应用依赖随机化末端连接物聚合酶链反应技术研究重铬酸钾对大鼠肺p53基因DNA损伤

Applying randomized terminal linker-dependent PCR to detect the DNA damage of p53 gene in rat

应用依赖随机化末端连接物PCR(RandomizedTerminalLinker-dependentPCR ,RDPCR)技术于体内试验的研究 ,从而检测特定基因的DNA损伤。腹腔注射重铬酸钾染毒大鼠 ,提取肺组织基因组DNA ,经p5 3基因外显子 7特异性引物P1重复直线延伸后与接头 (Linker)连接 ,用基因特异性引物P2和连接物引物经PCR扩增 ,电泳、转印 ,再与地高辛标记的探针杂交后显色。结果显示 ,在 2 0 .0mg kg和 40 .0mg kg剂量下 ,出现了两条清晰的杂交带 ,表明重铬酸钾可引起大鼠肺p5 3基因外显子 7的DNA损伤 ,且有两个损伤位点。此结果有助于进一步探讨六价铬化合物的致突变机制 。

In order to use the technique of Randomized Terminal Linker-dependent PCR(RDPCR) to detect DNA lesions of specific gene in vivo, rats were administered with potassium dichromate by i.p.injection and genomic DNA of lung were extracted. Single-stranded products were made by repeated primer extension, these products were ligated to a linker and thus amplified by primer P2 and PL. The final PCR products were detected by electrophoresis and Southern hybridization with DIG-labeled probe. The results showed that two hybridization bands for extron 7 of p53 gene were found with doseage of 20.0 mg/kg and 40.0 mg/kg indicating the DNA lesions of extron 7 of p53 gene, by potassium dichromate and there were two DNA lesion cites. The findings from this study provide evidences both for further investigation of mutational mechanism of hexavalent chromium and expansion on using RDPCR in toxicology.