Assessment of DNA damage and repair in specific genomic regions by quantitative immuno-coupled PCR.
Assessment of DNA damage and repair in specific genomic regions by quantitative immuno-coupled PCR.
复制标题
通过定量免疫偶联 PCR 评估特定基因组区域的 DNA 损伤和修复。
DOI：
10.1093/nar/22.12.2351
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发表时间：
1994
影响因子：
14.9
通讯作者：
Wani,AA
中科院分区：
文献类型：
--
作者：
Denissenko,MF;Venkatachalam,S;Yamasaki,EF;Wani,AA
在亚基因组水平上对DNA损伤和修复的精细分析表明，包括人类在内的哺乳动物细胞中DNA修复存在微观异质性。除了成熟的基于Southern杂交的方法来研究基因特异性DNA损伤和修复之外，还评估了利用PCR灵敏度的替代方法。后一种技术依赖于由于模板DNA的损伤而降低的PCR扩增。我们已经开发了一种新的定量分析相结合的选择性恢复的DNA损伤含有基因组片段的PCR扩增。从7，8-二羟基-抗-9，10-环氧-7，8，9，10-四氢苯并[a]芘（anti-BPDE）处理的人皮肤成纤维细胞中分离的DNA与多克隆抗体BP-1免疫沉淀。使用包含H-ras原癌基因密码子12周围149 bp靶区域的引物，通过PCR扩增的靶序列。用纳克量的基因组DNA观察到定量DNA损伤特异性反应。这种方法允许在每6.4kbp ras基因片段小于1anti-BPDE加合物的水平下分析初始DNA损伤。暴露于2 μManti-BPDE的修复熟练的GM 637细胞显示出从H-ras基因片段比从整个基因组更快地去除加合物。在GM 4429着色性干皮病（互补组A）细胞中，基因特异性修复不明显。所建立的技术可以扩展到定量测量任何已知序列的基因组区域中的不同DNA碱基损伤的修复。
Fine analysis of DNA damage and repair at the subgenomic level has indicated a microheterogeneity of DNA repair in mammalian cells, including human. In addition to the well established Southern hybridization-based approach to investigate gene-specific DNA damage and repair, alternative methods utilizing the sensitivity of PCR have been evaluated. The latter technique has relied on decreased PCR amplification due to damage in template DNA. We have developed a novel quantitative assay combining the selective recovery of DNA damage containing genomic fragments with the PCR amplification. DNA isolated from 7,8-dihydroxy-anti-9, 10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene (anti-BPDE) treated human skin fibro-blasts was immunoprecipitated with polyclonal antibody BP-1. Recovered target sequences were amplified by PCR using primers encompassing a 149 bp target region around codon 12 of the H-rasprotooncogene. Quantitative DNA damage specific response was observed with nanogram amounts of genomic DNA. This approach allowed analysis of the initial DNA damage at a level less than 1anti-BPDE adduct per 6.4 kbp ras gene fragment. Repair proficient GM637 cells exposed to 2 μManti-BPDE showed a faster removal of the adducts from the H-rasgene segment than from the genome overall. Gene-specific repair was not apparent in GM4429 xeroderma pigmentosum (complementation group A) cells. The established technique could be extended to the quantitative measurement of the repair of diverse DNA base lesions in any genomic region of known sequence.