumors share similar genomic profiles, and that cells in the primary tumor may tend to metastasize after acquiring metastasis-associated CNAs.

Developmental validation of the Quantifiler(®) HP and Trio Kits for human DNA quantification in forensic samples.

Science.gov (United States)

Holt, Allison; Wootton, Sharon Chao; Mulero, Julio J; Brzoska, Pius M; Langit, Emanuel; Green, Robert L

2016-03-01

The quantification of human genomic DNA is a necessary first step in the DNA casework sample analysis workflow. DNA quantification determines optimal sample input amounts for subsequent STR (short tandem repeat) genotyping procedures, as well as being a useful screening tool to identify samples most likely to provide probative genotypic evidence. To better mesh with the capabilities of newest-generation STR analysis assays, the Quantifiler(®) HP and Quantifiler(®) Trio DNA Quantification Kits were designed for greater detection sensitivity and more robust performance with samples that contain PCR inhibitors or degraded DNA. The new DNA quantification kits use multiplex TaqMan(®) assay-based fluorescent probe technology to simultaneously quantify up to three human genomic targets, allowing samples to be assessed for total human DNA, male contributor (i.e., Y-chromosome) DNA, as well as a determination of DNA degradation state. The Quantifiler HP and Trio Kits use multiple-copy loci to allow for significantly improved sensitivity compared to earlier-generation kits that employ single-copy target loci. The kits' improved performance provides better predictive ability for results with downstream, newest-generation STR assays, and their shortened time-to-result allows more efficient integration into the forensic casework analysis workflow. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

The 100th anniversary of the four-point probe technique: the role of probe geometries in isotropic and anisotropic systems

International Nuclear Information System (INIS)

Miccoli, I; Edler, F; Pfnür, H; Tegenkamp, C

2015-01-01

The electrical conductivity of solid-state matter is a fundamental physical property and can be precisely derived from the resistance measured via the four-point probe technique excluding contributions from parasitic contact resistances. Over time, this method has become an interdisciplinary characterization tool in materials science, semiconductor industries, geology, physics, etc, and is employed for both fundamental and application-driven research. However, the correct derivation of the conductivity is a demanding task which faces several difficulties, e.g. the homogeneity of the sample or the isotropy of the phases. In addition, these sample-specific characteristics are intimately related to technical constraints such as the probe geometry and size of the sample. In particular, the latter is of importance for nanostructures which can now be probed technically on very small length scales. On the occasion of the 100th anniversary of the four-point probe technique, introduced by Frank Wenner, in this review we revisit and discuss various correction factors which are mandatory for an accurate derivation of the resistivity from the measured resistance. Among others, sample thickness, dimensionality, anisotropy, and the relative size and geometry of the sample with respect to the contact assembly are considered. We are also able to derive the correction factors for 2D anisotropic systems on circular finite areas with variable probe spacings. All these aspects are illustrated by state-of-the-art experiments carried out using a four-tip STM/SEM system. We are aware that this review article can only cover some of the most important topics. Regarding further aspects, e.g. technical realizations, the influence of inhomogeneities or different transport regimes, etc, we refer to other review articles in this field. (topical review)

Bead-probe complex capture a couple of SINE and LINE family from genomes of two closely related species of East Asian cyprinid directly using magnetic separation

Science.gov (United States)

Tong, Chaobo; Guo, Baocheng; He, Shunping

2009-01-01

Background Short and long interspersed elements (SINEs and LINEs, respectively), two types of retroposons, are active in shaping the architecture of genomes and powerful tools for studies of phylogeny and population biology. Here we developed special protocol to apply biotin-streptavidin bead system into isolation of interspersed repeated sequences rapidly and efficiently, in which SINEs and LINEs were captured directly from digested genomic DNA by hybridization to bead-probe complex in solution instead of traditional strategy including genomic library construction and screening. Results A new couple of SINEs and LINEs that shared an almost identical 3'tail was isolated and characterized in silver carp and bighead carp of two closely related species. These SINEs (34 members), designated HAmo SINE family, were little divergent in sequence and flanked by obvious TSD indicated that HAmo SINE was very young family. The copy numbers of this family was estimated to 2 × 105 and 1.7 × 105 per haploid genome by Real-Time qPCR, respectively. The LINEs, identified as the homologs of LINE2 in other fishes, had a conserved primary sequence and secondary structures of the 3'tail region that was almost identical to that of HAmo SINE. These evidences suggest that HAmo SINEs are active and amplified recently utilizing the enzymatic machinery for retroposition of HAmoL2 through the recognition of higher-order structures of the conserved 42-tail region. We analyzed the possible structures of HAmo SINE that lead to successful amplification in genome and then deduced that HAmo SINE, SmaI SINE and FokI SINE that were similar in sequence each other, were probably generated independently and created by LINE family within the same lineage of a LINE phylogeny in the genomes of different hosts. Conclusion The presented results show the advantage of the novel method for retroposons isolation and a pair of young SINE family and its partner LINE family in two carp fishes, which strengthened

Bead-probe complex capture a couple of SINE and LINE family from genomes of two closely related species of East Asian cyprinid directly using magnetic separation

Directory of Open Access Journals (Sweden)

Guo Baocheng

2009-02-01

Full Text Available Abstract Background Short and long interspersed elements (SINEs and LINEs, respectively, two types of retroposons, are active in shaping the architecture of genomes and powerful tools for studies of phylogeny and population biology. Here we developed special protocol to apply biotin-streptavidin bead system into isolation of interspersed repeated sequences rapidly and efficiently, in which SINEs and LINEs were captured directly from digested genomic DNA by hybridization to bead-probe complex in solution instead of traditional strategy including genomic library construction and screening. Results A new couple of SINEs and LINEs that shared an almost identical 3'tail was isolated and characterized in silver carp and bighead carp of two closely related species. These SINEs (34 members, designated HAmo SINE family, were little divergent in sequence and flanked by obvious TSD indicated that HAmo SINE was very young family. The copy numbers of this family was estimated to 2 × 105 and 1.7 × 105 per haploid genome by Real-Time qPCR, respectively. The LINEs, identified as the homologs of LINE2 in other fishes, had a conserved primary sequence and secondary structures of the 3'tail region that was almost identical to that of HAmo SINE. These evidences suggest that HAmo SINEs are active and amplified recently utilizing the enzymatic machinery for retroposition of HAmoL2 through the recognition of higher-order structures of the conserved 42-tail region. We analyzed the possible structures of HAmo SINE that lead to successful amplification in genome and then deduced that HAmo SINE, SmaI SINE and FokI SINE that were similar in sequence each other, were probably generated independently and created by LINE family within the same lineage of a LINE phylogeny in the genomes of different hosts. Conclusion The presented results show the advantage of the novel method for retroposons isolation and a pair of young SINE family and its partner LINE family in two carp

Theoretical analysis of a dual-probe scanning tunneling microscope setup on graphene

DEFF Research Database (Denmark)

Settnes, Mikkel; Power, Stephen R.; Petersen, Dirch Hjorth

2014-01-01

Experimental advances allow for the inclusion of multiple probes to measure the transport properties of a sample surface. We develop a theory of dual-probe scanning tunneling microscopy using a Green's function formalism, and apply it to graphene. Sampling the local conduction properties at finite...... to different scattering processes. We compute the conductance maps of graphene systems with different edge geometries or height fluctuations to determine the effects of nonideal graphene samples on dual-probe measurements. © 2014 American Physical Society....

Volumetric water content measurement probes in earth-dam construction

Directory of Open Access Journals (Sweden)

Bardanis Michael

2016-01-01

Full Text Available Two frequency domain reflectometry (FDR probes have been used. They were used on compacted soils both in the laboratory and in the field. Measurements in the laboratory were intended for calibration. The range of densities and types of materials where insertion of the probes can be achieved was investigated first. The effect of sporadic presence of coarser grains and density on these calibrations, once insertion could be achieved, were investigated second. Measurements on laboratory prepared samples with the same moisture content were different when the sample was kept in the mould from when it was extruded from it. Also both these measurements were different from that in a sample of the same density but significantly larger in diameter. It was found that measurements with these probes are affected by dilation exhibited by soil around the rods of the probes during insertion. Readings immediately after insertion of the sensors on samples extruded from their moulds were the ones closer to measured values. These readings combined with total volume and mass obtained from sand-cone tests during the construction of an earth-dam allowed fairly accurate estimation of the dry unit weight but not the gravimetric water content.

Hyb-Seq: Combining Target Enrichment and Genome Skimming for Plant Phylogenomics

Directory of Open Access Journals (Sweden)

Kevin Weitemier

2014-08-01

Full Text Available Premise of the study: Hyb-Seq, the combination of target enrichment and genome skimming, allows simultaneous data collection for low-copy nuclear genes and high-copy genomic targets for plant systematics and evolution studies. Methods and Results: Genome and transcriptome assemblies for milkweed (Asclepias syriaca were used to design enrichment probes for 3385 exons from 768 genes (>1.6 Mbp followed by Illumina sequencing of enriched libraries. Hyb-Seq of 12 individuals (10 Asclepias species and two related genera resulted in at least partial assembly of 92.6% of exons and 99.7% of genes and an average assembly length >2 Mbp. Importantly, complete plastomes and nuclear ribosomal DNA cistrons were assembled using off-target reads. Phylogenomic analyses demonstrated signal conflict between genomes. Conclusions: The Hyb-Seq approach enables targeted sequencing of thousands of low-copy nuclear exons and flanking regions, as well as genome skimming of high-copy repeats and organellar genomes, to efficiently produce genome-scale data sets for phylogenomics.

Species-specific optical genosensors for the detection of mycotoxigenic Fusarium fungi in food samples

International Nuclear Information System (INIS)

Peltomaa, Riikka; Vaghini, Silvia; Patiño, Belén; Benito-Peña, Elena; Moreno-Bondi, María C.

2016-01-01

Plant-pathogenic Fusarium species, Fusarium verticillioides and Fusarium proliferatum, are the major producers of fumonisins which are one of the most common mycotoxins found in maize. Herein, we report the development of specific and sensitive genosensors for detecting these two closely related Fusarium species in food samples. The sensors are based on species-specific capture and detection probes, which bind to the intergenic spacer region of rDNA (IGS). Oligonucleotide functionalized magnetic microbeads are used to capture the target DNA which is then detected using biotinylated detection probes and a streptavidin-coupled label. The developed genosensors had detection limits of 1.8 pM and 3.0 pM for F. proliferatum and F. verticillioides, respectively, using synthetic DNA targets. Furthermore, the biosensors were used to analyze natural fungal contamination of commercial maize samples. After amplification of the genomic DNA the sensors detected the presence of the fungi, in accordance with previous results obtained with PCR. No cross-reactivity between F. verticillioides and F. proliferatum, or other fungi species tested, was observed. The developed biosensors can provide a valuable tool to evaluate the potential for mycotoxin contamination in conditions where detection of mycotoxins directly is challenging. - Highlights: • Optical genosensors detect fumonisin producing Fusarium species in maize samples. • Oligonucleotide probes designed on the intergenic spacer region of rDNA can distinguish between closely related species. • Sandwich hybridization assay with magnetic microbeads allows species-specific detection of Fusarium spp. directly from PCR.

Species-specific optical genosensors for the detection of mycotoxigenic Fusarium fungi in food samples

Energy Technology Data Exchange (ETDEWEB)

Peltomaa, Riikka; Vaghini, Silvia [Department of Analytical Chemistry, Faculty of Chemistry, Complutense University, Ciudad Universitaria s/n, Madrid 28040 (Spain); Patiño, Belén [Department of Microbiology III, Faculty of Biology, Complutense University, Ciudad Universitaria s/n, Madrid 28040 (Spain); Benito-Peña, Elena, E-mail: elenabp@ucm.es [Department of Analytical Chemistry, Faculty of Chemistry, Complutense University, Ciudad Universitaria s/n, Madrid 28040 (Spain); Moreno-Bondi, María C., E-mail: mcmbondi@ucm.es [Department of Analytical Chemistry, Faculty of Chemistry, Complutense University, Ciudad Universitaria s/n, Madrid 28040 (Spain)

2016-09-07

Plant-pathogenic Fusarium species, Fusarium verticillioides and Fusarium proliferatum, are the major producers of fumonisins which are one of the most common mycotoxins found in maize. Herein, we report the development of specific and sensitive genosensors for detecting these two closely related Fusarium species in food samples. The sensors are based on species-specific capture and detection probes, which bind to the intergenic spacer region of rDNA (IGS). Oligonucleotide functionalized magnetic microbeads are used to capture the target DNA which is then detected using biotinylated detection probes and a streptavidin-coupled label. The developed genosensors had detection limits of 1.8 pM and 3.0 pM for F. proliferatum and F. verticillioides, respectively, using synthetic DNA targets. Furthermore, the biosensors were used to analyze natural fungal contamination of commercial maize samples. After amplification of the genomic DNA the sensors detected the presence of the fungi, in accordance with previous results obtained with PCR. No cross-reactivity between F. verticillioides and F. proliferatum, or other fungi species tested, was observed. The developed biosensors can provide a valuable tool to evaluate the potential for mycotoxin contamination in conditions where detection of mycotoxins directly is challenging. - Highlights: • Optical genosensors detect fumonisin producing Fusarium species in maize samples. • Oligonucleotide probes designed on the intergenic spacer region of rDNA can distinguish between closely related species. • Sandwich hybridization assay with magnetic microbeads allows species-specific detection of Fusarium spp. directly from PCR.

Characterization of a Genomic Signature of Pregnancy in the Breast

Science.gov (United States)

Belitskaya-Lévy, Ilana; Zeleniuch-Jacquotte, Anne; Russo, Jose; Russo, Irma H.; Bordás, Pal; Åhman, Janet; Afanasyeva, Yelena; Johansson, Robert; Lenner, Per; Li, Xiaochun; de Cicco, Ricardo López; Peri, Suraj; Ross, Eric; Russo, Patricia A.; Santucci-Pereira, Julia; Sheriff, Fathima S.; Slifker, Michael; Hallmans, Göran; Toniolo, Paolo; Arslan, Alan A.

2012-01-01

The objective of the current study was to comprehensively compare the genomic profiles in the breast of parous and nulliparous postmenopausal women to identify genes that permanently change their expression following pregnancy. The study was designed as a two-phase approach. In the discovery phase, we compared breast genomic profiles of 37 parous with 18 nulliparous postmenopausal women. In the validation phase, confirmation of the genomic patterns observed in the discovery phase was sought in an independent set of 30 parous and 22 nulliparous postmenopausal women. RNA was hybridized to Affymetrix HG_U133 Plus 2.0 oligonucleotide arrays containing probes to 54,675 transcripts; scanned and the images analyzed using Affymetrix GCOS software. Surrogate variable analysis, logistic regression and significance analysis for microarrays were used to identify statistically significant differences in expression of genes. The False Discovery Rate (FDR) approach was used to control for multiple comparisons. We found that 208 genes (305 probe sets) were differentially expressed between parous and nulliparous women in both discovery and validation phases of the study at a FDR of 10% and with at least a 1.25-fold change. These genes are involved in regulation of transcription, centrosome organization, RNA splicing, cell cycle control, adhesion and differentiation. The results provide persuasive evidence that full-term pregnancy induces long-term genomic changes in the breast. The genomic signature of pregnancy could be used as an intermediate marker to assess potential chemopreventive interventions with hormones mimicking the effects of pregnancy for prevention of breast cancer. PMID:21622728

Functional RNA structures throughout the Hepatitis C Virus genome.

Science.gov (United States)

Adams, Rebecca L; Pirakitikulr, Nathan; Pyle, Anna Marie

2017-06-01

The single-stranded Hepatitis C Virus (HCV) genome adopts a set of elaborate RNA structures that are involved in every stage of the viral lifecycle. Recent advances in chemical probing, sequencing, and structural biology have facilitated analysis of RNA folding on a genome-wide scale, revealing novel structures and networks of interactions. These studies have underscored the active role played by RNA in every function of HCV and they open the door to new types of RNA-targeted therapeutics. Copyright © 2017 Elsevier B.V. All rights reserved.

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

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Liang Chi

2011-06-01

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

Impact of delay to cryopreservation on RNA integrity and genome-wide expression profiles in resected tumor samples.

Directory of Open Access Journals (Sweden)

Elodie Caboux

Full Text Available The quality of tissue samples and extracted mRNA is a major source of variability in tumor transcriptome analysis using genome-wide expression microarrays. During and immediately after surgical tumor resection, tissues are exposed to metabolic, biochemical and physical stresses characterized as "warm ischemia". Current practice advocates cryopreservation of biosamples within 30 minutes of resection, but this recommendation has not been systematically validated by measurements of mRNA decay over time. Using Illumina HumanHT-12 v3 Expression BeadChips, providing a genome-wide coverage of over 24,000 genes, we have analyzed gene expression variation in samples of 3 hepatocellular carcinomas (HCC and 3 lung carcinomas (LC cryopreserved at times up to 2 hours after resection. RNA Integrity Numbers (RIN revealed no significant deterioration of mRNA up to 2 hours after resection. Genome-wide transcriptome analysis detected non-significant gene expression variations of -3.5%/hr (95% CI: -7.0%/hr to 0.1%/hr; p = 0.054. In LC, no consistent gene expression pattern was detected in relation with warm ischemia. In HCC, a signature of 6 up-regulated genes (CYP2E1, IGLL1, CABYR, CLDN2, NQO1, SCL13A5 and 6 down-regulated genes (MT1G, MT1H, MT1E, MT1F, HABP2, SPINK1 was identified (FDR <0.05. Overall, our observations support current recommendation of time to cryopreservation of up to 30 minutes and emphasize the need for identifying tissue-specific genes deregulated following resection to avoid misinterpreting expression changes induced by warm ischemia as pathologically significant changes.

Utility of the pooling approach as applied to whole genome association scans with high-density Affymetrix microarrays

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Gray Joanna

2010-11-01

Full Text Available Abstract Background We report an attempt to extend the previously successful approach of combining SNP (single nucleotide polymorphism microarrays and DNA pooling (SNP-MaP employing high-density microarrays. Whereas earlier studies employed a range of Affymetrix SNP microarrays comprising from 10 K to 500 K SNPs, this most recent investigation used the 6.0 chip which displays 906,600 SNP probes and 946,000 probes for the interrogation of CNVs (copy number variations. The genotyping assay using the Affymetrix SNP 6.0 array is highly demanding on sample quality due to the small feature size, low redundancy, and lack of mismatch probes. Findings In the first study published so far using this microarray on pooled DNA, we found that pooled cheek swab DNA could not accurately predict real allele frequencies of the samples that comprised the pools. In contrast, the allele frequency estimates using blood DNA pools were reasonable, although inferior compared to those obtained with previously employed Affymetrix microarrays. However, it might be possible to improve performance by developing improved analysis methods. Conclusions Despite the decreasing costs of genome-wide individual genotyping, the pooling approach may have applications in very large-scale case-control association studies. In such cases, our study suggests that high-quality DNA preparations and lower density platforms should be preferred.

Ball assisted device for analytical surface sampling

Science.gov (United States)

ElNaggar, Mariam S; Van Berkel, Gary J; Covey, Thomas R

2015-11-03

A system for sampling a surface includes a sampling probe having a housing and a socket, and a rolling sampling sphere within the socket. The housing has a sampling fluid supply conduit and a sampling fluid exhaust conduit. The sampling fluid supply conduit supplies sampling fluid to the sampling sphere. The sampling fluid exhaust conduit has an inlet opening for receiving sampling fluid carried from the surface by the sampling sphere. A surface sampling probe and a method for sampling a surface are also disclosed.

Probing genomic diversity and evolution of Streptococcus suis serotype 2 by NimbleGen tiling arrays

Directory of Open Access Journals (Sweden)

Liao Hui

2011-05-01

Full Text Available Abstract Background Our previous studies revealed that a new disease form of streptococcal toxic shock syndrome (STSS is associated with specific Streptococcus suis serotype 2 (SS2 strains. To achieve a better understanding of the pathogenicity and evolution of SS2 at the whole-genome level, comparative genomic analysis of 18 SS2 strains, selected on the basis of virulence and geographic origin, was performed using NimbleGen tiling arrays. Results Our results demonstrate that SS2 isolates have highly divergent genomes. The 89K pathogenicity island (PAI, which has been previously recognized as unique to the Chinese epidemic strains causing STSS, was partially included in some other virulent and avirulent strains. The ABC-type transport systems, encoded by 89K, were hypothesized to greatly contribute to the catastrophic features of STSS. Moreover, we identified many polymorphisms in genes encoding candidate or known virulence factors, such as PlcR, lipase, sortases, the pilus-associated proteins, and the response regulator RevS and CtsR. On the basis of analysis of regions of differences (RDs across the entire genome for the 18 selected SS2 strains, a model of microevolution for these strains is proposed, which provides clues into Streptococcus pathogenicity and evolution. Conclusions Our deep comparative genomic analysis of the 89K PAI present in the genome of SS2 strains revealed details into how some virulent strains acquired genes that may contribute to STSS, which may lead to better environmental monitoring of epidemic SS2 strains.

Molecular beacon probes-base multiplex NASBA Real-time for detection of HIV-1 and HCV.

Science.gov (United States)

Mohammadi-Yeganeh, S; Paryan, M; Mirab Samiee, S; Kia, V; Rezvan, H

2012-06-01

Developed in 1991, nucleic acid sequence-based amplification (NASBA) has been introduced as a rapid molecular diagnostic technique, where it has been shown to give quicker results than PCR, and it can also be more sensitive. This paper describes the development of a molecular beacon-based multiplex NASBA assay for simultaneous detection of HIV-1 and HCV in plasma samples. A well-conserved region in the HIV-1 pol gene and 5'-NCR of HCV genome were used for primers and molecular beacon design. The performance features of HCV/HIV-1 multiplex NASBA assay including analytical sensitivity and specificity, clinical sensitivity and clinical specificity were evaluated. The analysis of scalar concentrations of the samples indicated that the limit of quantification of the assay was beacon probes detected all HCV genotypes and all major variants of HIV-1. This method may represent a relatively inexpensive isothermal method for detection of HIV-1/HCV co-infection in monitoring of patients.

Determination of organophosphorus pesticides in water samples by using a new sensitive luminescent probe of Eu (III) complex

Energy Technology Data Exchange (ETDEWEB)

Azab, Hassan A., E-mail: azab2@yahoo.com; Anwar, Z.M.; Rizk, M.A.; Khairy, Gasser M.; El-Asfoury, M.H.

2015-01-15

This work describes the application of fluorescence for investigating the interactions of Eu(III)-TAN-1,10 phenanthroline (where TAN=4,4,4-Trifluoro-1-(2-naphthyl)-1,3-butanedione) with pesticides Chlorpyrifos, Malathion, Endosulfan, Heptachlor. The complex was synthesized and characterized by elemental analysis, FTIR, x-ray spectroscopy, solid fluorescence and thermal analysis. The results indicated that the composition of this complex is [Eu(TAN){sub 2}(Phen)(H{sub 2}O){sub 2}]Cl. The luminescence properties of the complex in different solvents and at different pH values have been investigated. The results show that the complex exhibits more efficient luminescence at pH=7.5. The interactions of Eu-complex with different pesticides (Chlorpyrifos, Malathion, Endosulfan, and Heptachlor) in aqueous medium have been investigated by fluorescence measurements. The luminescence intensity of the probe is quenched by Malathion and enhanced by (Endosulfan, Heptachlor, and Chlorpyrifos). Direct methods for the determination of the pesticides under investigation have been developed using the luminescence variations of the probe in solution. The detection limits are 0.47, 1.02, 0.66, 0.64 µmol/L for Chlorpyrifos, Endosulfan, Heptachlor, and Malathion, respectively. The binding constants and thermodynamic parameters of the pesticides with probe were evaluated. The emission quantum yield (QY=0.71) of Eu(III)-complex was determined using tris (2,2'-bipyridyl) dichlororuthenium(II) hexahydrate. A thermodynamic analysis showed that the reaction is spontaneous with negative ΔG. Effect of some relevant interferents on the detection of pesticides has been investigated. The new method was applied to the determination of the pesticides in different types of water samples (tap, river, and waste water). - Highlights: • A new luminescent probe of Eu (III) complex has been developed for sensing some organophosphorus pesticides. • Four guest pesticides Chlorpyrifos, Malathion

Determination of organophosphorus pesticides in water samples by using a new sensitive luminescent probe of Eu (III) complex

International Nuclear Information System (INIS)

Azab, Hassan A.; Anwar, Z.M.; Rizk, M.A.; Khairy, Gasser M.; El-Asfoury, M.H.

2015-01-01

This work describes the application of fluorescence for investigating the interactions of Eu(III)-TAN-1,10 phenanthroline (where TAN=4,4,4-Trifluoro-1-(2-naphthyl)-1,3-butanedione) with pesticides Chlorpyrifos, Malathion, Endosulfan, Heptachlor. The complex was synthesized and characterized by elemental analysis, FTIR, x-ray spectroscopy, solid fluorescence and thermal analysis. The results indicated that the composition of this complex is [Eu(TAN) 2 (Phen)(H 2 O) 2 ]Cl. The luminescence properties of the complex in different solvents and at different pH values have been investigated. The results show that the complex exhibits more efficient luminescence at pH=7.5. The interactions of Eu-complex with different pesticides (Chlorpyrifos, Malathion, Endosulfan, and Heptachlor) in aqueous medium have been investigated by fluorescence measurements. The luminescence intensity of the probe is quenched by Malathion and enhanced by (Endosulfan, Heptachlor, and Chlorpyrifos). Direct methods for the determination of the pesticides under investigation have been developed using the luminescence variations of the probe in solution. The detection limits are 0.47, 1.02, 0.66, 0.64 µmol/L for Chlorpyrifos, Endosulfan, Heptachlor, and Malathion, respectively. The binding constants and thermodynamic parameters of the pesticides with probe were evaluated. The emission quantum yield (QY=0.71) of Eu(III)-complex was determined using tris (2,2'-bipyridyl) dichlororuthenium(II) hexahydrate. A thermodynamic analysis showed that the reaction is spontaneous with negative ΔG. Effect of some relevant interferents on the detection of pesticides has been investigated. The new method was applied to the determination of the pesticides in different types of water samples (tap, river, and waste water). - Highlights: • A new luminescent probe of Eu (III) complex has been developed for sensing some organophosphorus pesticides. • Four guest pesticides Chlorpyrifos, Malathion, Endosulfan, and

An Evanescent Field Optical Microscope. Scanning probe Microscopy

NARCIS (Netherlands)

van Hulst, N.F.; Segerink, Franciscus B.; Bölger, B.; Bölger, B.; Wickramasinghe, H. Kumar

1991-01-01

An Evanescent Field Optical Microscope (EFOM) is presented, which employs frustrated total internal reflection on a highly localized scale by means of a sharp dielectric tip. The coupling of the evanescent field to the sub-micrometer probe as a function of probe-sample distance, angle of incidence

Genotyping of Trypanosoma cruzi Sublineage in Human Samples from a North-East Argentina Area by Hybridization with DNA Probes and Specific Polymerase Chain Reaction (PCR)

Science.gov (United States)

Diez, Cristina; Lorenz, Virginia; Ortiz, Silvia; Gonzalez, Verónica; Racca, Andrea; Bontempi, Iván; Manattini, Silvia; Solari, Aldo; Marcipar, Iván

2010-01-01

We have evaluated blood samples of chronic and congenital Trypanosoma cruzi-infected patients from the city of Reconquista located in the northeast of Argentina where no information was previously obtained about the genotype of infecting parasites. Fourteen samples of congenital and 19 chronical patients were analyzed by hybridization with DNA probes of minicircle hypervariable regions (mHVR). In congenital patients, 50% had single infections with TcIId, 7% single infections with TcIIe, 29% mixed infections with TcIId/e, and 7% had mixed infections with TcIId/b and 7% TcIId/b, respectively. In Chronical patients, 52% had single infections with TcIId, 11% single infections with TcIIe, 26% had mixed infections with TcIId/e, and 11% had non-identified genotypes. With these samples, we evaluated the minicircle lineage-specific polymerase chain reaction assay (MLS-PCR), which involves a nested PCR to HVR minicircle sequences and we found a correlation with hybridization probes of 96.4% for TcIId and 54.8% for TcIIe. PMID:20064998

Deep ancestry of programmed genome rearrangement in lampreys.

Science.gov (United States)

Timoshevskiy, Vladimir A; Lampman, Ralph T; Hess, Jon E; Porter, Laurie L; Smith, Jeramiah J

2017-09-01

In most multicellular organisms, the structure and content of the genome is rigorously maintained over the course of development. However some species have evolved genome biologies that permit, or require, developmentally regulated changes in the physical structure and content of the genome (programmed genome rearrangement: PGR). Relatively few vertebrates are known to undergo PGR, although all agnathans surveyed to date (several hagfish and one lamprey: Petromyzon marinus) show evidence of large scale PGR. To further resolve the ancestry of PGR within vertebrates, we developed probes that allow simultaneous tracking of nearly all sequences eliminated by PGR in P. marinus and a second lamprey species (Entosphenus tridentatus). These comparative analyses reveal conserved subcellular structures (lagging chromatin and micronuclei) associated with PGR and provide the first comparative embryological evidence in support of the idea that PGR represents an ancient and evolutionarily stable strategy for regulating inherent developmental/genetic conflicts between germline and soma. Copyright © 2017 Elsevier Inc. All rights reserved.

Using Partial Genomic Fosmid Libraries for Sequencing CompleteOrganellar Genomes

Energy Technology Data Exchange (ETDEWEB)

McNeal, Joel R.; Leebens-Mack, James H.; Arumuganathan, K.; Kuehl, Jennifer V.; Boore, Jeffrey L.; dePamphilis, Claude W.

2005-08-26

Organellar genome sequences provide numerous phylogenetic markers and yield insight into organellar function and molecular evolution. These genomes are much smaller in size than their nuclear counterparts; thus, their complete sequencing is much less expensive than total nuclear genome sequencing, making broader phylogenetic sampling feasible. However, for some organisms it is challenging to isolate plastid DNA for sequencing using standard methods. To overcome these difficulties, we constructed partial genomic libraries from total DNA preparations of two heterotrophic and two autotrophic angiosperm species using fosmid vectors. We then used macroarray screening to isolate clones containing large fragments of plastid DNA. A minimum tiling path of clones comprising the entire genome sequence of each plastid was selected, and these clones were shotgun-sequenced and assembled into complete genomes. Although this method worked well for both heterotrophic and autotrophic plants, nuclear genome size had a dramatic effect on the proportion of screened clones containing plastid DNA and, consequently, the overall number of clones that must be screened to ensure full plastid genome coverage. This technique makes it possible to determine complete plastid genome sequences for organisms that defy other available organellar genome sequencing methods, especially those for which limited amounts of tissue are available.

Software engineering the mixed model for genome-wide association studies on large samples.

Science.gov (United States)

Zhang, Zhiwu; Buckler, Edward S; Casstevens, Terry M; Bradbury, Peter J

2009-11-01

Mixed models improve the ability to detect phenotype-genotype associations in the presence of population stratification and multiple levels of relatedness in genome-wide association studies (GWAS), but for large data sets the resource consumption becomes impractical. At the same time, the sample size and number of markers used for GWAS is increasing dramatically, resulting in greater statistical power to detect those associations. The use of mixed models with increasingly large data sets depends on the availability of software for analyzing those models. While multiple software packages implement the mixed model method, no single package provides the best combination of fast computation, ability to handle large samples, flexible modeling and ease of use. Key elements of association analysis with mixed models are reviewed, including modeling phenotype-genotype associations using mixed models, population stratification, kinship and its estimation, variance component estimation, use of best linear unbiased predictors or residuals in place of raw phenotype, improving efficiency and software-user interaction. The available software packages are evaluated, and suggestions made for future software development.

Design of planar microcoil-based NMR probe ensuring high SNR

Science.gov (United States)

Ali, Zishan; Poenar, D. P.; Aditya, Sheel

2017-09-01

A microNMR probe for ex vivo applications may consist of at least one microcoil, which can be used as the oscillating magnetic field (MF) generator as well as receiver coil, and a sample holder, with a volume in the range of nanoliters to micro-liters, placed near the microcoil. The Signal-to-Noise ratio (SNR) of such a probe is, however, dependent not only on its design but also on the measurement setup, and the measured sample. This paper introduces a performance factor P independent of both the proton spin density in the sample and the external DC magnetic field, and which can thus assess the performance of the probe alone. First, two of the components of the P factor (inhomogeneity factor K and filling factor η ) are defined and an approach to calculate their values for different probe variants from electromagnetic simulations is devised. A criterion based on dominant component of the magnetic field is then formulated to help designers optimize the sample volume which also affects the performance of the probe, in order to obtain the best SNR for a given planar microcoil. Finally, the P factor values are compared between different planar microcoils with different number of turns and conductor aspect ratios, and planar microcoils are also compared with conventional solenoids. These comparisons highlight which microcoil geometry-sample volume combination will ensure a high SNR under any external setup.

Design of planar microcoil-based NMR probe ensuring high SNR

Directory of Open Access Journals (Sweden)

Zishan Ali

2017-09-01

Full Text Available A microNMR probe for ex vivo applications may consist of at least one microcoil, which can be used as the oscillating magnetic field (MF generator as well as receiver coil, and a sample holder, with a volume in the range of nanoliters to micro-liters, placed near the microcoil. The Signal-to-Noise ratio (SNR of such a probe is, however, dependent not only on its design but also on the measurement setup, and the measured sample. This paper introduces a performance factor P independent of both the proton spin density in the sample and the external DC magnetic field, and which can thus assess the performance of the probe alone. First, two of the components of the P factor (inhomogeneity factor K and filling factor η are defined and an approach to calculate their values for different probe variants from electromagnetic simulations is devised. A criterion based on dominant component of the magnetic field is then formulated to help designers optimize the sample volume which also affects the performance of the probe, in order to obtain the best SNR for a given planar microcoil. Finally, the P factor values are compared between different planar microcoils with different number of turns and conductor aspect ratios, and planar microcoils are also compared with conventional solenoids. These comparisons highlight which microcoil geometry-sample volume combination will ensure a high SNR under any external setup.

Multiple-scanning-probe tunneling microscope with nanoscale positional recognition function.

Science.gov (United States)

Higuchi, Seiji; Kuramochi, Hiromi; Laurent, Olivier; Komatsubara, Takashi; Machida, Shinichi; Aono, Masakazu; Obori, Kenichi; Nakayama, Tomonobu

2010-07-01

Over the past decade, multiple-scanning-probe microscope systems with independently controlled probes have been developed for nanoscale electrical measurements. We developed a quadruple-scanning-probe tunneling microscope (QSPTM) that can determine and control the probe position through scanning-probe imaging. The difficulty of operating multiple probes with submicrometer precision drastically increases with the number of probes. To solve problems such as determining the relative positions of the probes and avoiding of contact between the probes, we adopted sample-scanning methods to obtain four images simultaneously and developed an original control system for QSPTM operation with a function of automatic positional recognition. These improvements make the QSPTM a more practical and useful instrument since four images can now be reliably produced, and consequently the positioning of the four probes becomes easier owing to the reduced chance of accidental contact between the probes.

Usability of human Infinium MethylationEPIC BeadChip for mouse DNA methylation studies.

Science.gov (United States)

Needhamsen, Maria; Ewing, Ewoud; Lund, Harald; Gomez-Cabrero, David; Harris, Robert Adam; Kular, Lara; Jagodic, Maja

2017-11-15

The advent of array-based genome-wide DNA methylation methods has enabled quantitative measurement of single CpG methylation status at relatively low cost and sample input. Whereas the use of Infinium Human Methylation BeadChips has shown great utility in clinical studies, no equivalent tool is available for rodent animal samples. We examined the feasibility of using the new Infinium MethylationEPIC BeadChip for studying DNA methylation in mouse. In silico, we identified 19,420 EPIC probes (referred as mEPIC probes), which align with a unique best alignment score to the bisulfite converted reference mouse genome mm10. Further annotation revealed that 85% of mEPIC probes overlapped with mm10.refSeq genes at different genomic features including promoters (TSS1500 and TSS200), 1st exons, 5'UTRs, 3'UTRs, CpG islands, shores, shelves, open seas and FANTOM5 enhancers. Hybridization of mouse samples to Infinium Human MethylationEPIC BeadChips showed successful measurement of mEPIC probes and reproducibility between inter-array biological replicates. Finally, we demonstrated the utility of mEPIC probes for data exploration such as hierarchical clustering. Given the absence of cost and labor convenient genome-wide technologies in the murine system, our findings show that the Infinium MethylationEPIC BeadChip platform is suitable for investigation of the mouse methylome. Furthermore, we provide the "mEPICmanifest" with genomic features, available to users of Infinium Human MethylationEPIC arrays for mouse samples.

Fluorescence suppression using wavelength modulated Raman spectroscopy in fiber-probe-based tissue analysis.

Science.gov (United States)

Praveen, Bavishna B; Ashok, Praveen C; Mazilu, Michael; Riches, Andrew; Herrington, Simon; Dholakia, Kishan

2012-07-01

In the field of biomedical optics, Raman spectroscopy is a powerful tool for probing the chemical composition of biological samples. In particular, fiber Raman probes play a crucial role for in vivo and ex vivo tissue analysis. However, the high-fluorescence background typically contributed by the auto fluorescence from both a tissue sample and the fiber-probe interferes strongly with the relatively weak Raman signal. Here we demonstrate the implementation of wavelength-modulated Raman spectroscopy (WMRS) to suppress the fluorescence background while analyzing tissues using fiber Raman probes. We have observed a significant signal-to-noise ratio enhancement in the Raman bands of bone tissue, which have a relatively high fluorescence background. Implementation of WMRS in fiber-probe-based bone tissue study yielded usable Raman spectra in a relatively short acquisition time (∼30  s), notably without any special sample preparation stage. Finally, we have validated its capability to suppress fluorescence on other tissue samples such as adipose tissue derived from four different species.

Horseradish peroxidase and antibody labeled gold nanoparticle probe for amplified immunoassay of ciguatoxin in fish samples based on capillary electrophoresis with electrochemical detection.

Science.gov (United States)

Zhang, Zhaoxiang; Liu, Ying; Zhang, Chaoying; Luan, Wenxiu

2015-03-01

This paper describes a new amplified immunoassay with horseradish peroxidase (HRP) and antibody (Ab) labeled gold nanoparticles (AuNPs) probe hyphenated to capillary electrophoresis (CE) with electrochemical (EC) detection for ultrasensitive determination of ciguatoxin CTX1B. AuNPs were conjugated with HRP and Ab, and then incubated with limited amount of CTX1B to produce immunocomplex. The immunoreactive sample was injected into capillary for CE separation and EC detection. Enhanced sensitivity was obtained by adopting the AuNPs as carriers of HRP and Ab at high HRP/Ab molar ratio. The calibration curve of CTX1B was in the range of 0.06-90 ng/mL. The detection limit was 0.045 ng/mL, which is 38-fold lower than that of HPLC-MS method for CTX1B analysis. The proposed method was successfully applied for the quantification of CTX1B in contamined fish samples by simultaneously labeling Ab and HRP on AuNPs. The amplified IA with HRP and Ab labeled AuNPs probe hyphenated to CE and EC detection provides a sensitive analytical approach for the determination of trace ciguatoxin in complex samples. Copyright © 2015 Elsevier Ltd. All rights reserved.

Electromechanical response of amorphous LaAlO3 thin film probed by scanning probe microscopies

Science.gov (United States)

Borowiak, Alexis S.; Baboux, Nicolas; Albertini, David; Vilquin, Bertrand; Saint Girons, Guillaume; Pelloquin, Sylvain; Gautier, Brice

2014-07-01

The electromechanical response of a 3 nm thick amorphous LaAlO3 layer obtained by molecular beam epitaxy has been studied using scanning probe microscopies. Although this kind of sample is not ferroelectric due to its amorphous nature, the resulting images are identical to what is generally obtained on truly ferroelectric samples probed by piezoresponse force microscopy: domains of apparently opposite polarisation are detected, and perfect, square shaped hysteresis loops are recorded. Moreover, written patterns are stable within 72 h. We discuss in the general case the possible origins of this behaviour in terms of charge injection, ionic conduction and motion of oxygen vacancies. In the case presented in this paper, since the writing process has been conducted with applied voltages lower than the injection threshold measured by conductive atomic force Microscopy, allowing to withdraw the hypothesis of charge injection in the sample, we propose that a bistable distribution of oxygen vacancies is responsible for this contrast.

Electromechanical response of amorphous LaAlO3 thin film probed by scanning probe microscopies

International Nuclear Information System (INIS)

Borowiak, Alexis S.; Baboux, Nicolas; Albertini, David; Gautier, Brice; Vilquin, Bertrand; Saint Girons, Guillaume; Pelloquin, Sylvain

2014-01-01

The electromechanical response of a 3 nm thick amorphous LaAlO 3 layer obtained by molecular beam epitaxy has been studied using scanning probe microscopies. Although this kind of sample is not ferroelectric due to its amorphous nature, the resulting images are identical to what is generally obtained on truly ferroelectric samples probed by piezoresponse force microscopy: domains of apparently opposite polarisation are detected, and perfect, square shaped hysteresis loops are recorded. Moreover, written patterns are stable within 72 h. We discuss in the general case the possible origins of this behaviour in terms of charge injection, ionic conduction and motion of oxygen vacancies. In the case presented in this paper, since the writing process has been conducted with applied voltages lower than the injection threshold measured by conductive atomic force Microscopy, allowing to withdraw the hypothesis of charge injection in the sample, we propose that a bistable distribution of oxygen vacancies is responsible for this contrast.

Three-way flexible cantilever probes for static contact

International Nuclear Information System (INIS)

Wang, Fei; Petersen, Dirch H; Hansen, Christian; Mortensen, Dennis; Friis, Lars; Hansen, Ole; Jensen, Helle V

2011-01-01

In micro four-point probe measurements, three-way flexible L-shaped cantilever probes show significant advantages over conventional straight cantilever probes. The L-shaped cantilever allows static contact to the sample surface which reduces the frictional wear of the cantilever tips. We analyze the geometrical design space that must be fulfilled for the cantilevers to obtain static contact with the test sample. The design space relates the spring constant tensor of the cantilevers to the minimal value of the static tip-to-sample friction coefficient. Using an approximate model, we provide the analytical calculation of the compliance matrix of the L-shaped cantilever. Compared to results derived from finite element model simulations, the theoretical model provides a good qualitative analysis while deviations for the absolute values are seen. From a statistical analysis, the deviation is small for cantilevers with low effective spring constants, while the deviation is significant for large spring constants where the quasi one-dimensional approximation is no longer valid

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

Indian Academy of Sciences (India)

2016-08-26

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

Herbarium genomics

DEFF Research Database (Denmark)

Bakker, Freek T.; Lei, Di; Yu, Jiaying

2016-01-01

Herbarium genomics is proving promising as next-generation sequencing approaches are well suited to deal with the usually fragmented nature of archival DNA. We show that routine assembly of partial plastome sequences from herbarium specimens is feasible, from total DNA extracts and with specimens...... up to 146 years old. We use genome skimming and an automated assembly pipeline, Iterative Organelle Genome Assembly, that assembles paired-end reads into a series of candidate assemblies, the best one of which is selected based on likelihood estimation. We used 93 specimens from 12 different...... correlation between plastome coverage and nuclear genome size (C value) in our samples, but the range of C values included is limited. Finally, we conclude that routine plastome sequencing from herbarium specimens is feasible and cost-effective (compared with Sanger sequencing or plastome...

The complete chloroplast genome sequence of Podocarpus lambertii: genome structure, evolutionary aspects, gene content and SSR detection.

Directory of Open Access Journals (Sweden)

Leila do Nascimento Vieira

Full Text Available BACKGROUND: Podocarpus lambertii (Podocarpaceae is a native conifer from the Brazilian Atlantic Forest Biome, which is considered one of the 25 biodiversity hotspots in the world. The advancement of next-generation sequencing technologies has enabled the rapid acquisition of whole chloroplast (cp genome sequences at low cost. Several studies have proven the potential of cp genomes as tools to understand enigmatic and basal phylogenetic relationships at different taxonomic levels, as well as further probe the structural and functional evolution of plants. In this work, we present the complete cp genome sequence of P. lambertii. METHODOLOGY/PRINCIPAL FINDINGS: The P. lambertii cp genome is 133,734 bp in length, and similar to other sequenced cupressophytes, it lacks one of the large inverted repeat regions (IR. It contains 118 unique genes and one duplicated tRNA (trnN-GUU, which occurs as an inverted repeat sequence. The rps16 gene was not found, which was previously reported for the plastid genome of another Podocarpaceae (Nageia nagi and Araucariaceae (Agathis dammara. Structurally, P. lambertii shows 4 inversions of a large DNA fragment ∼20,000 bp compared to the Podocarpus totara cp genome. These unexpected characteristics may be attributed to geographical distance and different adaptive needs. The P. lambertii cp genome presents a total of 28 tandem repeats and 156 SSRs, with homo- and dipolymers being the most common and tri-, tetra-, penta-, and hexapolymers occurring with less frequency. CONCLUSION: The complete cp genome sequence of P. lambertii revealed significant structural changes, even in species from the same genus. These results reinforce the apparently loss of rps16 gene in Podocarpaceae cp genome. In addition, several SSRs in the P. lambertii cp genome are likely intraspecific polymorphism sites, which may allow highly sensitive phylogeographic and population structure studies, as well as phylogenetic studies of species of

Genomic sovereignty and the African promise: mining the African genome for the benefit of Africa.

Science.gov (United States)

de Vries, Jantina; Pepper, Michael

2012-08-01

Scientific interest in genomics in Africa is on the rise with a number of funding initiatives aimed specifically at supporting research in this area. Genomics research on material of African origin raises a number of important ethical issues. A prominent concern relates to sample export, which is increasingly seen by researchers and ethics committees across the continent as being problematic. The concept of genomic sovereignty proposes that unique patterns of genomic variation can be found in human populations, and that these are commercially, scientifically or symbolically valuable and in need of protection against exploitation. Although it is appealing as a response to increasing concerns regarding sample export, there are a number of important conceptual problems relating to the term. It is not clear, for instance, whether it is appropriate that ownership over human genomic samples should rest with national governments. Furthermore, ethnic groups in Africa are frequently spread across multiple nation states, and protection offered in one state may not prevent researchers from accessing the same group elsewhere. Lastly, scientific evidence suggests that the assumption that genomic data is unique for population groups is false. Although the frequency with which particular variants are found can differ between groups, such genes or variants per se are not unique to any population group. In this paper, the authors describe these concerns in detail and argue that the concept of genomic sovereignty alone may not be adequate to protect the genetic resources of people of African descent.

Partial digestion with restriction enzymes of ultraviolet-irradiated human genomic DNA: a method for identifying restriction site polymorphisms

International Nuclear Information System (INIS)

Nobile, C.; Romeo, G.

1988-01-01

A method for partial digestion of total human DNA with restriction enzymes has been developed on the basis of a principle already utilized by P.A. Whittaker and E. Southern for the analysis of phage lambda recombinants. Total human DNA irradiated with uv light of 254 nm is partially digested by restriction enzymes that recognize sequences containing adjacent thymidines because of TT dimer formation. The products resulting from partial digestion of specific genomic regions are detected in Southern blots by genomic-unique DNA probes with high reproducibility. This procedure is rapid and simple to perform because the same conditions of uv irradiation are used for different enzymes and probes. It is shown that restriction site polymorphisms occurring in the genomic regions analyzed are recognized by the allelic partial digest patterns they determine

Hyb-Seq: Combining target enrichment and genome skimming for plant phylogenomics1

Science.gov (United States)

Weitemier, Kevin; Straub, Shannon C. K.; Cronn, Richard C.; Fishbein, Mark; Schmickl, Roswitha; McDonnell, Angela; Liston, Aaron

2014-01-01

• Premise of the study: Hyb-Seq, the combination of target enrichment and genome skimming, allows simultaneous data collection for low-copy nuclear genes and high-copy genomic targets for plant systematics and evolution studies. • Methods and Results: Genome and transcriptome assemblies for milkweed (Asclepias syriaca) were used to design enrichment probes for 3385 exons from 768 genes (>1.6 Mbp) followed by Illumina sequencing of enriched libraries. Hyb-Seq of 12 individuals (10 Asclepias species and two related genera) resulted in at least partial assembly of 92.6% of exons and 99.7% of genes and an average assembly length >2 Mbp. Importantly, complete plastomes and nuclear ribosomal DNA cistrons were assembled using off-target reads. Phylogenomic analyses demonstrated signal conflict between genomes. • Conclusions: The Hyb-Seq approach enables targeted sequencing of thousands of low-copy nuclear exons and flanking regions, as well as genome skimming of high-copy repeats and organellar genomes, to efficiently produce genome-scale data sets for phylogenomics. PMID:25225629

Imaginal discs--a new source of chromosomes for genome mapping of the yellow fever mosquito Aedes aegypti.

Directory of Open Access Journals (Sweden)

Maria V Sharakhova

2011-10-01

Full Text Available The mosquito Aedes aegypti is the primary global vector for dengue and yellow fever viruses. Sequencing of the Ae. aegypti genome has stimulated research in vector biology and insect genomics. However, the current genome assembly is highly fragmented with only ~31% of the genome being assigned to chromosomes. A lack of a reliable source of chromosomes for physical mapping has been a major impediment to improving the genome assembly of Ae. aegypti.In this study we demonstrate the utility of mitotic chromosomes from imaginal discs of 4(th instar larva for cytogenetic studies of Ae. aegypti. High numbers of mitotic divisions on each slide preparation, large sizes, and reproducible banding patterns of the individual chromosomes simplify cytogenetic procedures. Based on the banding structure of the chromosomes, we have developed idiograms for each of the three Ae. aegypti chromosomes and placed 10 BAC clones and a 18S rDNA probe to precise chromosomal positions.The study identified imaginal discs of 4(th instar larva as a superior source of mitotic chromosomes for Ae. aegypti. The proposed approach allows precise mapping of DNA probes to the chromosomal positions and can be utilized for obtaining a high-quality genome assembly of the yellow fever mosquito.

In Silico Genomic Fingerprints of the Bacillus anthracis Group Obtained by Virtual Hybridization

Directory of Open Access Journals (Sweden)

Hueman Jaimes-Díaz

2015-02-01

Full Text Available In this study we evaluate the capacity of Virtual Hybridization to identify between highly related bacterial strains. Eight genomic fingerprints were obtained by virtual hybridization for the Bacillus anthracis genome set, and a set of 15,264 13-nucleotide short probes designed to produce genomic fingerprints unique for each organism. The data obtained from each genomic fingerprint were used to obtain hybridization patterns simulating a DNA microarray. Two virtual hybridization methods were used: the Direct and the Extended method to identify the number of potential hybridization sites and thus determine the minimum sensitivity value to discriminate between genomes with 99.9% similarity. Genomic fingerprints were compared using both methods and phylogenomic trees were constructed to verify that the minimum detection value is 0.000017. Results obtained from the genomic fingerprints suggest that the distribution in the trees is correct, as compared to other taxonomic methods. Specific virtual hybridization sites for each of the genomes studied were also identified.

Unlabeled probes for the detection and typing of herpes simplex virus.

Science.gov (United States)

Dames, Shale; Pattison, David C; Bromley, L Kathryn; Wittwer, Carl T; Voelkerding, Karl V

2007-10-01

Unlabeled probe detection with a double-stranded DNA (dsDNA) binding dye is one method to detect and confirm target amplification after PCR. Unlabeled probes and amplicon melting have been used to detect small deletions and single-nucleotide polymorphisms in assays where template is in abundance. Unlabeled probes have not been applied to low-level target detection, however. Herpes simplex virus (HSV) was chosen as a model to compare the unlabeled probe method to an in-house reference assay using dual-labeled, minor groove binding probes. A saturating dsDNA dye (LCGreen Plus) was used for real-time PCR. HSV-1, HSV-2, and an internal control were differentiated by PCR amplicon and unlabeled probe melting analysis after PCR. The unlabeled probe technique displayed 98% concordance with the reference assay for the detection of HSV from a variety of archived clinical samples (n = 182). HSV typing using unlabeled probes was 99% concordant (n = 104) to sequenced clinical samples and allowed for the detection of sequence polymorphisms in the amplicon and under the probe. Unlabeled probes and amplicon melting can be used to detect and genotype as few as 10 copies of target per reaction, restricted only by stochastic limitations. The use of unlabeled probes provides an attractive alternative to conventional fluorescence-labeled, probe-based assays for genotyping and detection of HSV and might be useful for other low-copy targets where typing is informative.

Characterization of power induced heating and damage in fiber optic probes for near-field scanning optical microscopy

Science.gov (United States)

Dickenson, Nicholas E.; Erickson, Elizabeth S.; Mooren, Olivia L.; Dunn, Robert C.

2007-05-01

Tip-induced sample heating in near-field scanning optical microscopy (NSOM) is studied for fiber optic probes fabricated using the chemical etching technique. To characterize sample heating from etched NSOM probes, the spectra of a thermochromic polymer sample are measured as a function of probe output power, as was previously reported for pulled NSOM probes. The results reveal that sample heating increases rapidly to ˜55-60°C as output powers reach ˜50nW. At higher output powers, the sample heating remains approximately constant up to the maximum power studied of ˜450nW. The sample heating profiles measured for etched NSOM probes are consistent with those previously measured for NSOM probes fabricated using the pulling method. At high powers, both pulled and etched NSOM probes fail as the aluminum coating is damaged. For probes fabricated in our laboratory we find failure occurring at input powers of 3.4±1.7 and 20.7±6.9mW for pulled and etched probes, respectively. The larger half-cone angle for etched probes (˜15° for etched and ˜6° for pulled probes) enables more light delivery and also apparently leads to a different failure mechanism. For pulled NSOM probes, high resolution images of NSOM probes as power is increased reveal the development of stress fractures in the coating at a taper diameter of ˜6μm. These stress fractures, arising from the differential heating expansion of the dielectric and the metal coating, eventually lead to coating removal and probe failure. For etched tips, the absence of clear stress fractures and the pooled morphology of the damaged aluminum coating following failure suggest that thermal damage may cause coating failure, although other mechanisms cannot be ruled out.

Determination of paraquat in water samples using a sensitive fluorescent probe titration method.

Science.gov (United States)

Yao, Feihu; Liu, Hailong; Wang, Guangquan; Du, Liming; Yin, Xiaofen; Fu, Yunlong

2013-06-01

Paraquat (PQ), a nonselective herbicide, is non-fluorescent in aqueous solutions. Thus, its determination through direct fluorescent methods is not feasible. The supramolecular inclusion interaction of PQ with cucurbit[7]uril was studied by a fluorescent probe titration method. Significant quenching of the fluorescence intensity of the cucurbit[7]uril-coptisine fluorescent probe was observed with the addition of PQ. A new fluorescent probe titration method with high selectivity and sensitivity at the ng/mL level was developed to determine PQ in aqueous solutions with good precision and accuracy based on the significant quenching of the supramolecular complex fluorescence intensity. The proposed method was successfully used in the determination of PQ in lake water, tap water, well water, and ditch water in an agricultural area, with recoveries of 96.73% to 105.77%. The fluorescence quenching values (deltaF) showed a good linear relationship with PQ concentrations from 1.0 x 10(-8) to 1.2 x 10(-5) mol/L with a detection limit of 3.35 x 10(-9) mol/L. In addition, the interaction models of the supramolecular complexes formed between the host and the guest were established using theoretical calculations. The interaction mechanism between the cucurbit[7]uril and PQ was confirmed by 1H NMR spectroscopy.

CHARACTERIZING AND MODELING FERRITE-CORE PROBES

International Nuclear Information System (INIS)

Sabbagh, Harold A.; Murphy, R. Kim; Sabbagh, Elias H.; Aldrin, John C.

2010-01-01

In this paper, we accurately and carefully characterize a ferrite-core probe that is widely used for aircraft inspections. The characterization starts with the development of a model that can be executed using the proprietary volume-integral code, VIC-3D(c), and then the model is fitted to measured multifrequency impedance data taken with the probe in freespace and over samples of a titanium alloy and aluminum. Excellent results are achieved, and will be discussed.

Theory and practice of near-field thermal probes for microscopy and thermal analysis

International Nuclear Information System (INIS)

Hodges, C.S.

1999-03-01

Bacterial mats called biofilms that form on the surfaces of industrial steel pipes can cause corrosion of the pipe. Examining the steel surface of the corroded pipe usually involves removal of the biofilm using acid. This acid can also cause corrosion of the pipe so that the observed corrosion cracks and pits are the result of both the acid and the biofilm. It was thought that non-invasive examination of the corrosion caused by the biofilm may be obtained by using a thin wire bent into a loop that acts as both a heat source a nd a detector of heat, measuring the changes in heat flow out of the wire as the wire passes over the steel with the biofilm still present. This technique of using a heated probe to scan samples on a microscopic scale is called Scanning Thermal Microscopy (SThM) and uses an alternating current to produce a.c. thermal waves that emanate from the probe tip into the sample. The alternating current allows better signal-to-noise ratios and also selective depth imaging of the sample since the thermal wave penetrates into the sample a distance inversely proportional to the applied current frequency. Reversal in the contrast of SThM images on biofilms and subsequently all samples was observed as either the frequency or the amplitude of the temperature waves was altered. Whilst changing the time constant of the feedback circuit attached to the SThM probe did go some way to explain this effect, a full explanation is still wanting. Despite many efforts to image the biofilm/steel interface with the biofilm still present, often the biofilm was either too thick or too complicated to do this. A simpler thermal test sample is required to calibrate the thermal probe. In addition to SThM, one may select a point on a sample surface and ramp the temperature of the probe to obtain a Localised Thermal Analysis (LTA) temperature scan looking for melts, recrystallisations, glass transitions of the part of the sample in contact with the probe. This technique is a

A Taste of Algal Genomes from the Joint Genome Institute

Energy Technology Data Exchange (ETDEWEB)

Kuo, Alan; Grigoriev, Igor

2012-06-17

Algae play profound roles in aquatic food chains and the carbon cycle, can impose health and economic costs through toxic blooms, provide models for the study of symbiosis, photosynthesis, and eukaryotic evolution, and are candidate sources for bio-fuels; all of these research areas are part of the mission of DOE's Joint Genome Institute (JGI). To date JGI has sequenced, assembled, annotated, and released to the public the genomes of 18 species and strains of algae, sampling almost all of the major clades of photosynthetic eukaryotes. With more algal genomes currently undergoing analysis, JGI continues its commitment to driving forward basic and applied algal science. Among these ongoing projects are the pan-genome of the dominant coccolithophore Emiliania huxleyi, the interrelationships between the 4 genomes in the nucleomorph-containing Bigelowiella natans and Guillardia theta, and the search for symbiosis genes of lichens.

Nanoparticle sensor for label free detection of swine DNA in mixed biological samples

Energy Technology Data Exchange (ETDEWEB)

Ali, M E; Hashim, U [Institute of Nano Electronic Engineering (INNE), Universiti Malaysia Perlis, Lot 104-108, Tingkat 1, Block A, Taman Pertiwi Indah, Jalan Kangar-Alor Star, Seriab, 01000 Kangar, Perlis (Malaysia); Mustafa, S; Che Man, Y B; Yusop, M H M [Halal Products Research Institute, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor (Malaysia); Bari, M F [School of Materials Engineering, University Malaysia Perlis, Seriab 01000, Kangar, Perlis (Malaysia); Islam, Kh N [Department of Preclinical Sciences, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor (Malaysia); Hasan, M F, E-mail: uda@unimap.edu.my [Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor (Malaysia)

2011-05-13

We used 40 {+-} 5 nm gold nanoparticles (GNPs) as colorimetric sensor to visually detect swine-specific conserved sequence and nucleotide mismatch in PCR-amplified and non-amplified mitochondrial DNA mixtures to authenticate species. Colloidal GNPs changed color from pinkish-red to gray-purple in 2 mM PBS. Visually observed results were clearly reflected by the dramatic reduction of surface plasmon resonance peak at 530 nm and the appearance of new features in the 620-800 nm regions in their absorption spectra. The particles were stabilized against salt-induced aggregation upon the adsorption of single-stranded DNA. The PCR products, without any additional processing, were hybridized with a 17-base probe prior to exposure to GNPs. At a critical annealing temperature (55 {sup 0}C) that differentiated matched and mismatched base pairing, the probe was hybridized to pig PCR product and dehybridized from the deer product. The dehybridized probe stuck to GNPs to prevent them from salt-induced aggregation and retained their characteristic red color. Hybridization of a 27-nucleotide probe to swine mitochondrial DNA identified them in pork-venison, pork-shad and venison-shad binary admixtures, eliminating the need of PCR amplification. Thus the assay was applied to authenticate species both in PCR-amplified and non-amplified heterogeneous biological samples. The results were determined visually and validated by absorption spectroscopy. The entire assay (hybridization plus visual detection) was performed in less than 10 min. The LOD (for genomic DNA) of the assay was 6 {mu}g ml{sup -1} swine DNA in mixed meat samples. We believe the assay can be applied for species assignment in food analysis, mismatch detection in genetic screening and homology studies between closely related species.

Nanoparticle sensor for label free detection of swine DNA in mixed biological samples

International Nuclear Information System (INIS)

Ali, M E; Hashim, U; Mustafa, S; Che Man, Y B; Yusop, M H M; Bari, M F; Islam, Kh N; Hasan, M F

2011-01-01

We used 40 ± 5 nm gold nanoparticles (GNPs) as colorimetric sensor to visually detect swine-specific conserved sequence and nucleotide mismatch in PCR-amplified and non-amplified mitochondrial DNA mixtures to authenticate species. Colloidal GNPs changed color from pinkish-red to gray-purple in 2 mM PBS. Visually observed results were clearly reflected by the dramatic reduction of surface plasmon resonance peak at 530 nm and the appearance of new features in the 620-800 nm regions in their absorption spectra. The particles were stabilized against salt-induced aggregation upon the adsorption of single-stranded DNA. The PCR products, without any additional processing, were hybridized with a 17-base probe prior to exposure to GNPs. At a critical annealing temperature (55 0 C) that differentiated matched and mismatched base pairing, the probe was hybridized to pig PCR product and dehybridized from the deer product. The dehybridized probe stuck to GNPs to prevent them from salt-induced aggregation and retained their characteristic red color. Hybridization of a 27-nucleotide probe to swine mitochondrial DNA identified them in pork-venison, pork-shad and venison-shad binary admixtures, eliminating the need of PCR amplification. Thus the assay was applied to authenticate species both in PCR-amplified and non-amplified heterogeneous biological samples. The results were determined visually and validated by absorption spectroscopy. The entire assay (hybridization plus visual detection) was performed in less than 10 min. The LOD (for genomic DNA) of the assay was 6 μg ml -1 swine DNA in mixed meat samples. We believe the assay can be applied for species assignment in food analysis, mismatch detection in genetic screening and homology studies between closely related species.

Electromechanical response of amorphous LaAlO{sub 3} thin film probed by scanning probe microscopies

Energy Technology Data Exchange (ETDEWEB)

Borowiak, Alexis S.; Baboux, Nicolas; Albertini, David; Gautier, Brice, E-mail: brice.gautier@insa-lyon.fr [Institut des nanotechnologies de Lyon (INL), Institut National des Sciences Appliquées de Lyon, Université de Lyon, UMR CNRS 5270, 7 Avenue Capelle, F-69621 Villeurbanne Cedex (France); Vilquin, Bertrand; Saint Girons, Guillaume; Pelloquin, Sylvain [Institut des nanotechnologies de Lyon (INL), Ecole Centrale de Lyon, Université de Lyon, UMR CNRS 5270, 36 Avenue Guy de Collongues, F-69134 Ecully Cedex (France)

2014-07-07

The electromechanical response of a 3 nm thick amorphous LaAlO{sub 3} layer obtained by molecular beam epitaxy has been studied using scanning probe microscopies. Although this kind of sample is not ferroelectric due to its amorphous nature, the resulting images are identical to what is generally obtained on truly ferroelectric samples probed by piezoresponse force microscopy: domains of apparently opposite polarisation are detected, and perfect, square shaped hysteresis loops are recorded. Moreover, written patterns are stable within 72 h. We discuss in the general case the possible origins of this behaviour in terms of charge injection, ionic conduction and motion of oxygen vacancies. In the case presented in this paper, since the writing process has been conducted with applied voltages lower than the injection threshold measured by conductive atomic force Microscopy, allowing to withdraw the hypothesis of charge injection in the sample, we propose that a bistable distribution of oxygen vacancies is responsible for this contrast.

Probes for edge plasma studies of TFTR (invited)

International Nuclear Information System (INIS)

Manos, D.M.; Budny, R.V.; Kilpatrick, S.; Stangeby, P.; Zweben, S.

1986-01-01

Tokamak fusion test reactor (TFTR) probes are designed to study the interaction of the plasma with material surfaces such as the wall and limiters, and to study the transport of particles and energy between the core and edge. Present probe heads have evolved from prototypes in Princeton large torus (PLT), poloidal divertor experiment (PDX) [Princeton BETA experiment (PBX)], and the initial phase of TFTR operation. The newest heads are capable of making several simultaneous measurements and include Langmuir probes, heat flux probes, magnetic coils, rotating calorimeter fast ion probes, and sample exposure specimens. This paper describes these probe heads and presents some of the data they and their prototypes have acquired. The paper emphasizes measurement of transient plasma effects such as fast ion loss during auxiliary heating, the evolution of the edge plasma during heating, compression, and free expansion, and fluctuations in the edge plasma

Primary Multidrug Resistant Tuberculosis and Utility of Line Probe Assay for Its Detection in Smear-Positive Sputum Samples in a Tertiary Care Hospital in South India

Directory of Open Access Journals (Sweden)

Fahmiya Leena Yacoob

2016-01-01

Full Text Available In a high tuberculosis burdened country like India, rapid, cost-effective, and reliable diagnostic tools for tuberculosis are an urgent need of the hour to prevent inappropriate treatment strategies and further spread of resistance. This study aimed to estimate the proportion of new smear-positive tuberculosis cases with primary resistance to rifampicin and/or isoniazid as well as identify the common mutations associated with it. Sputum of 200 newly diagnosed smear-positive cases of 1+ score and above was directly subjected to Line Probe Assay using the GenoType MTBDRplus assay kit. All samples were inoculated onto solid media and 61 samples were inoculated in automated liquid culture also. The Line Probe Assay gave hundred percent interpretable results with 2.5% of the study population showing resistant pattern. Only 1% of the cases were primary multidrug resistant tuberculosis and 1.5% showed isoniazid monoresistance. S531L and C15T were the most common genetic mutations seen for rifampicin and isoniazid resistance, respectively. 40% had absent rpoB wild type 8 band indicating probable silent mutation after clinical correlation. The average turnaround time for Line Probe Assay was far less (3.8 days as compared to solid and liquid cultures (35.6 days and 13.5 days, resp..

Primary Multidrug Resistant Tuberculosis and Utility of Line Probe Assay for Its Detection in Smear-Positive Sputum Samples in a Tertiary Care Hospital in South India.

Science.gov (United States)

Yacoob, Fahmiya Leena; Philomina Jose, Beena; Karunakaran Lelitha, Sarada Devi; Sreenivasan, Sreelatha

2016-01-01

In a high tuberculosis burdened country like India, rapid, cost-effective, and reliable diagnostic tools for tuberculosis are an urgent need of the hour to prevent inappropriate treatment strategies and further spread of resistance. This study aimed to estimate the proportion of new smear-positive tuberculosis cases with primary resistance to rifampicin and/or isoniazid as well as identify the common mutations associated with it. Sputum of 200 newly diagnosed smear-positive cases of 1+ score and above was directly subjected to Line Probe Assay using the GenoType MTBDRplus assay kit. All samples were inoculated onto solid media and 61 samples were inoculated in automated liquid culture also. The Line Probe Assay gave hundred percent interpretable results with 2.5% of the study population showing resistant pattern. Only 1% of the cases were primary multidrug resistant tuberculosis and 1.5% showed isoniazid monoresistance. S531L and C15T were the most common genetic mutations seen for rifampicin and isoniazid resistance, respectively. 40% had absent rpoB wild type 8 band indicating probable silent mutation after clinical correlation. The average turnaround time for Line Probe Assay was far less (3.8 days) as compared to solid and liquid cultures (35.6 days and 13.5 days, resp.).

Sampling/classification of gasifier particulates

International Nuclear Information System (INIS)

Wegrzyn, J.

1984-01-01

A high temperature and pressure real time extractive sampling probe for particulate monitoring was built at Brookhaven National Laboratory and tested on Morgantown Energy Technology Center's 42 inch fixed bed gasifier. The probe was specifically designed for the conditions of highly loaded particulate and condensable streams, that exist at the outlet of a fixed bed gasifier. Some of the salient features of the probe are: porous tube gas injection, aerodynamic particle classification in the presence of condensable vapors, β gauge particle detection, and micro processor control. Three of the key design problems were the separation of the particles from the vapor without promoting condensation, the prevention of plugging, and real time monitoring. Some plugging did occur over the seven day sampling period, but by over pressurizing and back purging the clog was blown back into the process stream. The tests validate the proof of concept of the sampling probe and indicated that the particulate output from the bed came in the form of bursts (several minutes in duration) rather than in the form of a steady stream

Piezoresistor-equipped fluorescence-based cantilever probe for near-field scanning.

Science.gov (United States)

Kan, Tetsuo; Matsumoto, Kiyoshi; Shimoyama, Isao

2007-08-01

Scanning near-field optical microscopes (SNOMs) with fluorescence-based probes are promising tools for evaluating the optical characteristics of nanoaperture devices used for biological investigations, and this article reports on the development of a microfabricated fluorescence-based SNOM probe with a piezoresistor. The piezoresistor was built into a two-legged root of a 160-microm-long cantilever. To improve the displacement sensitivity of the cantilever, the piezoresistor's doped area was shallowly formed on the cantilever surface. A fluorescent bead, 500 nm in diameter, was attached to the bottom of the cantilever end as a light-intensity-sensitive material in the visible-light range. The surface of the scanned sample was simply detected by the probe's end being displaced by contact with the sample. Measuring displacements piezoresistively is advantageous because it eliminates the noise arising from the use of the optical-lever method and is free of any disturbance in the absorption or the emission spectrum of the fluorescent material at the probe tip. The displacement sensitivity was estimated to be 6.1 x 10(-6) nm(-1), and the minimum measurable displacement was small enough for near-field measurement. This probe enabled clear scanning images of the light field near a 300 x 300 nm(2) aperture to be obtained in the near-field region where the tip-sample distance is much shorter than the light wavelength. This scanning result indicates that the piezoresistive way of tip-sample distance regulation is effective for characterizing nanoaperture optical devices.

Scanning probe microscopy with vertically oriented cantilevers made easy

International Nuclear Information System (INIS)

Valdrè, G; Moro, D; Ulian, G

2012-01-01

Non-contact imaging in scanning probe microscopy (SPM) is becoming of great importance in particular for imaging biological matter and in general soft materials. Transverse dynamic force microscopy (TDFM) is an SPM-based methodology that exploiting a cantilever oriented in a vertical configuration with respect to the sample surface may work with very low tip to sample interaction forces. The probe is oscillated parallel to the sample surface, usually by a piezoelectric element. However, this methodology often requires complex microscope setups and detection systems, so it is usually developed in specific laboratories as a prototype microscope. Here, we present a very simple device that easily enables a commercial SPM head to be oriented in such a way to have the cantilever long axis perpendicular to the sample surface. No modifications of the SPM hardware and software are required and commercial available cantilevers can be used as probes. Performance tests using polystyrene spheres, muscovite crystallographic steps and DNA single molecules were successful and all resulted in agreement with other TDFM and SPM observations demonstrating the reliability of the device. (paper)

First-generation physical map of the Culicoides variipennis (Diptera: Ceratopogonidae) genome.

Science.gov (United States)

Nunamaker, R A; Brown, S E; McHolland, L E; Tabachnick, W J; Knudson, D L

1999-11-01

Recombinant cosmids labeled with biotin-11-dUTP or digoxigenin by nick translation were used as in situ hybridization probes to metaphase chromosomes of Culicoides variipennis (Coquillett). Paired fluorescent signals were detected on each arm of sister chromatids and were ordered along the 3 chromosomes. Thirty-three unique probes were mapped to the 3 chromosomes of C. variipennis (2n = 6): 7 to chromosome 1, 20 to chromosome 2, and 6 to chromosome 3. This work represents the first stage in generating a physical map of the genome of C. variipennis.

A microscale protein NMR sample screening pipeline

Energy Technology Data Exchange (ETDEWEB)

Rossi, Paolo; Swapna, G. V. T.; Huang, Yuanpeng J.; Aramini, James M. [State University of New Jersey, Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers (United States); Anklin, Clemens [Bruker Biospin Corporation (United States); Conover, Kenith; Hamilton, Keith; Xiao, Rong; Acton, Thomas B.; Ertekin, Asli; Everett, John K.; Montelione, Gaetano T., E-mail: guy@cabm.rutgers.ed [State University of New Jersey, Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers (United States)

2010-01-15

As part of efforts to develop improved methods for NMR protein sample preparation and structure determination, the Northeast Structural Genomics Consortium (NESG) has implemented an NMR screening pipeline for protein target selection, construct optimization, and buffer optimization, incorporating efficient microscale NMR screening of proteins using a micro-cryoprobe. The process is feasible because the newest generation probe requires only small amounts of protein, typically 30-200 {mu}g in 8-35 {mu}l volume. Extensive automation has been made possible by the combination of database tools, mechanization of key process steps, and the use of a micro-cryoprobe that gives excellent data while requiring little optimization and manual setup. In this perspective, we describe the overall process used by the NESG for screening NMR samples as part of a sample optimization process, assessing optimal construct design and solution conditions, as well as for determining protein rotational correlation times in order to assess protein oligomerization states. Database infrastructure has been developed to allow for flexible implementation of new screening protocols and harvesting of the resulting output. The NESG micro NMR screening pipeline has also been used for detergent screening of membrane proteins. Descriptions of the individual steps in the NESG NMR sample design, production, and screening pipeline are presented in the format of a standard operating procedure.

DESIGN OF THE CONTACT POTENTIALS DIFFERENCE PROBES

Directory of Open Access Journals (Sweden)

K. U. Pantsialeyeu

2016-01-01

Full Text Available The contact potential difference probes distinguished by great variety and produced mostly in the laboratory for specific experimental applications. As a rule, they consist of commercially available instrumentation, and have a number of disadvantages: large dimensions, complexity and high cost, small sensitivity, operating speed, noiseproof, etc. The purpose of this paper is to describe the basic approaches to design of the small dimension, complete contact potential difference probes, providing high sensitivity, operating speed, and noise immunity. In this paper the contact potential difference probe, which is a electrometer with dynamic capacitor plate at about 0.1–5 mm2 . These probes are could be used in scanning systems, such as a Scanning Kelvin Probe, as well as for controlling system of manufacturing processes, e.g. under friction. The design of such contact potential difference probes conducted using modern electronic components, unique circuitry and design solutions described in detail at paper. The electromechanical modulator applied for mechanical vibrations of the reference sample. To provide a high amplitude and phase stability the upgraded generator with Wien bridge was used instead traditional oscillation sensor. The preamplifier made on the base of modern operational amplifiers with femtoampere current input. The power of the preamplifier designed with «floating ground». It allows keeping the relation constant potential to the probe components when changing over a wide range the compensation voltage. The phase detector-integrator based on the electronic antiphase switches with the modulation frequency of the contact potential difference and the integrator. Fullwave phase detection would greatly increase the sensitivity of the probe. In addition, the application of the phase detection allows suppressing noise and crosstalk at frequencies different from the modulation frequency. The preamplifier and the reference sample

Measuring the homogeneity of Bi(2223)/Ag tapes by four-probe method and a Hall probe array

International Nuclear Information System (INIS)

Kovac, P.

1999-01-01

The nature of the BSCCO compound and application of the powder-in-tube technique usually lead to non-uniform quality across and/or along the ceramic fibres and finally to variations in the critical current and its irregular distribution in the Bi(2223)/Ag tape. Therefore, the gliding four-probe method and contactless field monitoring measurements have been used for homogeneity studies. The gliding potential contacts moved along the tape surface and a sensitive system based on an integrated Hall probe array containing 16 or 19 in-line probes supported by PC-compatible electronics with software allowed us to make a comparison of contact and contactless measurements at any elements of Bi(2223)/Ag sample. The results of both methods show very good correlation and the possibility of using a sensitive Hall probe array for monitoring the final quality of Bi(2223)/Ag tapes. (author)

Non-replication study of a genome-wide association study for hypertension and blood pressure in African Americans

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Kidambi Srividya

2012-04-01

Full Text Available Abstract Background A recent genome wide association study in 1017 African Americans identified several single nucleotide polymorphisms that reached genome-wide significance for systolic blood pressure. We attempted to replicate these findings in an independent sample of 2474 unrelated African Americans in the Milwaukee metropolitan area; 53% were women and 47% were hypertensives. Methods We evaluated sixteen top associated SNPs from the above genome wide association study for hypertension as a binary trait or blood pressure as a continuous trait. In addition, we evaluated eight single nucleotide polymorphisms located in two genes (STK-39 and CDH-13 found to be associated with systolic and diastolic blood pressures by other genome wide association studies in European and Amish populations. TaqMan MGB-based chemistry with fluorescent probes was used for genotyping. We had an adequate sample size (80% power to detect an effect size of 1.2-2.0 for all the single nucleotide polymorphisms for hypertension as a binary trait, and 1% variance in blood pressure as a continuous trait. Quantitative trait analyses were performed both by excluding and also by including subjects on anti-hypertensive therapy (after adjustments were made for anti-hypertensive medications. Results For all 24 SNPs, no statistically significant differences were noted in the minor allele frequencies between cases and controls. One SNP (rs2146204 showed borderline association (p = 0.006 with hypertension status using recessive model and systolic blood pressure (p = 0.02, but was not significant after adjusting for multiple comparisons. In quantitative trait analyses, among normotensives only, rs12748299 was associated with SBP (p = 0.002. In addition, several nominally significant associations were noted with SBP and DBP among normotensives but none were statistically significant. Conclusions This study highlights the importance of replication to confirm the validity of genome wide

Construction of a nurse shark (Ginglymostoma cirratum bacterial artificial chromosome (BAC library and a preliminary genome survey

Directory of Open Access Journals (Sweden)

Inoko Hidetoshi

2006-05-01

Full Text Available Abstract Background Sharks are members of the taxonomic class Chondrichthyes, the oldest living jawed vertebrates. Genomic studies of this group, in comparison to representative species in other vertebrate taxa, will allow us to theorize about the fundamental genetic, developmental, and functional characteristics in the common ancestor of all jawed vertebrates. Aims In order to obtain mapping and sequencing data for comparative genomics, we constructed a bacterial artificial chromosome (BAC library for the nurse shark, Ginglymostoma cirratum. Results The BAC library consists of 313,344 clones with an average insert size of 144 kb, covering ~4.5 × 1010 bp and thus providing an 11-fold coverage of the haploid genome. BAC end sequence analyses revealed, in addition to LINEs and SINEs commonly found in other animal and plant genomes, two new groups of nurse shark-specific repetitive elements, NSRE1 and NSRE2 that seem to be major components of the nurse shark genome. Screening the library with single-copy or multi-copy gene probes showed 6–28 primary positive clones per probe of which 50–90% were true positives, demonstrating that the BAC library is representative of the different regions of the nurse shark genome. Furthermore, some BAC clones contained multiple genes, making physical mapping feasible. Conclusion We have constructed a deep-coverage, high-quality, large insert, and publicly available BAC library for a cartilaginous fish. It will be very useful to the scientific community interested in shark genomic structure, comparative genomics, and functional studies. We found two new groups of repetitive elements specific to the nurse shark genome, which may contribute to the architecture and evolution of the nurse shark genome.

Construction of a nurse shark (Ginglymostoma cirratum) bacterial artificial chromosome (BAC) library and a preliminary genome survey.

Science.gov (United States)

Luo, Meizhong; Kim, Hyeran; Kudrna, Dave; Sisneros, Nicholas B; Lee, So-Jeong; Mueller, Christopher; Collura, Kristi; Zuccolo, Andrea; Buckingham, E Bryan; Grim, Suzanne M; Yanagiya, Kazuyo; Inoko, Hidetoshi; Shiina, Takashi; Flajnik, Martin F; Wing, Rod A; Ohta, Yuko

2006-05-03

Sharks are members of the taxonomic class Chondrichthyes, the oldest living jawed vertebrates. Genomic studies of this group, in comparison to representative species in other vertebrate taxa, will allow us to theorize about the fundamental genetic, developmental, and functional characteristics in the common ancestor of all jawed vertebrates. In order to obtain mapping and sequencing data for comparative genomics, we constructed a bacterial artificial chromosome (BAC) library for the nurse shark, Ginglymostoma cirratum. The BAC library consists of 313,344 clones with an average insert size of 144 kb, covering ~4.5 x 1010 bp and thus providing an 11-fold coverage of the haploid genome. BAC end sequence analyses revealed, in addition to LINEs and SINEs commonly found in other animal and plant genomes, two new groups of nurse shark-specific repetitive elements, NSRE1 and NSRE2 that seem to be major components of the nurse shark genome. Screening the library with single-copy or multi-copy gene probes showed 6-28 primary positive clones per probe of which 50-90% were true positives, demonstrating that the BAC library is representative of the different regions of the nurse shark genome. Furthermore, some BAC clones contained multiple genes, making physical mapping feasible. We have constructed a deep-coverage, high-quality, large insert, and publicly available BAC library for a cartilaginous fish. It will be very useful to the scientific community interested in shark genomic structure, comparative genomics, and functional studies. We found two new groups of repetitive elements specific to the nurse shark genome, which may contribute to the architecture and evolution of the nurse shark genome.

Calibration models for high enthalpy calorimetric probes.

Science.gov (United States)

Kannel, A

1978-07-01

The accuracy of gas-aspirated liquid-cooled calorimetric probes used for measuring the enthalpy of high-temperature gas streams is studied. The error in the differential temperature measurements caused by internal and external heat transfer interactions is considered and quantified by mathematical models. The analysis suggests calibration methods for the evaluation of dimensionless heat transfer parameters in the models, which then can give a more accurate value for the enthalpy of the sample. Calibration models for four types of calorimeters are applied to results from the literature and from our own experiments: a circular slit calorimeter developed by the author, single-cooling jacket probe, double-cooling jacket probe, and split-flow cooling jacket probe. The results show that the models are useful for describing and correcting the temperature measurements.

Performance of Molecular Inversion Probes (MIP) in Allele CopyNumber Determination

Energy Technology Data Exchange (ETDEWEB)

Wang, Yuker; Moorhead, Martin; Karlin-Neumann, George; Wang,Nicolas J.; Ireland, James; Lin, Steven; Chen, Chunnuan; Heiser, LauraM.; Chin, Koei; Esserman, Laura; Gray, Joe W.; Spellman, Paul T.; Faham,Malek

2007-05-14

We have developed a new protocol for using MolecularInversion Probes (MIP) to accurately and specifically measure allele copynumber (ACN). The new protocol provides for significant improvementsincluding the reduction of input DNA (from 2?g) by more than 25 fold (to75ng total genomic DNA), higher overall precision resulting in one orderof magnitude lower false positive rate, and greater dynamic range withaccurate absolute copy number up to 60 copies.

Preclinical detection of porcine circovirus type 2 infection using an ultrasensitive nanoparticle DNA probe-based PCR assay.

Directory of Open Access Journals (Sweden)

Yong Huang

Full Text Available Porcine circovirus type 2 (PCV2 has emerged as one of the most important pathogens affecting swine production globally. Preclinical identification of PCV2 is very important for effective prophylaxis of PCV2-associated diseases. In this study, we developed an ultrasensitive nanoparticle DNA probe-based PCR assay (UNDP-PCR for PCV2 detection. Magnetic microparticles coated with PCV2 specific DNA probes were used to enrich PCV2 DNA from samples, then gold nanoparticles coated with PCV2 specific oligonucleotides were added to form a sandwich nucleic acid-complex. After the complex was formed, the oligonucleotides were released and characterized by PCR. This assay exhibited about 500-fold more sensitive than conventional PCR, with a detection limit of 2 copies of purified PCV2 genomic DNA and 10 viral copies of PCV2 in serum. The assay has a wide detection range for all of PCV2 genotypes with reliable reproducibility. No cross-reactivity was observed from the samples of other related viruses including porcine circovirus type 1, porcine parvovirus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus and classical swine fever virus. The positive detection rate of PCV2 specific UNDP-PCR in 40 preclinical field samples was 27.5%, which appeared greater than that by conventional and real-time PCR and appeared application potency in evaluation of the viral loads levels of preclinical infection samples. The UNDP-PCR assay reported here can reliably rule out false negative results from antibody-based assays, provide a nucleic acid extraction free, specific, ultrasensitive, economic and rapid diagnosis method for preclinical PCV2 infection in field, which may help prevent large-scale outbreaks.

Correction for Measurement Error from Genotyping-by-Sequencing in Genomic Variance and Genomic Prediction Models

DEFF Research Database (Denmark)

Ashraf, Bilal; Janss, Luc; Jensen, Just

sample). The GBSeq data can be used directly in genomic models in the form of individual SNP allele-frequency estimates (e.g., reference reads/total reads per polymorphic site per individual), but is subject to measurement error due to the low sequencing depth per individual. Due to technical reasons....... In the current work we show how the correction for measurement error in GBSeq can also be applied in whole genome genomic variance and genomic prediction models. Bayesian whole-genome random regression models are proposed to allow implementation of large-scale SNP-based models with a per-SNP correction...... for measurement error. We show correct retrieval of genomic explained variance, and improved genomic prediction when accounting for the measurement error in GBSeq data...

Identifying the Basal Angiosperm Node in Chloroplast GenomePhylogenies: Sampling One's Way Out of the Felsenstein Zone

Energy Technology Data Exchange (ETDEWEB)

Leebens-Mack, Jim; Raubeson, Linda A.; Cui, Liying; Kuehl,Jennifer V.; Fourcade, Matthew H.; Chumley, Timothy W.; Boore, JeffreyL.; Jansen, Robert K.; dePamphilis, Claude W.

2005-05-27

While there has been strong support for Amborella and Nymphaeales (water lilies) as branching from basal-most nodes in the angiosperm phylogeny, this hypothesis has recently been challenged by phylogenetic analyses of 61 protein-coding genes extracted from the chloroplast genome sequences of Amborella, Nymphaea and 12 other available land plant chloroplast genomes. These character-rich analyses placed the monocots, represented by three grasses (Poaceae), as sister to all other extant angiosperm lineages. We have extracted protein-coding regions from draft sequences for six additional chloroplast genomes to test whether this surprising result could be an artifact of long-branch attraction due to limited taxon sampling. The added taxa include three monocots (Acorus, Yucca and Typha), a water lily (Nuphar), a ranunculid(Ranunculus), and a gymnosperm (Ginkgo). Phylogenetic analyses of the expanded DNA and protein datasets together with microstructural characters (indels) provided unambiguous support for Amborella and the Nymphaeales as branching from the basal-most nodes in the angiospermphylogeny. However, their relative positions proved to be dependent on method of analysis, with parsimony favoring Amborella as sister to all other angiosperms, and maximum likelihood and neighbor-joining methods favoring an Amborella + Nympheales clade as sister. The maximum likelihood phylogeny supported the later hypothesis, but the likelihood for the former hypothesis was not significantly different. Parametric bootstrap analysis, single gene phylogenies, estimated divergence dates and conflicting in del characters all help to illuminate the nature of the conflict in resolution of the most basal nodes in the angiospermphylogeny. Molecular dating analyses provided median age estimates of 161 mya for the most recent common ancestor of all extant angiosperms and 145 mya for the most recent common ancestor of monocots, magnoliids andeudicots. Whereas long sequences reduce variance in

One bacterial cell, one complete genome.

Directory of Open Access Journals (Sweden)

Tanja Woyke

2010-04-01

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

One Bacterial Cell, One Complete Genome

Energy Technology Data Exchange (ETDEWEB)

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

2010-04-26

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

RNA interactions in the 5' region of the HIV-1 genome

DEFF Research Database (Denmark)

Damgaard, Christian Kroun; Andersen, Ebbe Sloth; Knudsen, Bjarne

2004-01-01

The untranslated leader of the dimeric HIV-1 RNA genome is folded into a complex structure that plays multiple and essential roles in the viral replication cycle. Here, we have investigated secondary and tertiary structural elements within the 5' 744 nucleotides of the HIV-1 genome using...... a combination of bioinformatics, enzymatic probing, native gel electrophoresis, and UV-crosslinking experiments. We used a recently developed RNA folding algorithm (Pfold) to predict the common secondary structure of an alignment of 20 divergent HIV-1 sequences. Combining this analysis with biochemical data, we...

Custom-Designed MLPA Using Multiple Short Synthetic Probes Application to Methylation Analysis of Five Promoter CpG Islands in Tumor and Urine Specimens from Patients with Bladder Cancer

DEFF Research Database (Denmark)

Serizawa, R.R.; Ralfkiaer, U.; Dahl, C.

2010-01-01

Ligation of two oligonucleotide probes hybridized adjacently to a DNA template has been widely used for detection of genome alterations. The multiplex ligation-dependent probe amplification (MLPA) technique allows simultaneous screening of multiple target sequences in a single reaction by using p...

Continuously tunable nucleic acid hybridization probes.

Science.gov (United States)

Wu, Lucia R; Wang, Juexiao Sherry; Fang, John Z; Evans, Emily R; Pinto, Alessandro; Pekker, Irena; Boykin, Richard; Ngouenet, Celine; Webster, Philippa J; Beechem, Joseph; Zhang, David Yu

2015-12-01

In silico-designed nucleic acid probes and primers often do not achieve favorable specificity and sensitivity tradeoffs on the first try, and iterative empirical sequence-based optimization is needed, particularly in multiplexed assays. We present a novel, on-the-fly method of tuning probe affinity and selectivity by adjusting the stoichiometry of auxiliary species, which allows for independent and decoupled adjustment of the hybridization yield for different probes in multiplexed assays. Using this method, we achieved near-continuous tuning of probe effective free energy. To demonstrate our approach, we enforced uniform capture efficiency of 31 DNA molecules (GC content, 0-100%), maximized the signal difference for 11 pairs of single-nucleotide variants and performed tunable hybrid capture of mRNA from total RNA. Using the Nanostring nCounter platform, we applied stoichiometric tuning to simultaneously adjust yields for a 24-plex assay, and we show multiplexed quantitation of RNA sequences and variants from formalin-fixed, paraffin-embedded samples.

Inexpensive multiplexed library preparation for megabase-sized genomes.

Directory of Open Access Journals (Sweden)

Michael Baym

Full Text Available Whole-genome sequencing has become an indispensible tool of modern biology. However, the cost of sample preparation relative to the cost of sequencing remains high, especially for small genomes where the former is dominant. Here we present a protocol for rapid and inexpensive preparation of hundreds of multiplexed genomic libraries for Illumina sequencing. By carrying out the Nextera tagmentation reaction in small volumes, replacing costly reagents with cheaper equivalents, and omitting unnecessary steps, we achieve a cost of library preparation of $8 per sample, approximately 6 times cheaper than the standard Nextera XT protocol. Furthermore, our procedure takes less than 5 hours for 96 samples. Several hundred samples can then be pooled on the same HiSeq lane via custom barcodes. Our method will be useful for re-sequencing of microbial or viral genomes, including those from evolution experiments, genetic screens, and environmental samples, as well as for other sequencing applications including large amplicon, open chromosome, artificial chromosomes, and RNA sequencing.

Cognitive endophenotypes inform genome-wide expression profiling in schizophrenia.

Science.gov (United States)

Zheutlin, Amanda B; Viehman, Rachael W; Fortgang, Rebecca; Borg, Jacqueline; Smith, Desmond J; Suvisaari, Jaana; Therman, Sebastian; Hultman, Christina M; Cannon, Tyrone D

2016-01-01

We performed a whole-genome expression study to clarify the nature of the biological processes mediating between inherited genetic variations and cognitive dysfunction in schizophrenia. Gene expression was assayed from peripheral blood mononuclear cells using Illumina Human WG6 v3.0 chips in twins discordant for schizophrenia or bipolar disorder and control twins. After quality control, expression levels of 18,559 genes were screened for association with the California Verbal Learning Test (CVLT) performance, and any memory-related probes were then evaluated for variation by diagnostic status in the discovery sample (N = 190), and in an independent replication sample (N = 73). Heritability of gene expression using the twin design was also assessed. After Bonferroni correction (p schizophrenia patients, with comparable effect sizes in the same direction in the replication sample. For 41 of these 43 transcripts, expression levels were heritable. Nearly all identified genes contain common or de novo mutations associated with schizophrenia in prior studies. Genes increasing risk for schizophrenia appear to do so in part via effects on signaling cascades influencing memory. The genes implicated in these processes are enriched for those related to RNA processing and DNA replication and include genes influencing G-protein coupled signal transduction, cytokine signaling, and oligodendrocyte function. (c) 2015 APA, all rights reserved).

Analysis and Design of a Fiber-optic Probe for DNA Sensors Final Report CRADA No. TSB-1147-95

Energy Technology Data Exchange (ETDEWEB)

Molau, Nicole [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Vail, Curtis [Accu.Photonics, Inc., Ann Arbor, MI (United States)

2018-01-24

In 1995, a challenge in the field of genetics dealt with the acquisition of efficient DNA sequencing techniques for reading the 3 billion base-pairs that comprised the human genome. AccuPhotonics, Inc. proposed to develop and manufacture a state-of-the-art near-field scanning optical microscopy (NSOM) fiber-optic probe that was expected to increase probe efficiency by two orders of magnitude over the existing state-of-the-art and to improve resolution to 10Å. The detailed design calculation and optimization of electrical properties of the fiber-optic probe tip geometry would be performed at LLNL, using existing finite-difference time-domain (FDTD) electromagnetic (EM) codes.

Mobile Probing and Probes

DEFF Research Database (Denmark)

Duvaa, Uffe; Ørngreen, Rikke; Weinkouff Mathiasen, Anne-Gitte

2013-01-01

Mobile probing is a method, developed for learning about digital work situations, as an approach to discover new grounds. The method can be used when there is a need to know more about users and their work with certain tasks, but where users at the same time are distributed (in time and space......). Mobile probing was inspired by the cultural probe method, and was influenced by qualitative interview and inquiry approaches. The method has been used in two subsequent projects, involving school children (young adults at 15-17 years old) and employees (adults) in a consultancy company. Findings point...... to mobile probing being a flexible method for uncovering the unknowns, as a way of getting rich data to the analysis and design phases. On the other hand it is difficult to engage users to give in depth explanations, which seem easier in synchronous dialogs (whether online or face2face). The development...

Mobile Probing and Probes

DEFF Research Database (Denmark)

Duvaa, Uffe; Ørngreen, Rikke; Weinkouff, Anne-Gitte

2012-01-01

Mobile probing is a method, which has been developed for learning about digital work situations, as an approach to discover new grounds. The method can be used when there is a need to know more about users and their work with certain tasks, but where users at the same time are distributed (in time...... and space). Mobile probing was inspired by the cultural probe method, and was influenced by qualitative interview and inquiry approaches. The method has been used in two subsequent projects, involving school children (young adults at 15-17 years old) and employees (adults) in a consultancy company. Findings...... point to mobile probing being a flexible method for uncovering the unknowns, as a way of getting rich data to the analysis and design phases. On the other hand it is difficult to engage users to give in depth explanations, which seem easier in synchronous dialogs (whether online or face2face...

Isolation and characterization of DNA probes from a flow-sorted human chromosome 8 library that detect restriction fragment length polymorphism (RFLP).

Science.gov (United States)

Wood, S; Starr, T V; Shukin, R J

1986-01-01

We have used a recombinant DNA library constructed from flow-sorted human chromosome 8 as a source of single-copy human probes. These probes have been screened for restriction fragment length polymorphism (RFLP) by hybridization to Southern transfers of genomic DNA from five unrelated individuals. We have detected six RFLPs distributed among four probes after screening 741 base pairs for restriction site variation. These RFLPs all behave as codominant Mendelian alleles. Two of the probes detect rare variants, while the other two detect RFLPs with PIC values of .36 and .16. Informative probes will be useful for the construction of a linkage map for chromosome 8 and for the localization of mutant alleles to this chromosome. Images Fig. 1 PMID:2879441

Meta-analysis of genome-wide association from genomic prediction models

Science.gov (United States)

A limitation of many genome-wide association studies (GWA) in animal breeding is that there are many loci with small effect sizes; thus, larger sample sizes (N) are required to guarantee suitable power of detection. To increase sample size, results from different GWA can be combined in a meta-analys...

Magnetic Barkhausen Noise Measurements Using Tetrapole Probe Designs

Science.gov (United States)

McNairnay, Paul

A magnetic Barkhausen noise (MBN) testing system was developed for Defence Research and Development Canada (DRDC) to perform MBN measurements on the Royal Canadian Navy's Victoria class submarine hulls that can be correlated with material properties, including residual stress. The DRDC system was based on the design of a MBN system developed by Steven White at Queen's University, which was capable of performing rapid angular dependent measurements through the implementation of a flux controlled tetrapole probe. In tetrapole probe designs, the magnetic excitation field is rotated in the surface plane of the sample under the assumption of linear superposition of two orthogonal magnetic fields. During the course of this work, however, the validity of flux superposition in ferromagnetic materials, for the purpose of measuring MBN, was brought into question. Consequently, a study of MBN anisotropy using tetrapole probes was performed. Results indicate that MBN anisotropy measured under flux superposition does not simulate MBN anisotropy data obtained through manual rotation of a single dipole excitation field. It is inferred that MBN anisotropy data obtained with tetrapole probes is the result of the magnetic domain structure's response to an orthogonal magnetization condition and not necessarily to any bulk superposition magnetization in the sample. A qualitative model for the domain configuration under two orthogonal magnetic fields is proposed to describe the results. An empirically derived fitting equation, that describes tetrapole MBN anisotropy data, is presented. The equation describes results in terms of two largely independent orthogonal fields, and includes interaction terms arising due to competing orthogonally magnetized domain structures and interactions with the sample's magnetic easy axis. The equation is used to fit results obtained from a number of samples and tetrapole orientations and in each case correctly identifies the samples' magnetic easy axis.

New molecular markers and cytogenetic probes enable chromosome identification of wheat-Thinopyrum intermedium introgression lines for improving protein and gluten contents.

Science.gov (United States)

Li, Guangrong; Wang, Hongjin; Lang, Tao; Li, Jianbo; La, Shixiao; Yang, Ennian; Yang, Zujun

2016-10-01

New molecular markers were developed for targeting Thinopyrum intermedium 1St#2 chromosome, and novel FISH probe representing the terminal repeats was produced for identification of Thinopyrum chromosomes. Thinopyrum intermedium has been used as a valuable resource for improving the disease resistance and yield potential of wheat. A wheat-Th. intermedium ssp. trichophorum chromosome 1St#2 substitution and translocation has displayed superior grain protein and wet gluten content. With the aim to develop a number of chromosome 1St#2 specific molecular and cytogenetic markers, a high throughput, low-cost specific-locus amplified fragment sequencing (SLAF-seq) technology was used to compare the sequences between a wheat-Thinopyrum 1St#2 (1D) substitution and the related species Pseudoroegneria spicata (St genome, 2n = 14). A total of 5142 polymorphic fragments were analyzed and 359 different SLAF markers for 1St#2 were predicted. Thirty-seven specific molecular markers were validated by PCR from 50 randomly selected SLAFs. Meanwhile, the distribution of transposable elements (TEs) at the family level between wheat and St genomes was compared using the SLAFs. A new oligo-nucleotide probe named Oligo-pSt122 from high SLAF reads was produced for fluorescence in situ hybridization (FISH), and was observed to hybridize to the terminal region of 1St#L and also onto the terminal heterochromatic region of Th. intermedium genomes. The genome-wide markers and repetitive based probe Oligo-pSt122 will be valuable for identifying Thinopyrum chromosome segments in wheat backgrounds.

Successful application of FTA Classic Card technology and use of bacteriophage phi29 DNA polymerase for large-scale field sampling and cloning of complete maize streak virus genomes.

Science.gov (United States)

Owor, Betty E; Shepherd, Dionne N; Taylor, Nigel J; Edema, Richard; Monjane, Adérito L; Thomson, Jennifer A; Martin, Darren P; Varsani, Arvind

2007-03-01

Leaf samples from 155 maize streak virus (MSV)-infected maize plants were collected from 155 farmers' fields in 23 districts in Uganda in May/June 2005 by leaf-pressing infected samples onto FTA Classic Cards. Viral DNA was successfully extracted from cards stored at room temperature for 9 months. The diversity of 127 MSV isolates was analysed by PCR-generated RFLPs. Six representative isolates having different RFLP patterns and causing either severe, moderate or mild disease symptoms, were chosen for amplification from FTA cards by bacteriophage phi29 DNA polymerase using the TempliPhi system. Full-length genomes were inserted into a cloning vector using a unique restriction enzyme site, and sequenced. The 1.3-kb PCR product amplified directly from FTA-eluted DNA and used for RFLP analysis was also cloned and sequenced. Comparison of cloned whole genome sequences with those of the original PCR products indicated that the correct virus genome had been cloned and that no errors were introduced by the phi29 polymerase. This is the first successful large-scale application of FTA card technology to the field, and illustrates the ease with which large numbers of infected samples can be collected and stored for downstream molecular applications such as diversity analysis and cloning of potentially new virus genomes.

Theoretical analysis of a dual-probe scanning tunneling microscope setup on graphene.

Science.gov (United States)

Settnes, Mikkel; Power, Stephen R; Petersen, Dirch H; Jauho, Antti-Pekka

2014-03-07

Experimental advances allow for the inclusion of multiple probes to measure the transport properties of a sample surface. We develop a theory of dual-probe scanning tunneling microscopy using a Green's function formalism, and apply it to graphene. Sampling the local conduction properties at finite length scales yields real space conductance maps which show anisotropy for pristine graphene systems and quantum interference effects in the presence of isolated impurities. Spectral signatures in the Fourier transforms of real space conductance maps include characteristics that can be related to different scattering processes. We compute the conductance maps of graphene systems with different edge geometries or height fluctuations to determine the effects of nonideal graphene samples on dual-probe measurements.

Draft Genome Sequence of Hymenobacter sp. Strain AT01-02, Isolated from a Surface Soil Sample in the Atacama Desert, Chile

DEFF Research Database (Denmark)

Hansen, Anders Cai Holm; Paulino-Lima, Ivan Glaucio; Fujishima, Kosuke

2016-01-01

Here, we report the 5.09-Mb draft genome sequence of Hymenobacter sp. strain AT01-02, which was isolated from a surface soil sample in the Atacama Desert, Chile. The isolate is extremely resistant to UV-C radiation and is able to accumulate high intracellular levels of Mn/Fe....

Genome-wide identification of specific oligonucleotides using artificial neural network and computational genomic analysis

Directory of Open Access Journals (Sweden)

Chen Jiun-Ching

2007-05-01

Full Text Available Abstract Background Genome-wide identification of specific oligonucleotides (oligos is a computationally-intensive task and is a requirement for designing microarray probes, primers, and siRNAs. An artificial neural network (ANN is a machine learning technique that can effectively process complex and high noise data. Here, ANNs are applied to process the unique subsequence distribution for prediction of specific oligos. Results We present a novel and efficient algorithm, named the integration of ANN and BLAST (IAB algorithm, to identify specific oligos. We establish the unique marker database for human and rat gene index databases using the hash table algorithm. We then create the input vectors, via the unique marker database, to train and test the ANN. The trained ANN predicted the specific oligos with high efficiency, and these oligos were subsequently verified by BLAST. To improve the prediction performance, the ANN over-fitting issue was avoided by early stopping with the best observed error and a k-fold validation was also applied. The performance of the IAB algorithm was about 5.2, 7.1, and 6.7 times faster than the BLAST search without ANN for experimental results of 70-mer, 50-mer, and 25-mer specific oligos, respectively. In addition, the results of polymerase chain reactions showed that the primers predicted by the IAB algorithm could specifically amplify the corresponding genes. The IAB algorithm has been integrated into a previously published comprehensive web server to support microarray analysis and genome-wide iterative enrichment analysis, through which users can identify a group of desired genes and then discover the specific oligos of these genes. Conclusion The IAB algorithm has been developed to construct SpecificDB, a web server that provides a specific and valid oligo database of the probe, siRNA, and primer design for the human genome. We also demonstrate the ability of the IAB algorithm to predict specific oligos through

A Novel Pretreatment-Free Duplex Chamber Digital PCR Detection System for the Absolute Quantitation of GMO Samples.

Science.gov (United States)

Zhu, Pengyu; Wang, Chenguang; Huang, Kunlun; Luo, Yunbo; Xu, Wentao

2016-03-18

Digital polymerase chain reaction (PCR) has developed rapidly since it was first reported in the 1990s. However, pretreatments are often required during preparation for digital PCR, which can increase operation error. The single-plex amplification of both the target and reference genes may cause uncertainties due to the different reaction volumes and the matrix effect. In the current study, a quantitative detection system based on the pretreatment-free duplex chamber digital PCR was developed. The dynamic range, limit of quantitation (LOQ), sensitivity and specificity were evaluated taking the GA21 event as the experimental object. Moreover, to determine the factors that may influence the stability of the duplex system, we evaluated whether the pretreatments, the primary and secondary structures of the probes and the SNP effect influence the detection. The results showed that the LOQ was 0.5% and the sensitivity was 0.1%. We also found that genome digestion and single nucleotide polymorphism (SNP) sites affect the detection results, whereas the unspecific hybridization within different probes had little side effect. This indicated that the detection system was suited for both chamber-based and droplet-based digital PCR. In conclusion, we have provided a simple and flexible way of achieving absolute quantitation for genetically modified organism (GMO) genome samples using commercial digital PCR detection systems.

A Novel Pretreatment-Free Duplex Chamber Digital PCR Detection System for the Absolute Quantitation of GMO Samples

Directory of Open Access Journals (Sweden)

Pengyu Zhu

2016-03-01

Full Text Available Digital polymerase chain reaction (PCR has developed rapidly since it was first reported in the 1990s. However, pretreatments are often required during preparation for digital PCR, which can increase operation error. The single-plex amplification of both the target and reference genes may cause uncertainties due to the different reaction volumes and the matrix effect. In the current study, a quantitative detection system based on the pretreatment-free duplex chamber digital PCR was developed. The dynamic range, limit of quantitation (LOQ, sensitivity and specificity were evaluated taking the GA21 event as the experimental object. Moreover, to determine the factors that may influence the stability of the duplex system, we evaluated whether the pretreatments, the primary and secondary structures of the probes and the SNP effect influence the detection. The results showed that the LOQ was 0.5% and the sensitivity was 0.1%. We also found that genome digestion and single nucleotide polymorphism (SNP sites affect the detection results, whereas the unspecific hybridization within different probes had little side effect. This indicated that the detection system was suited for both chamber-based and droplet-based digital PCR. In conclusion, we have provided a simple and flexible way of achieving absolute quantitation for genetically modified organism (GMO genome samples using commercial digital PCR detection systems.

Results and Conclusions from the NASA Isokinetic Total Water Content Probe 2009 IRT Test

Science.gov (United States)

Reehorst, Andrew; Brinker, David

2010-01-01

The NASA Glenn Research Center has developed and tested a Total Water Content Isokinetic Sampling Probe. Since, by its nature, it is not sensitive to cloud water particle phase nor size, it is particularly attractive to support super-cooled large droplet and high ice water content aircraft icing studies. The instrument comprises the Sampling Probe, Sample Flow Control, and Water Vapor Measurement subsystems. Results and conclusions are presented from probe tests in the NASA Glenn Icing Research Tunnel (IRT) during January and February 2009. The use of reference probe heat and the control of air pressure in the water vapor measurement subsystem are discussed. Several run-time error sources were found to produce identifiable signatures that are presented and discussed. Some of the differences between measured Isokinetic Total Water Content Probe and IRT calibration seems to be caused by tunnel humidification and moisture/ice crystal blow around. Droplet size, airspeed, and liquid water content effects also appear to be present in the IRT calibration. Based upon test results, the authors provide recommendations for future Isokinetic Total Water Content Probe development.

Identification of genome-specific transcripts in wheat–rye translocation lines

Directory of Open Access Journals (Sweden)

Tong Geon Lee

2015-09-01

Full Text Available Studying gene expression in wheat–rye translocation lines is complicated due to the presence of homeologs in hexaploid wheat and high levels of synteny between wheat and rye genomes (Naranjo and Fernandez-Rueda, 1991 [1]; Devos et al., 1995 [2]; Lee et al., 2010 [3]; Lee et al., 2013 [4]. To overcome limitations of current gene expression studies on wheat–rye translocation lines and identify genome-specific transcripts, we developed a custom Roche NimbleGen Gene Expression microarray that contains probes derived from the sequence of hexaploid wheat, diploid rye and diploid progenitors of hexaploid wheat genome (Lee et al., 2014. Using the array developed, we identified genome-specific transcripts in a wheat–rye translocation line (Lee et al., 2014. Expression data are deposited in the NCBI Gene Expression Omnibus (GEO under accession number GSE58678. Here we report the details of the methods used in the array workflow and data analysis.

Characterization of the main error sources of chromatic confocal probes for dimensional measurement

International Nuclear Information System (INIS)

Nouira, H; El-Hayek, N; Yuan, X; Anwer, N

2014-01-01

Chromatic confocal probes are increasingly used in high-precision dimensional metrology applications such as roughness, form, thickness and surface profile measurements; however, their measurement behaviour is not well understood and must be characterized at a nanometre level. This paper provides a calibration bench for the characterization of two chromatic confocal probes of 20 and 350 µm travel ranges. The metrology loop that includes the chromatic confocal probe is stable and enables measurement repeatability at the nanometer level. With the proposed system, the major error sources, such as the relative axial and radial motions of the probe with respect to the sample, the material, colour and roughness of the measured sample, the relative deviation/tilt of the probe and the scanning speed are identified. Experimental test results show that the chromatic confocal probes are sensitive to these errors and that their measurement behaviour is highly dependent on them. (paper)

Genome Modeling System: A Knowledge Management Platform for Genomics.

Directory of Open Access Journals (Sweden)

Malachi Griffith

2015-07-01

Full Text Available In this work, we present the Genome Modeling System (GMS, an analysis information management system capable of executing automated genome analysis pipelines at a massive scale. The GMS framework provides detailed tracking of samples and data coupled with reliable and repeatable analysis pipelines. The GMS also serves as a platform for bioinformatics development, allowing a large team to collaborate on data analysis, or an individual researcher to leverage the work of others effectively within its data management system. Rather than separating ad-hoc analysis from rigorous, reproducible pipelines, the GMS promotes systematic integration between the two. As a demonstration of the GMS, we performed an integrated analysis of whole genome, exome and transcriptome sequencing data from a breast cancer cell line (HCC1395 and matched lymphoblastoid line (HCC1395BL. These data are available for users to test the software, complete tutorials and develop novel GMS pipeline configurations. The GMS is available at https://github.com/genome/gms.

Impact of Sample Type and DNA Isolation Procedure on Genomic Inference of Microbiome Composition

DEFF Research Database (Denmark)

Knudsen, Berith Elkær; Bergmark, Lasse; Munk, Patrick

2016-01-01

that in standard protocols. Based on this insight, we designed an improved DNA isolation procedure optimized for microbiome genomics that can be used for the three examined specimen types and potentially also for other biological specimens. A standard operating procedure is available from https://dx.doi.org/10......Explorations of complex microbiomes using genomics greatly enhance our understanding about their diversity, biogeography, and function. The isolation of DNA from microbiome specimens is a key prerequisite for such examinations, but challenges remain in obtaining sufficient DNA quantities required...... for certain sequencing approaches, achieving accurate genomic inference of microbiome composition, and facilitating comparability of findings across specimen types and sequencing projects. These aspects are particularly relevant for the genomics-based global surveillance of infectious agents and antimicrobial...

Short Communication Evaluation of an underwater biopsy probe for ...

African Journals Online (AJOL)

This study evaluated the use of a novel underwater biopsy probe designed to collect muscle and dermal tissue samples from large (170–220 cm total length), free-swimming bull sharks Carcharhinus leucas. The biopsy probe tissue retention rate was 87% after 23 trials, and the mean size of retained tissue was 310 mg (SD ...

Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

Energy Technology Data Exchange (ETDEWEB)

Greulich-Bode, Karin; Wang, Mei; Rhein, Andreas; Weier, Jingly; Weier, Heinz-Ulli

2008-12-16

Fluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100kb, careful probe selection and characterization are of paramount importance. We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific {approx}6kb plasmid onto an unusually small, {approx}55kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-?B2 locus. The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.

Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

Energy Technology Data Exchange (ETDEWEB)

Greulich-Bode, Karin M.; Wang, Mei; Rhein, Andreas P.; Weier, Jingly F.; Weier, Heinz-Ulli G.

2008-12-04

Fluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100kb, careful probe selection and characterization are of paramount importance. We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific {approx}6kb plasmid onto an unusually small, {approx}55kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-{kappa}B2 locus. The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.

Radioactive Probes on Ferromagnetic Surfaces

CERN Multimedia

2002-01-01

On the (broad) basis of our studies of nonmagnetic radioactive probe atoms on magnetic surfaces and at interfaces, we propose to investigate the magnetic interaction of magnetic probe atoms with their immediate environment, in particular of rare earth (RE) elements positioned on and in ferromagnetic surfaces. The preparation and analysis of the structural properties of such samples will be performed in the UHV chamber HYDRA at the HMI/Berlin. For the investigations of the magnetic properties of RE atoms on surfaces Perturbed Angular Correlation (PAC) measurements and Mössbauer Spectroscopy (MS) in the UHV chamber ASPIC (Apparatus for Surface Physics and Interfaces at CERN) are proposed.

Flux-focusing eddy current probe and rotating probe method for flaw detection

Science.gov (United States)

Wincheski, Buzz A.; Fulton, James P.; Nath, Shridhar C.; Simpson, John W.; Namkung, Min

1994-11-01

A flux-focusing electromagnetic sensor which uses a ferromagnetic flux-focusing lens simplifies inspections and increases detectability of fatigue cracks about circular fasteners and other circular inhomogeneities in high conductivity material. The unique feature of the device is the ferrous shield isolating a high-turn pick-up coil from an excitation coil. The use of the magnetic shield is shown to produce a null voltage output across the receiving coil in the presence of an unflawed sample. A redistribution of the current flow in the sample caused by the presence of flaws, however, eliminates the shielding condition and a large output voltage is produced, yielding a clear unambiguous flaw signal. By rotating the probe in a path around a circular fastener such as a rivet while maintaining a constant distance between the probe and the center of a rivet, the signal due to current flow about the rivet can be held constant. Any further changes in the current distribution, such as due to a fatigue crack at the rivet joint, can be detected as an increase in the output voltage above that due to the flow about the rivet head.

Targeted sequencing of large genomic regions with CATCH-Seq.

Directory of Open Access Journals (Sweden)

Kenneth Day

Full Text Available Current target enrichment systems for large-scale next-generation sequencing typically require synthetic oligonucleotides used as capture reagents to isolate sequences of interest. The majority of target enrichment reagents are focused on gene coding regions or promoters en masse. Here we introduce development of a customizable targeted capture system using biotinylated RNA probe baits transcribed from sheared bacterial artificial chromosome clone templates that enables capture of large, contiguous blocks of the genome for sequencing applications. This clone adapted template capture hybridization sequencing (CATCH-Seq procedure can be used to capture both coding and non-coding regions of a gene, and resolve the boundaries of copy number variations within a genomic target site. Furthermore, libraries constructed with methylated adapters prior to solution hybridization also enable targeted bisulfite sequencing. We applied CATCH-Seq to diverse targets ranging in size from 125 kb to 3.5 Mb. Our approach provides a simple and cost effective alternative to other capture platforms because of template-based, enzymatic probe synthesis and the lack of oligonucleotide design costs. Given its similarity in procedure, CATCH-Seq can also be performed in parallel with commercial systems.

Fabrication of tungsten probe for hard tapping operation in atomic force microscopy

Energy Technology Data Exchange (ETDEWEB)

Han, Guebum, E-mail: hanguebum@live.co.kr [Department of Physics and Optical Engineering, Rose-Hulman Institute of Technology, 5500 Wabash Avenue, Terre Haute, Indiana 47803 (United States); Department of Mechanical Design and Robot Engineering, Seoul National University of Science and Technology, 232 Gongneung-ro, Nowon-gu, Seoul 139-743 (Korea, Republic of); Ahn, Hyo-Sok, E-mail: hsahn@seoultech.ac.kr [Manufacturing Systems and Design Engineering Programme, Seoul National University of Science & Technology, 232 Gongneung-ro, Nowon-gu, Seoul 139-743 (Korea, Republic of)

2016-02-15

We propose a method of producing a tungsten probe with high stiffness for atomic force microscopy (AFM) in order to acquire enhanced phase contrast images and efficiently perform lithography. A tungsten probe with a tip radius between 20 nm and 50 nm was fabricated using electrochemical etching optimized by applying pulse waves at different voltages. The spring constant of the tungsten probe was determined by finite element analysis (FEA), and its applicability as an AFM probe was evaluated by obtaining topography and phase contrast images of a Si wafer sample partly coated with Au. Enhanced hard tapping performance of the tungsten probe compared with a commercial Si probe was confirmed by conducting hard tapping tests at five different oscillation amplitudes on single layer graphene grown by chemical vapor deposition (CVD). To analyze the damaged graphene sample, the test areas were investigated using tip-enhanced Raman spectroscopy (TERS). The test results demonstrate that the tungsten probe with high stiffness was capable of inducing sufficient elastic and plastic deformation to enable obtaining enhanced phase contrast images and performing lithography, respectively. - Highlights: • We propose a method of producing highly stiff tungsten probes for hard tapping AFM. • Spring constant of tungsten probe is determined by finite element method. • Enhanced hard tapping performance is confirmed. • Tip-enhanced Raman spectroscopy is used to identify damage to graphene.

Fabrication of tungsten probe for hard tapping operation in atomic force microscopy

International Nuclear Information System (INIS)

Han, Guebum; Ahn, Hyo-Sok

2016-01-01

We propose a method of producing a tungsten probe with high stiffness for atomic force microscopy (AFM) in order to acquire enhanced phase contrast images and efficiently perform lithography. A tungsten probe with a tip radius between 20 nm and 50 nm was fabricated using electrochemical etching optimized by applying pulse waves at different voltages. The spring constant of the tungsten probe was determined by finite element analysis (FEA), and its applicability as an AFM probe was evaluated by obtaining topography and phase contrast images of a Si wafer sample partly coated with Au. Enhanced hard tapping performance of the tungsten probe compared with a commercial Si probe was confirmed by conducting hard tapping tests at five different oscillation amplitudes on single layer graphene grown by chemical vapor deposition (CVD). To analyze the damaged graphene sample, the test areas were investigated using tip-enhanced Raman spectroscopy (TERS). The test results demonstrate that the tungsten probe with high stiffness was capable of inducing sufficient elastic and plastic deformation to enable obtaining enhanced phase contrast images and performing lithography, respectively. - Highlights: • We propose a method of producing highly stiff tungsten probes for hard tapping AFM. • Spring constant of tungsten probe is determined by finite element method. • Enhanced hard tapping performance is confirmed. • Tip-enhanced Raman spectroscopy is used to identify damage to graphene.

Band excitation method applicable to scanning probe microscopy

Science.gov (United States)

Jesse, Stephen [Knoxville, TN; Kalinin, Sergei V [Knoxville, TN

2010-08-17

Methods and apparatus are described for scanning probe microscopy. A method includes generating a band excitation (BE) signal having finite and predefined amplitude and phase spectrum in at least a first predefined frequency band; exciting a probe using the band excitation signal; obtaining data by measuring a response of the probe in at least a second predefined frequency band; and extracting at least one relevant dynamic parameter of the response of the probe in a predefined range including analyzing the obtained data. The BE signal can be synthesized prior to imaging (static band excitation), or adjusted at each pixel or spectroscopy step to accommodate changes in sample properties (adaptive band excitation). An apparatus includes a band excitation signal generator; a probe coupled to the band excitation signal generator; a detector coupled to the probe; and a relevant dynamic parameter extractor component coupled to the detector, the relevant dynamic parameter extractor including a processor that performs a mathematical transform selected from the group consisting of an integral transform and a discrete transform.

A highly selective and sensitive Tb3+-acetylacetone photo probe for the assessment of acetazolamide in pharmaceutical and serum samples

Science.gov (United States)

Youssef, A. O.

2018-04-01

A novel, simple, sensitive and selective spectrofluorimetric method was developed for the determination of Acetazolamide in pharmaceutical tablets and serum samples using photo probe Tb3+-ACAC. The Acetazolamide can remarkably quench the luminescence intensity of Tb3+-ACAC complex in DMSO at pH 6.8 and λex = 350 nm. The quenching of luminescence intensity of Tb3+-ACAC complex especially the electrical band at λem = 545 nm is used for the assessment of Acetazolamide in the pharmaceutical tablet and serum samples. The dynamic range found for the determination of Acetazolamide concentration is 4.49 × 10-9-1.28 × 10-7 mol L-1, and the limit of detection (LOD) and limit of quantification (LOQ) are (4.0 × 10-9 and 1.21 × 10-8) mol L-1, respectively.

Systems and methods for laser assisted sample transfer to solution for chemical analysis

Science.gov (United States)

Van Berkel, Gary J; Kertesz, Vilmos; Ovchinnikova, Olga S

2013-08-27

Systems and methods are described for laser ablation of an analyte from a specimen and capturing of the analyte in a dispensed solvent to form a testing solution. A solvent dispensing and extraction system can form a liquid microjunction with the specimen. The solvent dispensing and extraction system can include a surface sampling probe. The laser beam can be directed through the surface sampling probe. The surface sampling probe can also serve as an atomic force microscopy probe. The surface sampling probe can form a seal with the specimen. The testing solution including the analyte can then be analyzed using an analytical instrument or undergo further processing.

Molecular basis for the genome engagement by Sox proteins.

Science.gov (United States)

Hou, Linlin; Srivastava, Yogesh; Jauch, Ralf

2017-03-01

The Sox transcription factor family consists of 20 members in the human genome. Many of them are key determinants of cellular identities and possess the capacity to reprogram cell fates by pioneering the epigenetic remodeling of the genome. This activity is intimately tied to their ability to specifically bind and bend DNA alone or with other proteins. Here we discuss our current knowledge on how Sox transcription factors such as Sox2, Sox17, Sox18 and Sox9 'read' the genome to find and regulate their target genes and highlight the roles of partner factors including Pax6, Nanog, Oct4 and Brn2. We integrate insights from structural and biochemical studies as well as high-throughput assays to probe DNA specificity in vitro as well as in cells and tissues. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

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

Science.gov (United States)

Leister, Dario; Varotto, Claudio

2007-01-01

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

Probe for the mutagenic activity of the carcinogen 4-aminobiphenyl: synthesis and characterization of an M13mp10 genome containing the major carcinogen-DNA adduct at a unique site

International Nuclear Information System (INIS)

Lasko, D.D.; Basu, A.K.; Kadlubar, F.F.; Evans, F.E.; Lay, J.O. Jr.; Essigmann, J.M.

1987-01-01

The duplex genome of Escherichia coli virus M13mp10 was modified at a unique site to contain N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG/sup 8-ABP/), the major carcinogen-DNA adduct of the human bladder carcinogen 4-aminobiphenyl. A tetradeoxynucleotide containing a single dG/sup 8-ABP/ residue was synthesized by reacting 5'-d(TpGpCpA)-3' with N-acetoxy-N-(trifluoroacetyl)-4-aminobiphenyl, followed by high-performance liquid chromatography purification of the principal reaction product 5'-d(TpG/sup 8-ABP/pCpA)-3' (yield 15-30%). Characterization by fast atom bombardment mass spectrometry confirmed the structure as an intact 4-aminobiphenyl-modified tetranucleotide, while 1 H nuclear magnetic resonance spectroscopy established the site of substitution and the existence of ring stacking between the carcinogen residue and DNA bases. Experiments in which the tetranucleotides were 5' end labeled with [ 32 P]phosphate revealed the following: 1)the adducted oligomer, when incubated in a 1000-fold molar excess in the presence of T4 DNA ligase and ATP, was found to be incorporated into the gapped DNA molecules with an efficiency of approximately 30%, as compared to the unadducted d(pTpGpCpA), which was incorporated with 60% ligation efficiency; 2)radioactivity from the 5' end of each tetranucleotide was physically mapped to a restriction fragment that contained the PstI site and represented 0.2% of the genome; 3) the presence of the lesion within the PstI recognition site inhibited the ability of PstI to cleave the genome at this site; 4)in genomes in which ligation occurred, T4 DNA ligase was capable of covalently joining both modified and unmodified tetranucleotides to the gapped structures on both the 5' and the 3' ends with at least 90% efficiency. On the basis of these and other data, the dG/sup 8-ABP/-modified genome was judged to be a useful probe for investigation of site-specific mutagenesis in E. coli

3D site specific sample preparation and analysis of 3D devices (FinFETs) by atom probe tomography.

Science.gov (United States)

Kambham, Ajay Kumar; Kumar, Arul; Gilbert, Matthieu; Vandervorst, Wilfried

2013-09-01

With the transition from planar to three-dimensional device architectures such as Fin field-effect-transistors (FinFETs), new metrology approaches are required to meet the needs of semiconductor technology. It is important to characterize the 3D-dopant distributions precisely as their extent, positioning relative to gate edges and absolute concentration determine the device performance in great detail. At present the atom probe has shown its ability to analyze dopant distributions in semiconductor and thin insulating materials with sub-nm 3D-resolution and good dopant sensitivity. However, so far most reports have dealt with planar devices or restricted the measurements to 2D test structures which represent only limited challenges in terms of localization and site specific sample preparation. In this paper we will discuss the methodology to extract the dopant distribution from real 3D-devices such as a 3D-FinFET device, requiring the sample preparation to be carried out at a site specific location with a positioning accuracy ∼50 nm. Copyright © 2013 Elsevier B.V. All rights reserved.

A General Protocol for Temperature Calibration of MAS NMR Probes at Arbitrary Spinning Speeds

Science.gov (United States)

Guan, Xudong; Stark, Ruth E.

2010-01-01

A protocol using 207Pb NMR of solid lead nitrate was developed to determine the temperature of magic-angle spinning (MAS) NMR probes over a range of nominal set temperatures and spinning speeds. Using BioMAS and fastMAS probes with typical sample spinning rates of 8 and 35 kHz, respectively, empirical equations were devised to predict the respective sample temperatures. These procedures provide a straightforward recipe for temperature calibration of any MAS probe. PMID:21036557

Whole-genome DNA methylation status associated with clinical PTSD measures of OIF/OEF veterans

Science.gov (United States)

Hammamieh, R; Chakraborty, N; Gautam, A; Muhie, S; Yang, R; Donohue, D; Kumar, R; Daigle, B J; Zhang, Y; Amara, D A; Miller, S-A; Srinivasan, S; Flory, J; Yehuda, R; Petzold, L; Wolkowitz, O M; Mellon, S H; Hood, L; Doyle, F J; Marmar, C; Jett, M

2017-01-01

Emerging knowledge suggests that post-traumatic stress disorder (PTSD) pathophysiology is linked to the patients’ epigenetic changes, but comprehensive studies examining genome-wide methylation have not been performed. In this study, we examined genome-wide DNA methylation in peripheral whole blood in combat veterans with and without PTSD to ascertain differentially methylated probes. Discovery was initially made in a training sample comprising 48 male Operation Enduring Freedom (OEF)/Operation Iraqi Freedom (OIF) veterans with PTSD and 51 age/ethnicity/gender-matched combat-exposed PTSD-negative controls. Agilent whole-genome array detected ~5600 differentially methylated CpG islands (CpGI) annotated to ~2800 differently methylated genes (DMGs). The majority (84.5%) of these CpGIs were hypermethylated in the PTSD cases. Functional analysis was performed using the DMGs encoding the promoter-bound CpGIs to identify networks related to PTSD. The identified networks were further validated by an independent test set comprising 31 PTSD+/29 PTSD− veterans. Targeted bisulfite sequencing was also used to confirm the methylation status of 20 DMGs shown to be highly perturbed in the training set. To improve the statistical power and mitigate the assay bias and batch effects, a union set combining both training and test set was assayed using a different platform from Illumina. The pathways curated from this analysis confirmed 65% of the pool of pathways mined from training and test sets. The results highlight the importance of assay methodology and use of independent samples for discovery and validation of differentially methylated genes mined from whole blood. Nonetheless, the current study demonstrates that several important epigenetically altered networks may distinguish combat-exposed veterans with and without PTSD. PMID:28696412

Biosensor and chemical sensor probes for calcium and other metal ions

Science.gov (United States)

Vo-Dinh, Tuan; Viallet, Pierre

1996-01-01

The present invention relates to chemical sensor and biosensor probes for measuring low concentration of metals and metal ions in complex samples such as biological fluids, living cells, and environmental samples. More particularly the present invention relates to a gel-based Indo-1 and Fura-2 chemical sensor probes for the measurement of low concentrations of calcium, cadmium, magnesium and the like. Also disclosed is a detector device using the sensors of the present invention.

A DNA minor groove electronegative potential genome map based on photo-chemical probing

DEFF Research Database (Denmark)

Lindemose, Søren; Nielsen, Peter Eigil; Hansen, Morten

2011-01-01

The double-stranded DNA of the genome contains both sequence information directly relating to the protein and RNA coding as well as functional and structural information relating to protein recognition. Only recently is the importance of DNA shape in this recognition process being fully appreciat...

Comparing fixed sampling with minimizer sampling when using k-mer indexes to find maximal exact matches.

Science.gov (United States)

Almutairy, Meznah; Torng, Eric

2018-01-01

Bioinformatics applications and pipelines increasingly use k-mer indexes to search for similar sequences. The major problem with k-mer indexes is that they require lots of memory. Sampling is often used to reduce index size and query time. Most applications use one of two major types of sampling: fixed sampling and minimizer sampling. It is well known that fixed sampling will produce a smaller index, typically by roughly a factor of two, whereas it is generally assumed that minimizer sampling will produce faster query times since query k-mers can also be sampled. However, no direct comparison of fixed and minimizer sampling has been performed to verify these assumptions. We systematically compare fixed and minimizer sampling using the human genome as our database. We use the resulting k-mer indexes for fixed sampling and minimizer sampling to find all maximal exact matches between our database, the human genome, and three separate query sets, the mouse genome, the chimp genome, and an NGS data set. We reach the following conclusions. First, using larger k-mers reduces query time for both fixed sampling and minimizer sampling at a cost of requiring more space. If we use the same k-mer size for both methods, fixed sampling requires typically half as much space whereas minimizer sampling processes queries only slightly faster. If we are allowed to use any k-mer size for each method, then we can choose a k-mer size such that fixed sampling both uses less space and processes queries faster than minimizer sampling. The reason is that although minimizer sampling is able to sample query k-mers, the number of shared k-mer occurrences that must be processed is much larger for minimizer sampling than fixed sampling. In conclusion, we argue that for any application where each shared k-mer occurrence must be processed, fixed sampling is the right sampling method.

Comparing fixed sampling with minimizer sampling when using k-mer indexes to find maximal exact matches.

Directory of Open Access Journals (Sweden)

Meznah Almutairy

Full Text Available Bioinformatics applications and pipelines increasingly use k-mer indexes to search for similar sequences. The major problem with k-mer indexes is that they require lots of memory. Sampling is often used to reduce index size and query time. Most applications use one of two major types of sampling: fixed sampling and minimizer sampling. It is well known that fixed sampling will produce a smaller index, typically by roughly a factor of two, whereas it is generally assumed that minimizer sampling will produce faster query times since query k-mers can also be sampled. However, no direct comparison of fixed and minimizer sampling has been performed to verify these assumptions. We systematically compare fixed and minimizer sampling using the human genome as our database. We use the resulting k-mer indexes for fixed sampling and minimizer sampling to find all maximal exact matches between our database, the human genome, and three separate query sets, the mouse genome, the chimp genome, and an NGS data set. We reach the following conclusions. First, using larger k-mers reduces query time for both fixed sampling and minimizer sampling at a cost of requiring more space. If we use the same k-mer size for both methods, fixed sampling requires typically half as much space whereas minimizer sampling processes queries only slightly faster. If we are allowed to use any k-mer size for each method, then we can choose a k-mer size such that fixed sampling both uses less space and processes queries faster than minimizer sampling. The reason is that although minimizer sampling is able to sample query k-mers, the number of shared k-mer occurrences that must be processed is much larger for minimizer sampling than fixed sampling. In conclusion, we argue that for any application where each shared k-mer occurrence must be processed, fixed sampling is the right sampling method.

Comparing fixed sampling with minimizer sampling when using k-mer indexes to find maximal exact matches

Science.gov (United States)

Torng, Eric

2018-01-01

Bioinformatics applications and pipelines increasingly use k-mer indexes to search for similar sequences. The major problem with k-mer indexes is that they require lots of memory. Sampling is often used to reduce index size and query time. Most applications use one of two major types of sampling: fixed sampling and minimizer sampling. It is well known that fixed sampling will produce a smaller index, typically by roughly a factor of two, whereas it is generally assumed that minimizer sampling will produce faster query times since query k-mers can also be sampled. However, no direct comparison of fixed and minimizer sampling has been performed to verify these assumptions. We systematically compare fixed and minimizer sampling using the human genome as our database. We use the resulting k-mer indexes for fixed sampling and minimizer sampling to find all maximal exact matches between our database, the human genome, and three separate query sets, the mouse genome, the chimp genome, and an NGS data set. We reach the following conclusions. First, using larger k-mers reduces query time for both fixed sampling and minimizer sampling at a cost of requiring more space. If we use the same k-mer size for both methods, fixed sampling requires typically half as much space whereas minimizer sampling processes queries only slightly faster. If we are allowed to use any k-mer size for each method, then we can choose a k-mer size such that fixed sampling both uses less space and processes queries faster than minimizer sampling. The reason is that although minimizer sampling is able to sample query k-mers, the number of shared k-mer occurrences that must be processed is much larger for minimizer sampling than fixed sampling. In conclusion, we argue that for any application where each shared k-mer occurrence must be processed, fixed sampling is the right sampling method. PMID:29389989

Genome-wide and gene-based association studies of anxiety disorders in European and African American samples.

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Takeshi Otowa

Full Text Available Anxiety disorders (ADs are common mental disorders caused by a combination of genetic and environmental factors. Since ADs are highly comorbid with each other, partially due to shared genetic basis, studying AD phenotypes in a coordinated manner may be a powerful strategy for identifying potential genetic loci for ADs. To detect these loci, we performed genome-wide association studies (GWAS of ADs. In addition, as a complementary approach to single-locus analysis, we also conducted gene- and pathway-based analyses. GWAS data were derived from the control sample of the Molecular Genetics of Schizophrenia (MGS project (2,540 European American and 849 African American subjects genotyped on the Affymetrix GeneChip 6.0 array. We applied two phenotypic approaches: (1 categorical case-control comparisons (CC based upon psychiatric diagnoses, and (2 quantitative phenotypic factor scores (FS derived from a multivariate analysis combining information across the clinical phenotypes. Linear and logistic models were used to analyse the association with ADs using FS and CC traits, respectively. At the single locus level, no genome-wide significant association was found. A trans-population gene-based meta-analysis across both ethnic subsamples using FS identified three genes (MFAP3L on 4q32.3, NDUFAB1 and PALB2 on 16p12 with genome-wide significance (false discovery rate (FDR] <5%. At the pathway level, several terms such as transcription regulation, cytokine binding, and developmental process were significantly enriched in ADs (FDR <5%. Our approaches studying ADs as quantitative traits and utilizing the full GWAS data may be useful in identifying susceptibility genes and pathways for ADs.

A CTAB Procedure Of Total Genomic DNA Extraction For Medicinal Mushrooms

International Nuclear Information System (INIS)

Azhar Mohamad; Muhammad Hussaini Mohd Mustafa; Muhammad Hanif Azhari Noor; Rosnani Abdul Rashid; Hasan Hamdani Hasan Mutaat; Meswan Meskom; Mat Rasol Awang

2014-01-01

Medicinal mushroom is defined as mushrooms used in medicine or medical research. Isolation of intact, high-molecular-mass genomic DNA is essential for many molecular biology applications including Polymerase Chain Reaction (PCR), endonuclease restriction digestion, Southern blot analysis, and genomic library construction. The most important and prerequisite towards reliable molecular biology work is the total genomic DNA of a sample must be in good quality. Five freshly samples of medicinal mushroom were used in this work known as Auriculariapolytricha, Lentinus edode, Pleurotus sayorcaju, Sczhizopyllum commune and Ganodermalucidum. 5 mg of each sample were used to extraction the DNA, prepared in 3 replications and repeated twice. PCR based technique by using ISSR markers were used in checking the amplification ability of the total genomic extraction. A standard Doyle and Doyle protocol for genomic DNA extraction was modified in optimizing the total genomic DNA from the medicinal mushroom.The modification parameters were percentage of CTAB, incubation period and temperature. The results reveal that each sample required a certain combinations of time and period of incubation. Besides, percentage of CTAB in the buffer was found significant in giving a high yielding of extracted total genomic DNA. The extracted total genomic DNA from the medicinal mushroom yielded from 39.7 ng/ μl to 919.1 ng/ μl. The different yield among the samples found to be corresponded to polysaccharide content in the medicinal mushrooms. The objective of this works is to optimize total genomic DNA extraction of medicinal mushrooms towards a high quality intact genomic DNA for molecular activities. (author)

Laser Stimulated Genomic Exchange in Stem Cells. Laser Non-cloning Techniques

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Stefan, V. Alexander

2012-02-01

I propose a novel technique for a pluripotent stem cell generation. Genomic exchange is stimulated by the beat-wave free electron laser, (B-W FEL), frequency matching with the frequencies of the DNAootnotetextJ.D. Watson and F. H. C. Crick, Nature, 171, 737-738 (1953). eigen-oscillations. B-W FEL-1ootnotetextV. Stefan, B.I.Cohen, C. Joshi Science, 243,4890, (Jan 27,1989); Stefan, et al., Bull. APS. 32, No. 9, 1713 (1987); Stefan, APS March-2011, #S1.143; APS- March-2009, #K1.276. scans entire stem cell; B-W FEL-2 probes the chromosomes. The scanning and probing lasers: 300-500nm and 100-300nm, respectively; irradiances: the order-of-10s mW/cm^2 (above the threshold value for a particular gene structure); repetition rate of few-100s Hz. A variety of genetic-matching conditions can be arranged. Genomic glitches, (the cell nucleus transferootnotetextScott Noggle et al. Nature, 478, 70-75 (06 October 2011).), can be hedged by the use of lasers.

Methyl-CpG island-associated genome signature tags

Science.gov (United States)

Dunn, John J

2014-05-20

Disclosed is a method for analyzing the organismic complexity of a sample through analysis of the nucleic acid in the sample. In the disclosed method, through a series of steps, including digestion with a type II restriction enzyme, ligation of capture adapters and linkers and digestion with a type IIS restriction enzyme, genome signature tags are produced. The sequences of a statistically significant number of the signature tags are determined and the sequences are used to identify and quantify the organisms in the sample. Various embodiments of the invention described herein include methods for using single point genome signature tags to analyze the related families present in a sample, methods for analyzing sequences associated with hyper- and hypo-methylated CpG islands, methods for visualizing organismic complexity change in a sampling location over time and methods for generating the genome signature tag profile of a sample of fragmented DNA.

Errors in the calculation of sub-soil moisture probe by equivalent moisture content technique

International Nuclear Information System (INIS)

Lakshmipathy, A.V.; Gangadharan, P.

1982-01-01

The size of the soil sample required to obtain the saturation response, with a neutron moisture probe is quite large and this poses practical problems of handling and mixing large amounts of samples for absolute laboratory calibration. Hydrogenous materials are used as a substitute for water in the equivalent moisture content technique, for calibration of soil moisture probes. In this it is assumed that only hydrogen of the bulk sample is responsible for the slowing down of fast neutrons and the slow neutron countrate is correlated to equivalent water content by considering the hydrogen density of sample. It is observed that the higher atomic number elements present in water equivalent media also affect the response of the soil moisture probe. Hence calculations, as well as experiments, were undertaken to know the order of error introduced by this technique. The thermal and slow neutron flux distribution around the BF 3 counter of a sub-soil moisture probe is calculated using three group diffusion theory. The response of the probe corresponding to different equivalent moisture content of hydrogenous media, is calculated taking into consideration the effective length of BF 3 counter. Soil with hydrogenous media such as polyethylene, sugar and water are considered for calculation, to verify the suitability of these materials as substitute for water during calibration of soil moisture probe. Experiments were conducted, to verify the theoretically calculated values. (author)

Development of Thinopyrum ponticum-specific molecular markers and FISH probes based on SLAF-seq technology.

Science.gov (United States)

Liu, Liqin; Luo, Qiaoling; Teng, Wan; Li, Bin; Li, Hongwei; Li, Yiwen; Li, Zhensheng; Zheng, Qi

2018-05-01

Based on SLAF-seq, 67 Thinopyrum ponticum-specific markers and eight Th. ponticum-specific FISH probes were developed, and these markers and probes could be used for detection of alien chromatin in a wheat background. Decaploid Thinopyrum ponticum (2n = 10x = 70) is a valuable gene reservoir for wheat improvement. Identification of Th. ponticum introgression would facilitate its transfer into diverse wheat genetic backgrounds and its practical utilization in wheat improvement. Based on specific-locus-amplified fragment sequencing (SLAF-seq) technology, 67 new Th. ponticum-specific molecular markers and eight Th. ponticum-specific fluorescence in situ hybridization (FISH) probes have been developed from a tiny wheat-Th. ponticum translocation line. These newly developed molecular markers allowed the detection of Th. ponticum DNA in a variety of materials specifically and steadily at high throughput. According to the hybridization signal pattern, the eight Th. ponticum-specific probes could be divided into two groups. The first group including five dispersed repetitive sequence probes could identify Th. ponticum chromatin more sensitively and accurately than genomic in situ hybridization (GISH). Whereas the second group having three tandem repetitive sequence probes enabled the discrimination of Th. ponticum chromosomes together with another clone pAs1 in wheat-Th. ponticum partial amphiploid Xiaoyan 68.

A specific DNA probe which identifies Babesia bovis in whole blood.

Science.gov (United States)

Petchpoo, W; Tan-ariya, P; Boonsaeng, V; Brockelman, C R; Wilairat, P; Panyim, S

1992-05-01

A genomic library of Babesia bovis DNA from the Mexican strain M was constructed in plasmid pUN121 and cloned in Escherichia coli. Several recombinants which hybridized strongly to radioactively labeled B. bovis genomic DNA in an in situ screening were selected and further analyzed for those which specifically hybridized to B. bovis DNA. It was found that pMU-B1 had the highest sensitivity, detecting 25 pg of purified B. bovis DNA, and 300 parasites in 10 microliters of whole infected blood, or 0.00025% parasitemia. pMU-B1 contained a 6.0 kb B. bovis DNA insert which did not cross-hybridize to Babesia bigemina, Trypanosoma evansi, Plasmodium falciparum, Anaplasma marginale, Boophilus microplus and cow DNA. In the Southern blot analysis of genomic DNA, pMU-B1 could differentiate between two B. bovis geographic isolates, Mexican strain M and Thai isolate TS4. Thus, the pMU-B1 probe will be useful in the diagnosis of Babesia infection in cattle and ticks, and in the differentiation of B. bovis strains.

Developments in Scanning Hall Probe Microscopy

Science.gov (United States)

Chouinard, Taras; Chu, Ricky; David, Nigel; Broun, David

2009-05-01

Low temperature scanning Hall probe microscopy is a sensitive means of imaging magnetic structures with high spatial resolution and magnetic flux sensitivity approaching that of a Superconducting Quantum Interference Device. We have developed a scanning Hall probe microscope with novel features, including highly reliable coarse positioning, in situ optimization of sensor-sample alignment and capacitive transducers for linear, long range positioning measurement. This has been motivated by the need to reposition accurately above fabricated nanostructures such as small superconducting rings. Details of the design and performance will be presented as well as recent progress towards time-resolved measurements with sub nanosecond resolution.

Advanced Whole-Genome Sequencing and Analysis of Fetal Genomes from Amniotic Fluid.

Science.gov (United States)

Mao, Qing; Chin, Robert; Xie, Weiwei; Deng, Yuqing; Zhang, Wenwei; Xu, Huixin; Zhang, Rebecca Yu; Shi, Quan; Peters, Erin E; Gulbahce, Natali; Li, Zhenyu; Chen, Fang; Drmanac, Radoje; Peters, Brock A

2018-04-01

Amniocentesis is a common procedure, the primary purpose of which is to collect cells from the fetus to allow testing for abnormal chromosomes, altered chromosomal copy number, or a small number of genes that have small single- to multibase defects. Here we demonstrate the feasibility of generating an accurate whole-genome sequence of a fetus from either the cellular or cell-free DNA (cfDNA) of an amniotic sample. cfDNA and DNA isolated from the cell pellet of 31 amniocenteses were sequenced to approximately 50× genome coverage by use of the Complete Genomics nanoarray platform. In a subset of the samples, long fragment read libraries were generated from DNA isolated from cells and sequenced to approximately 100× genome coverage. Concordance of variant calls between the 2 DNA sources and with parental libraries was >96%. Two fetal genomes were found to harbor potentially detrimental variants in chromodomain helicase DNA binding protein 8 ( CHD8 ) and LDL receptor-related protein 1 ( LRP1 ), variations of which have been associated with autism spectrum disorder and keratosis pilaris atrophicans, respectively. We also discovered drug sensitivities and carrier information of fetuses for a variety of diseases. We were able to elucidate the complete genome sequence of 31 fetuses from amniotic fluid and demonstrate that the cfDNA or DNA from the cell pellet can be analyzed with little difference in quality. We believe that current technologies could analyze this material in a highly accurate and complete manner and that analyses like these should be considered for addition to current amniocentesis procedures. © 2018 American Association for Clinical Chemistry.

Field protocols for the genomic era

Directory of Open Access Journals (Sweden)

N Bulatova

2009-08-01

Full Text Available For many decades karyotype was the only source of overall genomic information obtained from species of mammal. However, approaches have been developed in recent years to obtain molecular and ultimately genomic information directly from the extracted DNA of an organism. Molecular data have accumulated hugely for mammalian taxa. The growing volume of studies should motivate field researchers to collect suitable samples for molecular analysis from various species across all their ranges. This is the reason why we here include a molecular sampling procedure within a field work protocol, which also includes more traditional (including cytogenetic techniques. In this way we hope to foster the development of molecular and genomic studies in non-standard wild mammals.

Harnessing Whole Genome Sequencing in Medical Mycology.

Science.gov (United States)

Cuomo, Christina A

2017-01-01

Comparative genome sequencing studies of human fungal pathogens enable identification of genes and variants associated with virulence and drug resistance. This review describes current approaches, resources, and advances in applying whole genome sequencing to study clinically important fungal pathogens. Genomes for some important fungal pathogens were only recently assembled, revealing gene family expansions in many species and extreme gene loss in one obligate species. The scale and scope of species sequenced is rapidly expanding, leveraging technological advances to assemble and annotate genomes with higher precision. By using iteratively improved reference assemblies or those generated de novo for new species, recent studies have compared the sequence of isolates representing populations or clinical cohorts. Whole genome approaches provide the resolution necessary for comparison of closely related isolates, for example, in the analysis of outbreaks or sampled across time within a single host. Genomic analysis of fungal pathogens has enabled both basic research and diagnostic studies. The increased scale of sequencing can be applied across populations, and new metagenomic methods allow direct analysis of complex samples.