Effects of the T4 bacteriophage gene 32 product on the efficiency and fidelity of DNA amplification using T4 DNA polymerase.
Two bacteriophage DNA polymerases (Pol), T4 Pol and modified T7 Pol, were used to catalyze DNA amplification in vitro by PCR, and their efficiency and fidelity in DNA amplification were examined in the presence and absence of the T4 bacteriophage gene 32-encoded protein (SSB32). The SSB32 protein significantly improved the efficiency of amplification by T4 Pol. Examination of the amplified DNA by denaturing gradient gel electrophoresis (DGGE) revealed that the protein also reduced the rates of error produced by T4 Pol during PCR, from 6.3 x 10(-6) to 2.0 x 10(-6) errors per base duplication after 10(11)-fold amplification. This protein also improved, but to a lesser extent, the fidelity of modified T7 Pol, from 1.80 x 10(-5) to 1.15 x 10(-5) errors per base duplication. High fidelity polymerase chain reaction (hifi-PCR) is needed for studies requiring isolation of mutant sequences present as only a small fraction of the wild type in the amplified DNA. Although several thermostable Pol are currently available for use in automated PCR, their fidelity was found to be significantly lower than that of the thermosensitive T4 Pol. Therefore, T4 Pol is useful for studies requiring hifi-PCR, although this enzyme needs to be added in the reaction mixture during every cycle of PCR.
Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, PA 15238.
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Sandhu
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Keohavong
D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D014764:Viral Proteins / Q000378:metabolism*
D004268:DNA-Binding Proteins; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; C060035:gp32 protein, Enterobacteria phage T4; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase; D004259:DNA-Directed DNA Polymerase