This proposal is based on the premise that a hot start is beneficial or necessary for efficient and reliable amplification of most PCR reactions, particularly the most demanding PCR reactions. A heat switch can be added to PCR protocols by any of several manual or built-in methods, all of which have drawbacks such as narrow conditions, cross-contamination, and/or lack of applicability to long PCR. The investigators propose development of a Taq polymerase mutant that is heat-switchable by virtue of cold-sensitive mutations that will be introduced. Proposed is the development of a mutant Taq DNA polymerase that is much less active than the wild-type at room (reaction setup) temperature, yet normally active at 65-70 degrees, and still resistant to 95 degrees. The investigators will sequence the mutant genes (expected to harbor more than one mutation each from the mutagenesis) and test single mutations to identify ones that confer cold-sensitivity. Finally they will purify mutant and combined-mutant enzymes, to see if demanding PCR (short and long) that requires a hot start can be improved by their use under a broad range of conditions.
|Kermekchiev, Milko B; Tzekov, Anatoly; Barnes, Wayne M (2003) Cold-sensitive mutants of Taq DNA polymerase provide a hot start for PCR. Nucleic Acids Res 31:6139-47|