MUTATION AND SNP IDENTIFICATION WITHOUT SEQUENCING

本项目的目的是开发一种基于固定化错配结合蛋白(IMBP)和短(20-30mer)合成寡核苷酸的高度精细的等位基因/突变鉴定方法。将短寡核苷酸与IMBP结合使用，可以同时、但独立地检测聚合酶链式反应扩增子中的几个突变或多态位点，并且只需一次聚合酶链式反应。该方法避免了聚合酶链式反应错误的问题，并允许长扩增，这在以前造成了IMBP的背景问题。将准备试剂盒，以识别绵羊Pron蛋白基因与瘙痒病易感性相关的三个密码子上的等位基因，并准确识别P53肿瘤抑制基因的一系列关键突变。这项技术在临床诊断和基因组学中立即得到应用，可以在很大程度上消除使用测序来识别已知等位基因/突变或多态的需要。建议的商业应用：IMBP突变/多态检测的短寡核苷酸方法的商业应用包括临床诊断，该方法在很大程度上可以取代测序来识别已知突变。此外，该方法将对涉及多态鉴定和分布的遗传学研究以及人类序列变异和疾病相关基因的发现做出重大贡献。

The aim of this project is to develop a highly refined method of allele/mutation identification based on the use of Immobilized Mismatch Binding Protein (IMBP) and short (20-30mer) synthetic oligonucleotides. The use of short oligos with IMBP allows the simultaneous, but independent examination of several mutant or polymorphic sites in a PCR amplicon and with a single PCR amplification. The method avoids problems of PCR errors and allows long amplicons, which heretofore have created background problems with IMBP. Kits will be prepared to identify alleles at three codons of the sheep prion protein gene related to scrapie susceptibility and to precisely identify a collection of key mutations in the p53 tumor suppressor gene. The technology has immediate applications in clinical diagnostics and genomics and can largely eliminate the need to use sequencing for identification of known alleles/mutations or polymorphisms. PROPOSED COMMERCIAL APPLICATIONS: Commercial applications of a short oligo method of IMBP mutation/polymorphism detection include clinical diagnostics, where the method can largely replace sequencing for identification of known mutations. In addition, the method will be a major contributor to genetic studies involving polymorphism identification and distribution as well as studies of human sequence variation and the discovery of disease- associated genes.