Inhibitor-Resistant Thermostable DNA Polymerases for PCR

描述(申请人提供)：基于DNA的分析和诊断程序越来越多地利用聚合酶链式反应扩增技术的能力，以快速和准确地提供有关生物样本的信息，通常是高通量格式。一些关键的方法也依赖于对扩增反应的定量分析。然而，许多类型的组织、血液和其他临床样本可以用来产生模板DNA用于PCR扩增，其中含有DNA聚合酶的内源性抑制物。这些抑制物会产生假阴性结果，严重干扰分析。这些抑制物包括血液中的肝素和Heine，以及粪便中的胆盐。此外，一些用于DNA提取或检测的试剂抑制了PCR反应。我们建议使用定向进化和专利筛选技术的组合来创造耐热的DNA聚合酶变种，这些变种对常见的PCR抑制剂具有抵抗力。我们的固相微克隆磁共振仪(MCI)技术在分析诱变的酶文库方面优于传统的液相方法，因为它能够在每次分析中筛选3,000-10,000个变异体，同时最大限度地减少所需底物的数量。通过创造特定用途的聚合酶变种来抵抗抑制剂，这项技术将为聚合酶链式反应用户提供经济有效和节省时间的试剂，从而提高他们分析的可靠性。

DESCRIPTION (provided by applicant): DNA-based analytical and diagnostic procedures are increasingly exploiting the ability of PCR amplification technology to rapidly and accurately provide information about biological samples, often in a high-throughput format. Several key methods also rely on quantitative analysis of the amplification reactions. However, many types of tissue, blood and other clinical samples which can be used to generate template DNA for PCR amplification contain endogenous inhibitors of DNA polymerases. These inhibitors can seriously interfere with the analysis by creating false negative results. Such inhibitors include heparin and heine in blood, and bile salts in feces. In addition, a number of reagents used for DNA extraction or detection inhibit PCR reactions. We propose to use a combination of directed evolution and patented screening technology to create thermostable DNA polymerase variants that are resistant to common PCR inhibitors. Our sotid-phase MicroColonylmager (MCI) Technology is superior to conventional liquid-phase methods for analyzing mutagenized libraries of enzymes because it enables screening of 3,000-10,000 variants per assay while at the same time minimizing the amount of substrate required. By creating application-specific polymerase variants engineered to resist inhibitors, this technology will provide PCR users with cost-effective and time-saving reagents that will improve the reliability of their assays.