Nucleic Acids Research, 2003, Vol. 31, No. 21 6139-6147
© 2003 Oxford University Press
Cold-sensitive mutants of Taq DNA polymerase provide a hot start for PCR
DNA Polymerase Technology Inc., 1508 South Grand Avenue, St Louis, MO 63104, USA and Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA
*To whom correspondence should be addressed at Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA. Tel: +1 314 362 3351; Fax: +1 253 369 3024; Email: wayne{at}barnes1.wustl.edu
Correspondence may also be addressed to Milko B. Kermekchiev. Tel: +1 314 771 5566; Fax: +1 314 771 5581; Email: milko@klentaq.com
Although the thermophilic bacterium Thermus aquaticus grows optimally at 70°C and cannot grow at moderate temperatures, its DNA polymerase I has significant activity at 20–37°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°C yet retain apparently normal activity at 68°C and resistance at 95°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.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. B. Kermekchiev, L. I. Kirilova, E. E. Vail, and W. M. Barnes Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples Nucleic Acids Res., April 1, 2009; 37(5): e40 - e40. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. V. Lebedev, N. Paul, J. Yee, V. A. Timoshchuk, J. Shum, K. Miyagi, J. Kellum, R. I. Hogrefe, and G. Zon Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance Nucleic Acids Res., November 1, 2008; 36(20): e131 - e131. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Barker, S. Oehler, and B. Muller-Hill "Cold-Sensitive" Mutants of the Lac Repressor J. Bacteriol., March 1, 2007; 189(5): 2174 - 2175. [Abstract] [Full Text] [PDF]
|
|