Huang H, Keohavong P
Department of Environmental and Occupational Health, University of Pittsburgh, PA 15238, USA.
DNA Cell Biol. 1996 Jul;15(7):589-94. doi: 10.1089/dna.1996.15.589.
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.
在存在或不存在T4噬菌体基因32蛋白（gp32）的情况下，利用变性梯度凝胶电泳（DGGE）检测了天然（野生型，wt）和核酸外切酶缺陷型（exo-）Deep Vent DNA聚合酶在聚合酶链反应（PCR）进行DNA扩增过程中诱导的错误率和突变。发现gp32可降低野生型Deep Vent的错误率，但对核酸外切酶缺陷型Deep Vent的错误率没有影响。在不存在和存在gp32时，天然形式每碱基复制的平均错误数分别为8.0×10⁻⁵和6.0×10⁻⁵。对于核酸外切酶缺陷型，在不存在和存在gp32时，每碱基复制的错误率分别为2.0×10⁻⁴和2.2×10⁻⁴。对天然Deep Vent产生的突变进行检测表明，A/T到G/C的转换占主导，这与我们早期对源自其他嗜热细菌的DNA聚合酶的研究结果一致。这些结果表明，通过将野生型Deep Vent与gp32联合使用，可以实现高保真PCR。
