DNA damage reduces Taq DNA polymerase fidelity and PCR amplification efficiency
DNA damage reduces Taq DNA polymerase fidelity and PCR amplification efficiency
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DOI：
10.1016/j.bbrc.2007.01.169
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发表时间：
2007-04-06
影响因子：
3.1
通讯作者：
Denvir, James
中科院分区：
文献类型：
--
作者：
Sikorsky, Jan A.;Primerano, Donald A.;Denvir, James
DNA损伤阻断DNA聚合酶进展并增加错误编码。在这项研究中，我们评估了特定病变对Taq DNA聚合酶保真度和扩增效率的影响。在8-氧代-7，8-二氢-2 '-脱氧鸟苷（8-oxodG）存在下，Taq DNA聚合酶插入dCMP和较小程度的dAMP。8-氧代-7，8-二氢-2 '-脱氧腺苷（8-oxodA）指导dTMP的掺入，并引起在其他系统中未观察到的明显n - 1缺失。脱碱基损伤的存在导致dAMP掺入和n - 1缺失。此外，我们还引入了平均修饰效率（MME）作为一种更精确的方法来确定受损模板的PCR扩增效率。使用这种方法，我们能够量化含有8-oxodG（单个或多个）、8-oxodA或无碱基位点的模板的扩增效率的降低。由于MME方法可以检测到扩增效率的小幅降低，因此它可能有助于比较环境降解或存档DNA样本的损伤程度。(c)2007爱思唯尔公司All rights reserved.
DNA damage blocks DNA polymerase progression and increases miscoding. In this study, we assessed the effects of specific lesions on Taq DNA polymerase fidelity and amplification efficiency. In the presence of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), Taq DNA polymerase inserted dCMP and to a lesser extent dAMP. 8-Oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA) instructed the incorporation of dTMP and caused a pronounced n - 1 deletion not observed in other systems. The presence of an abasic lesion led to dAMP incorporation and n - 1 deletions. In addition, we introduce the mean modified efficiency (MME) as a more precise method for determining PCR amplification efficiency of damaged templates. Using this method, we were able to quantify reductions in amplification efficiency of templates containing 8-oxodG (single or multiple), 8-oxodA, or abasic sites. Because the MME method can detect small reductions in amplification efficiency, it may be useful in comparing the extent of damage in environmentally degraded or archival DNA specimens. (c) 2007 Elsevier Inc. All rights reserved.