Newcomb T G, Allen K J, Tkeshelashvili L, Loeb L A
Department of Pathology, The Joseph Gottstein Memorial Cancer Research Laboratory, University of Washington, Seattle, WA 98195, USA.
Mutat Res. 1999 Jun 1;427(1):21-30. doi: 10.1016/S0027-5107(99)00075-5.
DNA lesions caused by reactive oxygen species (ROS) are considered to be one of the major contributors to DNA damage and mutagenesis. In this study, we developed a modification of allele-specific PCR to detect CC-->TT mutations caused by oxidative damage. These tandem mutations have been previously demonstrated to be indicative of oxygen damage in the absence of UV-irradiation. Using a CC target site in the rat DNA polymerase beta (pol beta) gene and a thermostable restriction enzyme that cuts the wild type sequence but not the TT mutation, we demonstrate that the TT mutation can be preferentially amplified from plasmid DNA damaged by oxygen radicals but not other DNA-damaging agents. We evaluated the potential utility of this assay in screening for mutations in cells and in analyzing those that arise during clonal proliferation in carcinogenesis.
由活性氧（ROS）引起的DNA损伤被认为是导致DNA损伤和诱变的主要因素之一。在本研究中，我们对等位基因特异性PCR进行了改进，以检测由氧化损伤引起的CC→TT突变。这些串联突变先前已被证明在无紫外线照射的情况下可指示氧损伤。利用大鼠DNA聚合酶β（polβ）基因中的CC靶位点和一种能切割野生型序列但不能切割TT突变的热稳定限制性酶，我们证明TT突变可以从被氧自由基损伤的质粒DNA中优先扩增出来，而不能从其他DNA损伤剂损伤的质粒DNA中扩增出来。我们评估了该检测方法在筛选细胞突变以及分析致癌过程中克隆增殖期间出现的突变方面的潜在效用。
