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 MicroCotonylmager (,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.
Thesaurus Terms: DNA directed DNA polymerase, polymerase chain reaction, protein engineering, technology /technique development, thermostability chemical synthesis, cholanate compound, combinatorial chemistry, directed evolution, enzyme inhibitor, heme, heparin biotechnology, genetic manipulation, high throughput technology, molecular cloning, nucleic acid sequence
