Showing papers on "Exome sequencing published in 2001"

Optimization of experimental conditions for RNA-based sequencing of MLH1 and MSH2 genes.

Abstract: The most sensitive technique for the detection of germline mutations is exon by exon sequencing of the gene under investigation using genomic DNA as a template for analysis. This approach, however, has cost and sensitivity limitations that can, at least in part, be overcome by RNA-based analysis. Germline mutations of MLH1 and MSH2 are the most frequent cause of the inherited susceptibility to colorectal and other epithelial cancers known as hereditary non-polyposis colorectal cancer (HNPCC). We compared the analysis of the MLH1 and MSH2 genes using mRNA and genomic DNA as starting material from 21 HNPCC patients. All samples were investigated by RT-PCR, sequencing of cDNA and simultaneous sequencing of genomic DNA. The cDNA was generated using specific primers complementary to the ends of MLH1 and MSH2 genes, respectively. Mutations in MLH1 and MSH2 were detected in 11 out of 21 unrelated patients. In 10 out of 11 cases, mutations were detected independently of the type of primers used for reverse transcription (RT). One novel missense mutation (K751R) in MLH1 was detected using this method. One nonsense mutation (E205X) in MSH2 was only detectable when RT was performed using MSH2 gene-specific primers. Shorter PCR products indicative of alternatively spliced transcripts were not observed when MLH1 or MSH2 specific cDNA RT primers were employed to generate template, except in one case where exon skipping was observed for exons 9 and 10. In this report we demonstrate that primers specific for RT of MLH1 and MSH2 are crucial for increasing the sensitivity of cDNA analysis. DNA sequencing using RNA as a basis for template construction may be a valuable and economical alternative to genomic DNA sequencing. Hum Mutat 17:52–60, 2001. © 2001 Wiley-Liss, Inc.

Detection of Single Nucleotide Polymorphisms in Cattle Genes Using Automated DNA Sequencing and Dideoxy Fingerprinting

Abstract: Single nucleotide polymorphisms (SNPs) are the most common form of DNA sequence polymorphisms and mutation in vertebrates. Any detection approach must involve the determination of DNA sequence and allele frequencies which are important for linkage analysis studies and high-throughput genotyping methods. In this experiment two SNPa detection methods, direct automated DNA sequencing and dideoxy Fingerprinting (ddF) were used to detect SNPs in Insulin-like Growth Factor Binding Protein-3 gene (IGFBP3) and Growth Hormone gene (GH) in cattle. Using direct automated DNA sequencing 2 SNPs repressnting C/T and G/T and 2 SNPs representing A/C and C/T were identified in IGFBP-3 gene and GH gene, respectively. Dideoxynucleotide qaunosine triphosphate (ddGTP) used as the chain terminator in ddf method generated the fingerprint corresponding to only the G/T mutation in IGFBP-3 gene. The frequencies of the minor allele were estimated by the ratio of peak heights from chromatograms of sequencing traces generated from the pooled DNA. The results indicated that 20% of allele frequency of the minor allele should be present in the population in order to detect informative SNPs.

Mutation detection by cycle sequencing.

Abstract: Candidate genes are screened for mutations by a DNA sequencing procedure known as cycle sequencing. First, a segment of the candidate gene is PCR amplified from the genomic DNA of an affected individual. The PCR product is then subjected to multiple rounds of further amplification in a thermal cycler using a heat-stable DNA polymerase in the presence of different dideoxynucleotides and a radiolabeled primer. The resulting 32P-labeled sequence reaction products are fractionated on a denaturing polyacrylamide gel and visualized by autoradiography. DNA segments on the order of 200 bp from 10 to 30 individuals can be screened on each gel. Cycle sequencing eliminates the need to subclone genomic fragments or PCR products, which makes it a much simpler method than conventional sequencing for identifying mutations.

Systematic sequencing of complex genomes

Abstract: Biology and medicine are in the midst of a revolution, the full extent of which will probably not be realized for many years to come. The catalyst for this revolution is the Human Genome Project1 and related activities that aim to develop improved technologies for analysing DNA, to generate detailed information about the genomes of numerous organisms, and to establish powerful experimental and computational approaches for studying genome structure and function. The past few years have seen a remarkable crescendo in accomplishments related to DNA sequencing, with genome sequences being generated for several key experimental organisms, including a yeast (Saccharomyces cerevisiae), a nematode (Caenorhabditis elegans), a fly (Drosophila melanogaster), a plant (Arabidopsis thaliana) and the human (Homo sapiens). Collectively, the generation of these sequence data and others is launching the ‘sequence-based era’of biomedical research. Associated with the above accomplishments has been the refinement of existing strategies for genome sequencing, as well as the development of new ones. Among these are approaches that make extensive use of large-insert clones and associated physical maps, some that take a whole-genome approach without using clone-based physical maps, and others that use a hybrid strategy that involves elements of the other two. Each of these general strategies for genome sequencing is described in this review. There are many potential uses of genome-sequence data. In some cases, a detailed and accurate sequencebased ‘blueprint’ of a genome is required (for example, to establish a comprehensive gene catalogue and/or to gain insight into long-range genome organization), whereas in other cases, an incomplete survey will suffice (for example, to acquire information about the repetitive sequences in a genome and/or to carry out simple, non-comprehensive comparisons to sequences from other organisms). Importantly, the intended use(s) of genome-sequence data must be carefully considered when choosing a specific sequencing strategy and defining the end point of a particular project. These issues, as well as the plans for future sequencing initiatives by the Human Genome Project, are also discussed.

Mutation detection by sequencing and TaqMan-allele specific amplification

Abstract: Mutation detection in various genetic disorders has been facilitated by recent advances in nucleotide sequencing technology. An introduction of capillary electrophoresis enabled sequencing analysis to be performed in clinical service laboratory. On the other hand, previously identified known mutations can be readily detected by TaqMan-allele specific amplification, which has been developed in our laboratory.