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Robin B Gasser - One of the best experts on this subject based on the ideXlab platform.
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Single-Strand Conformation Polymorphism (SSCP) for the analysis of genetic variation
Nature Protocols, 2006Co-Authors: Robin B Gasser, Neil B Chilton, Bronwyn E. Campbell, Aaron Jex, Domenico Otranto, Claudia Cafarchia, Ian Beveridge, Xing-quan ZhuAbstract:The accurate analysis of genetic variation has major implications in many areas of biomedical research, including the identification of infectious agents (such as parasites), the diagnosis of infections, and the detection of unknown or known disease-causing mutations. Mutation scanning methods, including PCR-coupled Single-Strand Conformation Polymorphism (SSCP), have significant advantages over many other nucleic acid techniques for the accurate analysis of allelic and mutational sequence variation. The present protocol describes the SSCP method of analysis, including all steps from the small-scale isolation of genomic DNA and PCR amplification of target sequences, through to the gel-based separation of amplicons and scanning for mutations by SSCP (either by the analysis of radiolabeled amplicons in mutation detection enhancement (MDE) gels or by non-isotopic SSCP using precast GMA gels). The subsequent sequence analysis of polymorphic bands isolated from gels is also detailed. The SSCP protocol can readily detect point mutations for amplicon sizes of up to 450–500 bp, and usually takes 1–2 days to carry out. This user-friendly, low-cost, potentially high-throughput platform has demonstrated the utility to study a wide range of pathogens and diseases, and has the potential to be applied to any gene of any organism.
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Genetic analysis of Trichinella populations by 'cold' Single-Strand Conformation Polymorphism analysis.
Veterinary Parasitology, 2005Co-Authors: Robin B Gasser, Youssef G. Abs El-osta, Dante S. Zarlenga, Edoardo PozioAbstract:A non-isotopic Single-Strand Conformation Polymorphism ('cold' SSCP) technique has been assessed for the analysis of sequence variability in the expansion segment 5 (ES5) of domain IV and the D3 domain of nuclear ribosomal DNA within and/or among isolates and individual muscle (first-stage) larvae representing all currently recognized species/genotypes of Trichinella. Data are consistent with the ability of cold SSCP to identify intra-specific as well as inter-specific variability among Trichinella genotypes. The cold SSCP approach should be applicable to a range of other genetic markers for comparative studies of Trichinella populations globally.
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screening for haplotypic variability within oesophagostomum bifurcum nematoda employing a single strand Conformation Polymorphism approach
Molecular and Cellular Probes, 2002Co-Authors: Johanna M De Gruijter, A M Polderman, Robin B GasserAbstract:Abstract Genetic markers in the mitochondrial genome have proven useful for population genetic studies because of their maternal inheritance and relatively high evolutionary rates. In this study, we exploited the high resolution capacity of PCR-coupled Single-Strand Conformation Polymorphism (SSCP) to screen for sequence variation in part of the cytochrome c oxidase subunit 1 gene (p cox 1) among individuals of the parasitic nematode, Oesophagostomum bifurcum from human or Mona monkey hosts from Africa. SSCP analysis revealed distinct profiles among some of the individuals, and subsequent sequence analysis of representative samples defined 10 different haplotypes. For comparative purposes, the p cox 1 sequences for representatives of four other species of Oesophagostomum from livestock were included. While there were high levels (11·5–13·7%) of sequence difference among the latter species, there was no fixed nucleotide difference between O. bifurcum individuals from humans and those from monkeys. The data support the proposal that O. bifurcum from the two primate hosts represents a single species and that the haplotypic variability in p cox 1 represents population variation. The results reinforce the usefulness of the SSCP-sequencing approach for studying genetic variation in nematode populations using mitochondrial markers.
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Applications of Single-Strand Conformation Polymorphism (SSCP) to taxonomy, diagnosis, population genetics and molecular evolution of parasitic nematodes.
Veterinary Parasitology, 2001Co-Authors: Robin B Gasser, Neil B ChiltonAbstract:The analysis of genetic variation in parasitic nematodes has important implications for studying aspects of taxonomy, diagnosis, population genetics, drug resistance and molecular evolution. This article highlights some applications of PCR-based Single-Strand Conformation Polymorphism (SSCP) for the analysis of sequence variation in individual parasites (and their populations) to address some of these areas. It also describes the principles and advantages of SSCP, and provides some examples for future applications in parasitology.
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genotyping taenia tapeworms by single strand Conformation Polymorphism of mitochondrial dna
Electrophoresis, 1999Co-Authors: Robin B Gasser, Wayne G WoodsAbstract:To overcome limitations in identifying tapeworms of the genus Taenia by traditional approaches, we have established a Single-Strand Conformation Polymorphism (SSCP) method utilizing two different regions of mitochondrial (mt) DNA as targets. The NADH dehydrogenase 1 and the cytochrome c oxidase subunit I genes were amplified from genomic DNA by polymerase chain reaction (PCR), denatured and subjected to electrophoresis in mutation detection enhancement gels. SSCP analysis achieved delineation among eight different species of Taenia from different hosts based on characteristic profiles and enabled the detection of intraspecific variability in profiles for some taxa. This SSCP-based typing method has important implications for taxonomy, diagnosis and for studying the genetic structure of Taenia populations.
Jeanjacques Godon - One of the best experts on this subject based on the ideXlab platform.
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Microbial diversity in Tunisian olive fermentation brine as evaluated by small subunit rRNA - Single strand Conformation Polymorphism analysis
International Journal of Food Microbiology, 2008Co-Authors: Mohamed Chamkha, Sami Sayadi, Valérie Bru-adan, Jeanjacques GodonAbstract:The microbial diversity of a Tunisian olive fermentation brine was analysed using a culture-independent approach based on the polymerase chain reaction-single strand Conformation Polymorphism (PCR-SSCP). SSCP patterns show a remarkably simple microbial community but higher for bacterial community than for the eukaryotic community. This study did not show the presence of archaeal populations. After PCR amplification, two small subunit (SSU) rRNA clone libraries of Bacteria and Eucarya populations were established. Three bacteria and only one eukaryotic phylotype were identified. Two dominant bacteria showed 100% phylogenetic similarity to the 16S rRNA sequences of Lactobacillus plantarum and Lactobacillus collinoides and represent 85% of bacterial community. The third bacteria phylotype was phylogenetically close related to the 16S rRNA sequence of a moderately halophilic bacterium belonging to the class gamma Proteobacteria. The dominant eukaryotic phylotype was identified as a Pichia membranaefaciens.
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Monitoring of a pyrite-oxidising bacterial population using DNA Single-Strand Conformation Polymorphism and microscopic techniques
Applied Microbiology and Biotechnology, 2002Co-Authors: Fabienne Battaglia-brunet, Jeanjacques Godon, Manuel Clarens, Patrick D'hugues, S Foucher, D MorinAbstract:The Single-Strand Conformation Polymorphism (SSCP) technique was used to study the evolution of a bacterial consortium during the batch oxidation of a cobaltiferous pyrite in two types of bio-reactor: a bubble column and a classical stirred tank. Sequencing 16S rDNA revealed the presence of three organisms affiliated to Leptospirillum ferrooxidans, Acidithiobacillus thiooxidans and Sulfobacillus thermosulfidooxidans, respectively. Attempts were made to determine the proportions of bacteria attached to solid particles or freely suspended in the medium using a combination of PCR-SSCP and a microscopic technique. Ac. thiooxidans-related bacteria were dominant in the liquid during the early phase of the batch, but were later supplanted by L. ferrooxidans-related bacteria. L. ferrooxidans-related organisms were always in the majority on the solids. The growth of S. thermosulfidooxidans-related bacteria seemed to be favoured by the bubble-column reactor.
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monitoring of activity dynamics of an anaerobic digester bacterial community using 16s rrna polymerase chain reaction single strand Conformation Polymorphism analysis
Environmental Microbiology, 2000Co-Authors: Celine Delbes, R Moletta, Jeanjacques GodonAbstract:Summary The influence of parameter changes on the bacterial community of a laboratory-scale anaerobic digester fed with glucose was investigated using a cultureindependent approach based on Single-Strand Conformation Polymorphism (SSCP) analysis of total 16S rDNA and 16S rRNA amplification products. With the
Kenshi Hayashi - One of the best experts on this subject based on the ideXlab platform.
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Single‐strand Conformation Polymorphism analysis by perpendicular temperature‐gradient gel electrophoresis
Electrophoresis, 1995Co-Authors: Kokichi Sugano, Noriko Fukayama, Hisanao Ohkura, Yukio Shimosato, Yasushi Yamada, Inoue Tamotsu, Takao Sekiya, Kenshi HayashiAbstract:Using a newly developed temperature-gradient gel electrophoresis apparatus, mutations in the K-ras oncogene and p53 tumor suppressor gene were analyzed for Single-Strand Conformation Polymorphism (SSCP). The mobilities of Single-Stranded DNAs, carrying various mutations, change--depending on the gel temperature during electrophoresis. Therefore, temperature-gradient Single-Strand Conformation Polymorphism (TG-SSCP) analysis can provide useful information concerning the optimum temperature for SSCP and may also be used to screen for various mutations or Polymorphisms in a single electrophoretic run.
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Detection of DNA aberrations in human cancers by Single-Strand Conformation Polymorphism analysis of polymerase chain reaction products.
Tohoku Journal of Experimental Medicine, 1992Co-Authors: Yoshinori Murakami, Kenshi Hayashi, Youiohi Suzuki, Yosuke Kishimoto, Setsuo Hirohashi, Takao SekiyaAbstract:MURAKAMI, Y., SUZUKI, Y., KISHIMOTO, Y., HIROHASHI, S., HAYASHI, K, and SEKIYA, T. Detection of DNA Aberrations in Human Cancers by Single-Strand Conformation Polymorphism Analysis of Polymerase Chain Reaction Products. Tohoku J. Exp. Med., 1992, 168 (2), 247-255-We have developed a simple, sensitive method, Single-Strand Conformation Polymorphism (SSCP) analysis, to detect a single nucleotide substitution in a DNA fragment amplified and labeled by the polymerase chain reaction (PCR). Mobility shift of Single-Stranded DNAs due to their specific Conformations on non-denaturing polyacrylamide gel electrophoresis can reveal DNA aberrations. By the PCR-SSCP analysis of DNAs from surgical specimens of human cancers, mutated ras genes (17%) and aberrations of tumor suppressor p53 gene (53%) including loss of one of the two alleles and a mutation in the remaining allele were detected in lung carcinomas and aberrations of both of the p53 and retinoblastoma (RB) genes were detected exclusively in advanced hepatocellular carcinomas.-SSCP analysis; lung carcinoma; hepatocellular carcinoma; ras gene; p53 gene; RB gene
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f sscp fluorescence based polymerase chain reaction single strand Conformation Polymorphism pcr sscp analysis
Genome Research, 1992Co-Authors: Reiko Makino, Takao Sekiya, H Yazyu, Y Kishimoto, Kenshi HayashiAbstract:: A fluorescence-based method for polymerase chain reaction-Single-Strand Conformation Polymorphism (PCR-SSCP) analysis, F-SSCP, was developed in which the target sequence is amplified by the PCR using fluorescent primers. The amplified products are then heat-denatured and applied to a water-jacket controlled gel in an automated DNA sequencer. The separated strands are detected as laser-excited fluorescence at the bottom of the gel, and mutations are detected as shifts in the position of the peaks in the fluorogram. The system does not involve radioactivity, and the conditions of electrophoresis are more strictly controlled than in the previous system, which relied on ambient air-cooling to maintain the gel at a constant temperature. The nature of the output data allows direct quantitative interpretation, and so the relative abundance of each allele in a mixture of two or more alleles can easily be estimated. The application of F-SSCP for detection of mutations and loss of heterozygosities of p53 in tumor tissues is reported.
Takao Sekiya - One of the best experts on this subject based on the ideXlab platform.
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Nucleotide sequence changes in human genome: detection by Single-Strand Conformation Polymorphism analysis
Proceedings of the Japan Academy Series B, 2004Co-Authors: Takao SekiyaAbstract:Mobility shift of Single-Stranded DNA molecules with a single-base substitution in polyacrylamide gel electrophoresis due to a change of secondary and tertiary structures provides a simple, sensitive method, Single-Strand Conformation Polymorphism (SSCP) analysis, for detection of nucleotide sequence changes in DNA. The method with the quite unique principle can detect single-nucleotide substitutions, insertions or deletions of a short nucleotide sequence and loss of genes in human cancers and other genetic diseases. The great progress of the Human Genome Project has revealed thousands of genes associated with these diseases and led to an increasing need for detection of mutations and SNPs in large numbers of DNA samples. The recent development of high-throughput SSCP technologies will enable to meet this need even in a clinical setting. (Communicated by Shoji SHIBATA, M.J.A., Feb. 12, 2004)
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Identification of mycobacteria by nonradioisotopic Single-Strand Conformation Polymorphism analysis
Diagnostic Microbiology and Infectious Disease, 1995Co-Authors: Yutaka Tokue, Kokichi Sugano, Hisanao Ohkura, Yukio Shimosato, Takeshi Noda, Daizo Saito, Tadao Kakizoe, Takao SekiyaAbstract:Clinical isolates of mycobacteria were identified to species levels using nonradioisotopic Single-Strand Conformation Polymorphism (non-RI SSCP) analysis of 16S rRNA gene fragments amplified by polymerase chain reaction with primers common to all of mycobacterial species. The method is based on a hypervariable region within the 16S rRNA in mycobacteria, which is characterized by species-specific nucleotide sequences. A total of 92 mycobacterial strains (Mycobacterium tuberculosis, M. avium, M. gordonae, M. intracellulare, M. kansasii, M. chelonae, M. nonchromogenicum, M. xenopi, and unidentified strain) were studied. They were classified into nine types of pattern showing Single-Strand DNA bands having different mobilities. Each strain was shown in the species-specific mobility by non-RI SSCP analysis. The results of non-RI SSCP analysis were identical to those of standard biochemical methods and 16S rRNA sequencing.
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Single‐strand Conformation Polymorphism analysis by perpendicular temperature‐gradient gel electrophoresis
Electrophoresis, 1995Co-Authors: Kokichi Sugano, Noriko Fukayama, Hisanao Ohkura, Yukio Shimosato, Yasushi Yamada, Inoue Tamotsu, Takao Sekiya, Kenshi HayashiAbstract:Using a newly developed temperature-gradient gel electrophoresis apparatus, mutations in the K-ras oncogene and p53 tumor suppressor gene were analyzed for Single-Strand Conformation Polymorphism (SSCP). The mobilities of Single-Stranded DNAs, carrying various mutations, change--depending on the gel temperature during electrophoresis. Therefore, temperature-gradient Single-Strand Conformation Polymorphism (TG-SSCP) analysis can provide useful information concerning the optimum temperature for SSCP and may also be used to screen for various mutations or Polymorphisms in a single electrophoretic run.
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Detection of DNA aberrations in human cancers by Single-Strand Conformation Polymorphism analysis of polymerase chain reaction products.
Tohoku Journal of Experimental Medicine, 1992Co-Authors: Yoshinori Murakami, Kenshi Hayashi, Youiohi Suzuki, Yosuke Kishimoto, Setsuo Hirohashi, Takao SekiyaAbstract:MURAKAMI, Y., SUZUKI, Y., KISHIMOTO, Y., HIROHASHI, S., HAYASHI, K, and SEKIYA, T. Detection of DNA Aberrations in Human Cancers by Single-Strand Conformation Polymorphism Analysis of Polymerase Chain Reaction Products. Tohoku J. Exp. Med., 1992, 168 (2), 247-255-We have developed a simple, sensitive method, Single-Strand Conformation Polymorphism (SSCP) analysis, to detect a single nucleotide substitution in a DNA fragment amplified and labeled by the polymerase chain reaction (PCR). Mobility shift of Single-Stranded DNAs due to their specific Conformations on non-denaturing polyacrylamide gel electrophoresis can reveal DNA aberrations. By the PCR-SSCP analysis of DNAs from surgical specimens of human cancers, mutated ras genes (17%) and aberrations of tumor suppressor p53 gene (53%) including loss of one of the two alleles and a mutation in the remaining allele were detected in lung carcinomas and aberrations of both of the p53 and retinoblastoma (RB) genes were detected exclusively in advanced hepatocellular carcinomas.-SSCP analysis; lung carcinoma; hepatocellular carcinoma; ras gene; p53 gene; RB gene
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f sscp fluorescence based polymerase chain reaction single strand Conformation Polymorphism pcr sscp analysis
Genome Research, 1992Co-Authors: Reiko Makino, Takao Sekiya, H Yazyu, Y Kishimoto, Kenshi HayashiAbstract:: A fluorescence-based method for polymerase chain reaction-Single-Strand Conformation Polymorphism (PCR-SSCP) analysis, F-SSCP, was developed in which the target sequence is amplified by the PCR using fluorescent primers. The amplified products are then heat-denatured and applied to a water-jacket controlled gel in an automated DNA sequencer. The separated strands are detected as laser-excited fluorescence at the bottom of the gel, and mutations are detected as shifts in the position of the peaks in the fluorogram. The system does not involve radioactivity, and the conditions of electrophoresis are more strictly controlled than in the previous system, which relied on ambient air-cooling to maintain the gel at a constant temperature. The nature of the output data allows direct quantitative interpretation, and so the relative abundance of each allele in a mixture of two or more alleles can easily be estimated. The application of F-SSCP for detection of mutations and loss of heterozygosities of p53 in tumor tissues is reported.
Henrik Bjørn - One of the best experts on this subject based on the ideXlab platform.
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distinguishing oesophagostomum dentatum from oesophagostomum quadrispinulatum developmental stages by a single strand Conformation Polymorphism method
International Journal for Parasitology, 1998Co-Authors: Robin B Gasser, Wayne G Woods, Henrik BjørnAbstract:Abstract At some life-cycle stages, it is impossible to distinguish between the two species of porcine nodular worm, Oesophagostomum dentatum and Oesophagostomum quadrispinulatum , using morphological features. A PCR-based Single-Strand Conformation Polymorphism technique was established to overcome this limitation. The rDNA region spanning the second internal transcribed spacer was amplified by PCR from genomic DNA from morphologically well-defined adult worms. The PCR products were then denatured and subjected to electrophoresis in a non-denaturing gel matrix. Single-Strand Conformation Polymorphism analysis of the products generated characteristic and reproducible patterns for each of the two species and allowed their unequivocal delineation. The Single-Strand Conformation Polymorphism was also applied effectively to assess the purity of nine laboratory-maintained cultures of infective third-stage larvae believed to be monospecific for O. dentatum or O. quadrispinulatum . The analysis showed that all six O. dentatum cultures were indeed monospecific, whereas the three cultures believed to be monospecific for O. quadrispinulatum were either a mixture of O. dentatum and O. quadrispinulatum larvae or pure O. dentatum larvae. These findings demonstrated the usefulness of the Single-Strand Conformation Polymorphism approach for the routine monitoring of the purity of parasite lines and indicated its value for studies on the population biology of porcine nodular worms.
