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目的 采用cDNA代表性差异分析法 ,分离二羟环氧苯并芘诱导恶性转化的细胞与对照组细胞之间的差异表达基因。方法 先从细胞中提取mRNA并合成双链cDNA ,双链DNA以Sau3AI内切酶消化 ,连接接头并经PCR扩增。以BPDE诱导恶变细胞的cDNA为“检测子”、以对照组细胞cDNA为“驱赶子”进行消减杂交。结果 经 3轮消减杂交后分离出 5个cDNA片段 ,在琼脂糖凝胶上清晰显示 2 10bp以下的 5条条带 ,这些片段分别与总RNA作Northern印迹杂交证实它们均来自BPDE诱变的细胞。结论 cDNA代表性差异分析法是筛选化学致癌相关基因的有效、灵敏的方法
OBJECTIVE: To isolate differentially expressed genes between malignant transformed cells and control cells induced by dihydroxybenzophenone by cDNA differential analysis. Methods mRNA was extracted from cells and double-stranded cDNA was synthesized. Double stranded DNA was digested with Sau3AI endonuclease, ligated and amplified by PCR. The cDNA of induced malignant cells was detected by BPDE as a “detector”, and the cDNA of control cells was used as a “drive-by” for subtractive hybridization. Results Five cDNA fragments were isolated after three rounds of subtractive hybridization and five bands below 2 10bp were clearly displayed on agarose gel. Northern blotting of these fragments with total RNA respectively confirmed that they were all from BPDE-mutated cells . Conclusion The cDNA representative difference analysis is an effective and sensitive method for screening chemical carcinogenesis-related genes