MutS based SNP detection, genome scanning and rare sequence enrichment

基于MutS的SNP检测、基因组扫描和稀有序列富集

• 批准号: 7220122
• 负责人: ROBERT E WAGNER
• 金额: $ 10万
• 依托单位: GENE CHECK, INC.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7220122
• 关键词: Affinity; Amino Acids; Antibodies; Area; Bacteria; Binding; Binding Proteins; Biological Assay; Biotin; Chromosome Mapping; Clinical; Complex; Condition; DNA; Detection; Development; Diagnostic; Disease; Escherichia coli; Genes; Genetic; Genetic Variation; Genome; Genome Scan; Genomics; Genotype; Goals; Hereditary Disease; Human; In Vitro; Infectious Agent; Intention; Link; Malignant Neoplasms; Medicine; Methods; Mutation; Mutation Detection; Normal Cell; Numbers; Oligonucleotides; Pharmacogenetics; Pharmacotherapy; Point Mutation; Polymerase Chain Reaction; Polymorphism Analysis; Prion Diseases; Prions; Production; Range; Relative (related person); Research; Resistance; Sampling; Scanning; Scrapie; Sheep; Single Nucleotide Polymorphism; Solutions; Somatic Mutation; Syndrome; System; Technology; Testing; Tissues; Toxic effect; Variant; base; biotin-streptavidin complex; gene discovery; genome wide association study; improved; in vivo; research study; response; trait; tumor

DESCRIPTION (provided by applicant): MutS based SNP detection, genome scanning and rare sequence enrichment The goal of this project is to develop MutS based assays for SNP detection, rare sequence detection, rare sequence isolation and genome scanning for SNP discovery. The approach will be to use end-blocking of heteroduplexes, which has been shown to improve the stability of MutS binding. Initial experiments will involve SNP detection and rare SNP detection and isolation using synthetic oligonucleotides and will utilize Gene Check's Immobilized Mismatch Binding Protein method of SNP detection. These experiments will also determine if end- blocking improves MutS binding to mismatches that are poorly bound in vivo and in vitro without end-blocking. Three different end-blocks will be tested (four-way DNA junctions, streptavidin/biotin complexes and antibody/biotin complexes)and MutS will be obtained from three different bacteria (E. coli, T. aquaticus and T. thermophilis). Successful conditions will be applied to PCR amplicons for SNP and rare SNP detection by amplifying genomic DNA of known genotypes mixed in various ratios. Finally, bacterial genomic DNA will be used in experiments testing end-blocking in applications for genomic scanning for unknown SNPs and for direct genotyping of known SNPs without amplification. MutS based SNP detection, genome scanning and rare sequence enrichment Development of a MutS based method of SNP detection and discovery and rare SNP and sequence detection and isolation will help meet an increasing need for SNP discovery and whole genome scanning in the move toward personalized medicine. The ability to detect rare SNPs and sequences will have immediate clinical impact, both for humans in such areas as diagnostic scanning for rare mutations among normal cells as in tumor margins, and in veterinary diagnostics by allowing rapid and inexpensive analysis of pooled samples for infectious agents and genetic diseases or production traits.

本项目的目标是开发用于SNP检测、稀有序列检测、稀有序列分离和基因组扫描的基于MutS的测定，以用于SNP发现。该方法将使用异源双链体的末端封闭，这已被证明可以提高MutS结合的稳定性。最初的实验将涉及使用合成寡核苷酸的SNP检测和罕见SNP检测和分离，并将利用Gene Check的SNP检测的固定化错配结合蛋白方法。这些实验还将确定末端封闭是否改善MutS与错配的结合，所述错配在没有末端封闭的情况下在体内和体外结合不良。将测试三种不同的末端封闭物（四向DNA连接、链霉亲和素/生物素复合物和抗体/生物素复合物），并从三种不同的细菌（大肠杆菌）中获得MutS。coli、T. aquaticus和T.热膨胀）。通过扩增以各种比例混合的已知基因型的基因组DNA，将成功的条件应用于SNP和罕见SNP检测的PCR扩增子。最后，细菌基因组DNA将用于测试末端封闭的实验，用于未知SNP的基因组扫描和已知SNP的直接基因分型而无需扩增。基于MutS的SNP检测、基因组扫描和稀有序列富集基于MutS的SNP检测和发现以及稀有SNP和序列检测和分离的方法的开发将有助于满足在向个性化医学发展的过程中对SNP发现和全基因组扫描的日益增长的需求。检测罕见SNP和序列的能力将具有直接的临床影响，无论是在诸如诊断扫描肿瘤边缘中正常细胞中的罕见突变的人类领域，还是在兽医诊断中，通过允许快速和廉价地分析合并样品的传染性因子和遗传疾病或生产性状。