DETECTION OF PLATINUM-DNA ADDUCTS BY P-32 POSTLABELING
DETECTION OF PLATINUM-DNA ADDUCTS BY P-32 POSTLABELING
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DOI：
10.1093/nar/23.8.1300
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发表时间：
1995-04-25
影响因子：
14.9
通讯作者：
SARIS, CP
中科院分区：
文献类型：
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作者：
BLOMMAERT, FA;SARIS, CP
我们建立了一种灵敏的P-32后标记方法，用于在体外和体内检测DNA中双功能链内交联d（Pt-GpG）和d（Pt-ApG）。酶消化DNA后，使用强阳离子交换色谱法从未铂化的产物中纯化带正电荷的铂加合物。随后，样品用氰化物脱铂，因为铂化的二核苷酸是多核苷酸激酶的非常差的底物。使用Sep-pak C18柱除去过量的氰化物，随后对所得的二核苷单磷酸d（GpG）和d（ApG）进行后标记。通过组合TLC和HPLC程序对标记后混合物进行分析。良好的相关性与现有的方法（原子吸收光谱法，免疫细胞化学和ELISA）被发现在体外和体内顺铂处理的DNA样品。在10 μ g DNA样品中，检测限为1加合物/10（7）个核苷酸。
We developed a sensitive P-32-postlabeling method for the detection of bifunctional intrastrand crosslinks d(Pt-GpG) and d(Pt-ApG) in DNA in vitro and in vivo. After enzymatic digestion of DNA the positively charged platinum adducts were purified from unplatinated products, using strong cation exchange chromatography. Subsequently the samples were deplatinated with cyanide, because platinated dinucleotides are very poor substrates for polynucleotide kinase. The excess of cyanide was removed using Sep-pak C18 cartridges, and the resulting dinucleoside monophosphates d(GpG) and d(ApG) were subsequently postlabelled. Analysis of the postlabelling mixture was performed by a combined TLC and HPLC-procedure. Good correlations with existing methods (AAS, immunocytochemistry and ELISA) were found in DNA samples treated in vitro and in vivo with cis- or carboplatin. The detection limit of the assay was 1 adduct/10(7) nucleotides in a 10 mu g DNA sample.