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Chimeric thermostable DNA polymerases with reverse transcriptase and attenuated 3'-5' exonuclease activity
Biochemistry (2006)
  • Nancy J Schoenbrunner
  • Olga Budker
  • Ellen Fiss
  • David Gelfand
  • Rachel Shahinian
  • Chris Sigua
Abstract
The synthesis of accurate, full-length cDNA from low-abundance RNA and the subsequent PCR amplification under conditions which provide amplicon that contains minimal mutations remain a difficult molecular biological process. Many of the challenges associated with performing sensitive, long RT/PCR have been alleviated by using a mixture of DNA polymerases. These mixtures have typically contained a DNA polymerase devoid of 3'-5' exonuclease, or "proofreading", activity blended with a small amount of an Archaea DNA polymerase possessing 3'-5' exonuclease activity, since reverse transcriptases lack 3'-5' exonuclease activity and generally have low fidelity. To create a DNA polymerase with efficient reverse transcriptase and 3'-5' exonuclease activity, a family of mutant DNA polymerases with a range of attenuated 3'-5' exonuclease activities was constructed from a chimeric DNA polymerase derived from Thermus species Z05 and Thermotoga maritima DNA polymerases. These "designer" DNA polymerases were fashioned using structure-based tools to identify amino acid residues involved in the substrate-binding site of the exonuclease domain of a thermostable DNA polymerase. Mutation of some of these residues resulted in proteins in which DNA polymerase activity was unaffected, while proofreading activity ranged from 60% of the wild-type level to undetectable levels. Kinetic characterization of the exonuclease activity indicated that the mutations affected catalysis much more than binding. On the basis of their specificity constants (kcat/KM), the mutant enzymes have a 5-15-fold stronger preference for a double-stranded mismatched substrate over a single-stranded substrate than the wild-type DNA polymerase, a desirable attribute for RT/PCR. The utility of these enzymes was evaluated in a RT/PCR assay to generate a 1.7 kb amplicon from HIV-1 RNA
Publication Date
Fall October 24, 2006
Citation Information
Nancy J Schoenbrunner, Olga Budker, Ellen Fiss, David Gelfand, et al.. "Chimeric thermostable DNA polymerases with reverse transcriptase and attenuated 3'-5' exonuclease activity" Biochemistry Vol. 45 Iss. 42 (2006)
Available at: http://works.bepress.com/olga_budker/1/