A POLYMERASE CHAIN REACTION-BASED METHOD TO DETECT CISPLATIN ADDUCTS IN SPECIFIC GENES
A POLYMERASE CHAIN REACTION-BASED METHOD TO DETECT CISPLATIN ADDUCTS IN SPECIFIC GENES
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DOI：
10.1093/nar/19.22.6209
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发表时间：
1991-11-25
影响因子：
14.9
通讯作者：
EASTMAN, A
中科院分区：
文献类型：
--
作者：
JENNERWEIN, MM;EASTMAN, A
DNA中的每一个大的损伤都可能抑制Taq DNA聚合酶，从而减少聚合酶链反应中产生的扩增。我们研究了使用这种抑制来量化抗癌药物顺铂产生的DNA损伤的可行性。产物通过电泳然后溴化乙锭染色来检测。通过在扩增反应中加入[P-32]dCTP并随后评估掺入的放射性来进行定量。在体外铂化DNA基因组DNA至确定的水平，并用产生150、750或2，000个碱基对片段的引物扩增。PCR的抑制程度与每个大小片段中DNA铂化的预测水平一致，表明聚合酶被每个顺铂诱导的损伤抑制。该方法用于检测顺铂诱导的CHO细胞腺嘌呤磷酸核糖转移酶基因的损伤。将细胞与0-125 μ M顺铂一起孵育2小时，纯化DNA并进行PCR。在用75 μ M顺铂孵育的细胞的DNA中观察到2kbp片段扩增的显著减少。抑制的程度与原子吸收法测量的整个基因组中DNA损伤的数量密切相关。在150个碱基片段的扩增中没有检测到变化，因此可以将其用于归一化DNA样品之间任何变化的数据。该检测方法与目前用于分析基因特异性损伤的其他方法具有相同的灵敏度。该测定法的优点在于，它避免了对特异性核酸内切酶复合物的需要，以识别和切割DNA加合物，如以前在分析特定基因组序列中的损伤时所需的。
Every bulky lesion in DNA can potentially inhibit the Taq DNA polymerase and thereby decrease the amplification produced in the polymerase chain reaction. We investigated the feasibility of using this inhibition to quantify DNA lesions produced by the anticancer drug cisplatin. Products were detected by electrophoresis followed by ethidium bromide staining. Quantitation was obtained by including [P-32]dCTP in the amplification reaction and subsequently assessing the incorporated radioactivity. Hamster genomic DNA was platinated in vitro to defined levels and amplified with primers that produce either a 150, 750 or 2,000 base pair fragment. The degree of inhibition of PCR agreed with the predicted level of DNA platination in each size of fragment, suggesting that the polymerase was inhibited by every cisplatin-induced lesion. This method was used to detect cisplatin-induced lesions in the adenine phosphoribosyltransferase gene of CHO cells. Cells were incubated with 0-125-mu-M cisplatin for 2 h, the DNA was purified and subjected to PCR. A significant decrease in amplification of the 2 kbp fragment was observed in DNA from cells incubated with cisplatin at 75-mu-M. The degree of inhibition agreed closely with the amount of DNA damage in the overall genome as measured by atomic absorption. No change was detected in amplification of the 150 base fragment which can therefore be used to normalize data for any variations between DNA samples. This assay has the same sensitivity as other methods currently used for the analysis of gene-specific damage. The advantage of this assay is that it obviates the need for specific endonuclease complexes to recognize and cleave DNA adducts as previously required when analyzing damage in specific genomic sequences.