MUTATION AND SNP IDENTIFICATION WITHOUT SEQUENCING

本项目的目的是开发一种高度精细的等位基因/突变鉴定方法，该方法基于使用固定化错配结合蛋白（IMBP）和短（20- 30聚体）合成寡核苷酸。短寡核苷酸与IMBP的使用允许同时但独立地检查PCR扩增子中的几个突变体或多态性位点，并且具有单一PCR扩增。该方法避免了PCR错误的问题，并允许长扩增子，这在此之前已经产生了IMBP的背景问题。将制备试剂盒以鉴定与羊瘙痒病易感性相关的绵羊朊蛋白基因的三个密码子的等位基因，并精确鉴定p53肿瘤抑制基因中的关键突变的集合。该技术在临床诊断和基因组学中具有直接应用，并且可以在很大程度上消除使用测序来鉴定已知等位基因/突变或多态性的需要。拟议的商业应用：IMBP突变/多态性检测的短寡核苷酸方法的商业应用包括临床诊断，其中该方法可以在很大程度上取代用于鉴定已知突变的测序。此外，该方法将是涉及多态性鉴定和分布的遗传学研究以及人类序列变异研究和疾病相关基因发现的主要贡献者。

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.