ENHANCED PCR FIDELITY AND SPECIFICITY

描述（改编自研究者摘要）： 本工作旨在探索错配结合蛋白的应用 来自极端嗜热真细菌Aquifex pyrophilus（Apy）， 海栖热袍菌（Thermotoga maritima，Tma），以改善突变检测， 通过增强的等位基因特异性PCR扩增检测多态性。 基本 技术是错配特异性TaqMan PCR。 A TaqMan寡核苷酸 （oligo）是结合模板的不可延伸的oligo，热稳定的， 错配结合蛋白将与TaqMan寡核苷酸之间的错配结合， 和模板，热稳定的错配结合蛋白将结合到 错配并阻止DNA聚合。 因此，匹配突变体的引物 等位基因将选择对扩增产物，同样的选择采取 放置在每个周期，允许几何选择。 前提是 这种简单的封闭管技术用于检测大量的 过量的正常等位基因将在以下研究中具有重要应用： 癌症和癌症流行病学。 错配结合蛋白-异源双链复合物的特异性和比率 将确定错配和凸起环处的形成和解离 以解释DNA聚合和TaqMan抑制之间的竞争。 通过匹配和错配TaqMan引物-模板的DNA聚合 将研究复合物以优化DNA聚合酶的选择， 错配结合蛋白和TaqMan寡核苷酸设计， 敏感性和等位基因特异性。 错配结合蛋白对引物检测等位基因特异性的影响 PCR将主要研究其改进PCR引物的潜力 一般的特殊性。 将研究它们对PCR保真度的影响 确定错配特异性TaqMan PCR的特异性上限 并在一般情况下提高PCR保真度， 稳定可靠的PCR克隆。

DESCRIPTION (Adapted from the Investigator's Abstract): The primary goal of this work is to explore the application of mismatch binding proteins derived from the hyperthermophilic eubacteria Aquifex pyrophilus (Apy) and Thermotoga maritima (Tma) to improve detection of mutations and polymorphisms through enhanced allele-specific PCR amplification. The basic technology is a mismatch-specific TaqMan PCR. A TaqMan oligonucleotide (oligo) is an unextendable oligo which binds to a template, thermostable mismatch-binding proteins will bind to the mismatch between the TaqMan oligo and template, thermostable mismatch-binding proteins will bind to the mismatch and prevent DNA polymerization. Thus a primer matching a mutant allele will select against amplification product, the same selection takes place at each cycle, permitting geometric selection. The hypothesis is that this simple closed tube technology for detecting mutant alleles in a vast excess of normal alleles will have important applications in the study of cancer and cancer epidemiology. The specificity and rate of mismatch binding protein-heteroduplex complex formation and dissociation at mismatches and bulge loops will be determined to explain the competition between DNA polymerization and TaqMan inhibition. DNA polymerization through matched and mismatched TaqMan primer-template complexes will be investigated to optimize the choice of DNA polymerases, mismatch binding proteins and TaqMan oligo design in terms of both PCR sensitivity and allele specificity. The effect of mismatch binding proteins on primer-detected allele-specific PCR will be investigated primarily for their potential to improve PCR primer specificity in general. Their effect in PCR fidelity will be investigated to determine the upper limit on specificity of mismatch-specific TaqMan PCR and to improve PCR fidelity in general, with applications to reduce PCR stuttering and faithful PCR cloning.