Fidelity and predominant mutations produced by deep vent wild-type and exonuclease-deficient DNA polymerases during in vitro DNA amplification.
Denaturing gradient gel electrophoresis (DGGE) was used to examine error rates and mutations induced by native (wt) and exonuclease-deficient (exo-) Deep Vent DNA polymerases during DNA amplification by polymerase chain reaction (PCR), in the presence or absence of the T4 bacteriophage gene 32 protein (gp32).gp32 was found to decrease the error rate of the wt, but not that of the exo-, Deep Vent. The average errors per base duplication for the native form were 8.0 x 10(-5) and 6.0 x 10(-5) in the absence and presence of gp32, respectively. For the exo- form, the error rates were 2.0 x 10(-4) and 2.2 x 10(-4) errors per base duplication in the absence and presence of gp32, respectively. Examination of mutations produced by native Deep Vent showed that A/T to G/C transition predominated, consistent with the results of our earlier studies with DNA polymerases derived from other thermophilic bacteria. These results indicate that PCR with high fidelity can be achieved by using wt Deep Vent in combination with gp32.
Huang
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H
Department of Environmental and Occupational Health, University of Pittsburgh, PA 15238, USA.
Keohavong
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P
D001483:Base Sequence; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D004586:Electrophoresis; D005092:Exonucleases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction / Q000379:methods*; D014764:Viral Proteins / Q000378:metabolism*
D004268:DNA-Binding Proteins; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; C088944:Deep Vent DNA polymerase; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases