【摘要】 目的简化RDPCR建立程序,并在核酸序列水平精确定位DNA损伤。方法培养TK6细胞,抽提基因组DNA,制备k-ras基因外显子2的单链探针。扩增k-ras基因外显子2(以下称短片段)。酶切短片段和基因组DNA构建损伤模型,前者经依赖随机化末端连接物PCR(RDPCR)扩增,每一步产物作凝胶电泳,终产物与单链探针杂交显色,并进行测序;后者以短片段建立的实验条件进行RDPCR扩增,产物与单链探针杂交显色,并进行测序。结果凝胶电泳可见酶切短片段在RDPCR中每一步骤的目的产物条带;酶切短片段和酶切基因组DNA的RDPCR产物均在相应位置出现杂交条带,测序证实损伤均位于HinfI在k-ras外显子2上的酶切位点。结论短片段建立RDPCR的实验条件简单易行;并首次利用RDPCR检测DNA损伤精确定位于核酸序列水平的碱基位置。更多还原

【Abstract】 Objective To simplify the establishing procedure of RDPCR and detect the DNA lesion on the precise position at the level of nucleic acid. Methods The TK6 cell was cultured and its genomic DNA was extracted and prepared to the single-stranded probes of the exon 2 of k-ras gene. The exon 2 of k-ras gene was amplified,called short fragment. The digestion of the genomic DNA and the short fragment were treated by the endonuclease HinfI.To the former, the products come from the every step of RDPCR were imaged after gel electrophoresis(GE) and the end products was taken coloration after southern hybridization with the single-stranded probes of the exon 2 of k-ras gene as well as were sequenced after re-amplified. To the latter, the genomic DNA were treated as the former except the GE. Results From the image of GE,the straps could be seen clearly that come from every step of amplifying of short fragments which were cleaved by the endonuclease HinfI in RDPCR. The clear hybridized bands caused by the products of RDPCR were seen at the expectant position. The sequencing analysis showed that the position was linked by the linker was just the restriction site of HinfI. Conclusion Results indicated that RDPCR could be easily established by the short fragment method. This may be the first time detect the precise position of DNA lesion at the level of nucleic acid by RDPCR.更多还原