FIDELITY OF DNA-POLYMERASES IN DNA AMPLIFICATION
FIDELITY OF DNA-POLYMERASES IN DNA AMPLIFICATION
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DOI：
10.1073/pnas.86.23.9253
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发表时间：
1989-12-01
影响因子：
11.1
通讯作者：
THILLY, WG
中科院分区：
文献类型：
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作者：
KEOHAVONG, P;THILLY, WG
采用变性梯度凝胶电泳（DGGE）技术对聚合酶链反应（PCR）扩增产物进行分离和纯化。该策略允许直接计数和鉴定由聚合酶的T4、修饰的T7、Klenow片段产生的点突变。通过DGGE将不正确合成的序列与野生型分离为突变体/野生型异源双链体，并使用异源双链体分数计算平均错误率（突变/碱基重复）。在人次黄嘌呤/鸟嘌呤磷酸核糖基转移酶（HPRT）基因的外显子3的104-碱基对低温解链结构域中诱导的错误率为3.4. times. 10-5对于修饰的T7，1.3 ×10-4对于Klenow片段，和2.1 × 10 - 4。10-4在106倍扩增后的Taq聚合酶。T4 DNA聚合酶的错误率不超过3倍。10-6每个碱基重复的错误。对主要突变进行测序，发现是G. cntdot的转换。C到A. cntdot. T表示T4和修饰的T7 DNA聚合酶，和A. T至G. cntdot. C表示Taq聚合酶。Klenow片段诱导了2个和4个碱基对的可能的转换和缺失。
Denaturing gradient gel electrophoresis (DGGE) was used to separate and isolate the products of DNA amplification by polymerase chain reaction (PCR). The strategy permitted direct enumeration and identification of point mutations created by T4, modified T7, Klenow fragment of polymerases. Incorrectly synthesized sequences were separated from the wild type by DGGE as mutant/wild-type heteroduplexes and the heteroduplex fraction was used to calculate the average error rate (mutations per base duplication). The error rate induced in the 104-base-pair low-temperature melting domain of exon 3 of the human hypoxanthine/guanine phosphoribosyltransferase (HPRT) gene was .apprxeq. 3.4 .times. 10-5 for modified T7, 1.3 .times. 10-4 for Klenow fragment, and 2.1 .times. 10-4 for Taq polymerases after a 106-fold amplification. The error rate for T4 DNA polymerase was not more than 3 .times. 10-6 error per base duplication. The predominant mutations were sequenced and found to be transitions of G .cntdot. C to A .cntdot. T for T4 and modified T7 DNA polymerases, and A .cntdot. T to G .cntdot. C for Taq polymerase. Klenow fragment induced both possible transitions and deletions of 2 and 4 base pairs.