Cold-sensitive mutants of Taq DNA polymerase provide a hot start for PCR

Although the thermophilic bacterium Thermus aquaticus grows optimally at 70degreesC and cannot grow at moderate temperatures, its DNA polymerase I has significant activity at 20-37degreesC. This activity is a bane to some PCRs, since it catalyzes non-specific priming. We report mutations of Klentaq (an N-terminal deletion variant) DNA polymerase that have markedly reduced activity at 37degreesC yet retain apparently normal activity at 68degreesC and resistance at 95degreesC. The first four of these mutations are clustered on the outside surface of the enzyme, nowhere near the active site, but at the hinge point of a domain that has been proposed to move at each cycle of nucleotide incorporation. We show that the novel cold-sensitive mutants can provide a hot start for PCR and exhibit slightly improved fidelity.