Mapping oxidative DNA damage using ligation-mediated polymerase chain reaction technology.
Mapping oxidative DNA damage using ligation-mediated polymerase chain reaction technology.
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DOI：
10.1006/meth.2000.1055
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发表时间：
2000-10
期刊：
影响因子：
4.8
通讯作者：
H. Rodriguez;S. Akman;G. Holmquist;G. Wilson;W. J. Driggers;S. Ledoux
中科院分区：
文献类型：
--
作者：
H. Rodriguez;S. Akman;G. Holmquist;G. Wilson;W. J. Driggers;S. Ledoux
活性氧诱导 DNA 中氧化碱基形成药典。通过使用来自大肠杆菌的两种碱基切除修复糖基化酶、Fpg 糖基化酶和核酸内切酶 III 的组合进行消化，可以在这些修饰碱基的大部分位点上切割 DNA。通过基因组测序技术、连接介导的聚合酶链式反应 (LMPCR)，可以沿着测序凝胶放射自显影图以核苷酸分辨率绘制所得糖基酶依赖性 5'-磷酰基末端的频率。在培养的大鼠细胞中，线粒体 DNA 中内源氧化碱基的频率足够高，大约每 100 kb 1 个氧化碱基，可以通过 LMPCR 从 0.1 微克总细胞 DNA 制剂中直接定位。核 DNA 的内源性氧化碱基损伤频率较低，无法从 1 微克总细胞 DNA 制剂中进行绘制。对 300 µg 限制性内切酶消化的基因组 DNA 中的 PGK1 和 p53 基因进行制备性凝胶电泳，结果显示这些基因富集了 25 倍，并且在内切酶消化后进行 LMPCR，给出了足够的信号来绘制用 50 µM H2O2 处理的人类细胞的氧化碱基频率。
Reactive oxygen species induce a pharmacopoeia of oxidized bases in DNA. DNA can be cleaved at most of the sites of these modified bases by digestion with a combination of two base excision repair glycosylases from Escherichia coli, Fpg glycosylase, and endonuclease III. The frequency of the resulting glycosylase-dependent 5'-phosphoryl ends can be mapped at nucleotide resolution along a sequencing gel autoradiogram by a genomic sequencing technique, ligation-mediated polymerase chain reaction (LMPCR). In cultured rat cells, the frequency of endogenous oxidized bases in mitochondrial DNA is sufficiently high, about one oxidized base per 100 kb, to be directly mapped from 0.1 microg of total cellular DNA preparations by LMPCR. Nuclear DNA has a lower frequency of endogenous oxidative base damage which cannot be mapped from 1-microg preparations of total cellular DNA. Preparative gel electrophoresis of the PGK1 and p53 genes from 300 microg of restriction endonuclease-digested genomic DNA showed a 25-fold enrichment for the genes and, after endonuclease digestion followed by LMPCR, gave sufficient signal to map the frequency of oxidized bases from human cells treated with 50 microM H2O2.