Cold-sensitive mutants of Taq DNA polymerase provide a hot start for PCR.
Although the thermophilic bacterium Thermus aquaticus grows optimally at 70 degrees C and cannot grow at moderate temperatures, its DNA polymerase I has significant activity at 20-37 degrees C. 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 37 degrees C yet retain apparently normal activity at 68 degrees C and resistance at 95 degrees C. 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.
Kermekchiev
Milko B
MB
DNA Polymerase Technology Inc., 1508 South Grand Avenue, St Louis, MO 63104, USA.
Tzekov
Anatoly
A
Barnes
Wayne M
WM
D001665:Binding Sites; D020134:Catalytic Domain; D003080:Cold Temperature; D004261:DNA Replication; D004795:Enzyme Stability; D015723:Gene Library; D006358:Hot Temperature; D016296:Mutagenesis / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D019914:Taq Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013824:Thermus / Q000201:enzymology* / Q000235:genetics
D019914:Taq Polymerase