The Polymerase Chain Reaction (PCR) is one of the most widely used techniques in molecular biology. Unfortunately, thermocycling at elevated temperatures (60o to 95oC) causes extensive thermal damage to DNA;in particular, A+G depurination. In vivo, apurinic sites are repaired by AP lyase enzymes, which cleave DNA to form elongatable 3'OH ends. However, in vitro PCR reactions generally lack DNA repair enzymes. Since apurinic sites are not removed, DNA synthesis terminates. In this Phase I Research grant application, enzymology is described for DNA repair- proficient PCR. A novel thermostable enzyme mixture will be developed, using recombinant Thermotoga maritima AP lyase, PyroPhage 3173 polymerase, and an engineered pyrophosphatase. In DNA repair-proficient PCR, sites of A+G depurination are enzymatically hydrolysed to 3'OH ends during thermocycling, so that thermally damaged DNA strands are continually converted into elongatable molecules. As a result, it is hypothesized that the yield, sensitivity, and amplicon length of PCR can be increased. PDF Creator - PDF4Free v2.0 www.pdf4free.com
The Polymerase Chain Reaction (PCR) is one of the most widely used techniques in molecular medicine for the diagnosis of heritable and infectious diseases. However, the high-temperature reaction conditions employed during PCR result in thermal damage to DNA. The proposed research describes a repair-proficient enzyme mixture to improve the reaction yield, sensitivity, and throughput of DNA-based diagnostics. PDF Creator - PDF4Free v2.0 www.pdf4free.com
