核酸序列水平定位DNA损伤的实验研究

Experiment Research on the Location of DNA Lesion in the Nucleic Acid Sequence

目的研究在核酸序列水平定位DNA损伤的方法。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。酶切基因组DNA构建损伤模型,应用依赖随机化末端连接物PCR(RDPCR)进行Southern blot,对产物进行测序。结果酶切DNA的RDPCR产物在相应位置出现杂交条带,测序证实损伤位于Hinf在k-ras外显子2的酶切位点。结论将RDPCR和测序技术结合在一起,可在核酸水平上准确定位DNA损伤的碱基位置。

Objective To research on establishing a method that can detect the DNA lesion at the level of nucleic acid. Methods The TK6 cell was cultured and treated, the genomic DNA was extracted, and the strand cleavage was induced by the endonuclease Hinf Ⅰ . Then the genomic DNA was amplified by randomized terminal linker-dependent PCR （RDPCR）, and hybridized by Southern hybridization with the single-stranded probes of the exon 2 of k-ras gene. The PCR products were sequenced. Results The clear hybridized band from the products of RDPCR was seen at the expectant position cut by Hinf Ⅰ . The sequencing analysis showed that the position of DNA lesion linked by the linker was just the restriction site of Hinf Ⅰ . Conclusion It can he used to detect the DNA lesion at the level of nucleic acid sequence with the combination of sequence and RDPCR technologies.