Inhibitor-Resistant Thermostable DNA Polymerases for PCR

用于 PCR 的耐抑制剂耐热 DNA 聚合酶

• 批准号: 6743019
• 负责人: WILLIAM JOSEPH COLEMAN
• 金额: $ 12.88万
• 依托单位: KAIROS SCIENTIFIC, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6743019
• 关键词: DNA directed DNA polymerase; biotechnology; chemical synthesis; cholanate compound; combinatorial chemistry; directed evolution; enzyme inhibitors; genetic manipulation; heme; heparin; high throughput technology; molecular cloning; nucleic acid sequence; polymerase chain reaction; protein engineering; technology /technique development; thermostability

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.

描述（由申请人提供）：基于DNA的分析和诊断程序越来越多地利用PCR扩增技术的能力，以快速准确地提供有关生物样品的信息，通常以高通量的形式。几种关键方法也依赖于扩增反应的定量分析。然而，许多类型的组织、血液和其它临床样品可用于产生PCR扩增的模板DNA，它们含有DNA聚合酶的内源性抑制剂。这些抑制剂会产生假阴性结果，严重干扰分析。这些抑制剂包括血液中的肝素和海涅，以及粪便中的胆盐。此外，许多用于DNA提取或检测的试剂抑制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建耐常见PCR抑制剂的热稳定DNA聚合酶变体。我们的固相MicroColonylmager（MCI）技术上级于分析酶突变文库的传统液相方法，因为它能够在每次测定中筛选3，000 - 10，000个变体，同时最大限度地减少所需底物的量。通过创建应用特定的聚合酶变体来抵抗抑制剂，该技术将为PCR用户提供具有成本效益和节省时间的试剂，从而提高其检测的可靠性。