cDNA代表性差异分析法筛选二羟环氧苯并芘致癌相关基因片段

Isolation of Genes Related to Malignant Transformation Induced by Dihydroxyepoxy Benzo Pyrene with cDNA Representational Difference Analysis

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。

Objective To isolate the differentially expressed genes between transformed cells by dihydroxyepoxy benzo pyrene and controls.Methods mRNAs from these cells were extracted and double stranded cDNAs were prepared.Double stranded cDNAs were digested with Sau3A I,adaptor ligated,and amplified by PCR.cDNA from dihydroxyepoxy benzo pyrene-treated cells was used as a 'tester',and cDNA from controls as a 'driver',driving the process of subtraction.Results Five differential cDNA fragments were isolated by cDNA representational difference analysis (cDNA-RDA) after three rounds of subtractive hybridization.They showed five distinctive bands below 210bp on agarose gel.While these differential cDNA fragments were hybridized to total RNA by Northern Blot,it was found that they really came from the BPDE-treated cells.Conclusions cDNA-RDA was an effective,sentitive and specific technique to screen the candidates of genes related to chemical carcinogenesis.