The sensitivity of gene detection in blood specimens in important clinical and forensic PCR applications is limited due to inhibitory blood substances which reduce amplification efficiency and may lead to false negative results. Different pre-PCR treatments that are being used to overcome this inhibitory effect significantly increase the duration and cost of the tests. Our project started with the idea to use the in vitro evolution approach to develop KlenTaq polymerase mutants that are highly resistant to the blood inhibition, which will allow a faster and lower-cost direct PCR analysis of blood samples. We also proposed to select KlenTaq mutants with rapid DMA elongation, which will reduce the time of the amplification cycle. Both types of mutant enzymes were obtained and characterized in the Phase I of the project. KlenTaq mutants combining the two selected phenotypes were also obtained. In Phase II we propose to further evolve the obtained mutant enzymes by determining the best amino acid substitutions at the codons responsible for the above mutations. To this end, we will use saturating site- directed mutagenisis. Next, using the same technique, we plan to transfer these amino acid changes into the full-length Taq enzyme, thus expanding the scope of PCR applications. Finally we will try to combine the two novel qualities of Taq with the cold-sensitive mutants of the enzyme designed for highly specific hot-start PCR already achieved by our company. The resultant triple-quality enzymes will be included in PCR kits specialized for detection of clinically important targets including HIV and Hepatitis B. Until now, the diagnosis of infectious diseases and genetic disorders has required costly and time- consuming procedures. We believe our new products will provide improved accuracy, efficiency, and lower cost of these tests, which will benefit the public by making them more affordable in the U.S. and worldwide. ? ?
| Zhang, Zhian; Kermekchiev, Milko B; Barnes, Wayne M (2010) Direct DNA amplification from crude clinical samples using a PCR enhancer cocktail and novel mutants of Taq. J Mol Diagn 12:152-61 |
