Effect of DNA damage on PCR amplification efficiency with the relative threshold cycle method
Effect of DNA damage on PCR amplification efficiency with the relative threshold cycle method
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DOI：
10.1016/j.bbrc.2004.08.168
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发表时间：
2004-10-22
影响因子：
3.1
通讯作者：
Denvir, J
中科院分区：
文献类型：
--
作者：
Sikorsky, JA;Primerano, DA;Denvir, J
用于量化DNA损伤的聚合酶停止测定假设单个病变足以阻止聚合酶的进展。为了测试特异性病变对PCR扩增效率的影响，我们使用real-time PCR扩增了含有正常或修饰DNA碱基的90个碱基寡核苷酸，并测定了每个模板的相对阈值周期扩增效率。我们发现，虽然含有单个8-氧-7,8-二氢-2'-脱氧鸟苷（8-oxodG）的模板的扩增效率没有明显受到干扰，但单个8-氧-7,8-二氢-2'-脱氧腺苷、碱基或顺式胸苷二聚体的存在会显著降低扩增效率。此外，虽然含有两个相隔13个碱基的8-oxodGs的模板和未修饰的模板扩增，但两个串联8-oxodGs的存在极大地阻碍了扩增。从这些发现，我们得出结论，聚合酶进展的减少取决于损伤的类型和损伤在模板内的相对位置。(C) 2004爱思唯尔公司版权所有。
Polymerase stop assays used to quantify DNA damage assume that single lesions are sufficient to block polymerase progression. To test the effect of specific lesions on PCR amplification efficiency, we amplified synthetic 90 base oligonucleotides containing normal or modified DNA bases using real-time PCR and determined the relative threshold cycle amplification efficiency of each template. We found that while the amplification efficiencies of templates containing a single 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were not significantly perturbed, the presence of a single 8-oxo-7,8-dihydro-2'-deoxyadenosine, abasic site, or a cis-syn thymidine dimer dramatically reduced amplification efficiency. In addition, while templates containing two 8-oxodGs separated by 13 bases amplified as well as the unmodified template, the presence of two tandem 8-oxodGs substantially hindered amplification. From these findings, we conclude that the reduction in polymerase progression is dependent on the type of damage and the relative position of lesions within the template. (C) 2004 Elsevier Inc. All rights reserved.