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目的 :构建人胃癌抑制消减cDNA文库 ,为进一步大批量筛选、克隆胃癌特异性表达的基因奠定基础。方法 :从胃癌和癌旁正常组织分离poly(A) +RNA ,经反转录后 ,利用抑制消减杂交(SSH)方法 ,通过两轮杂交和两次抑制PCR构建了两种组织间差异表达基因的cDNA消减文库。结果与结论 :挑取 80 0个克隆进行PCR扩增检测是否有插入片段 ,结果显示其中 750个克隆有插入片段 ,片段大小范围为 2 50～ 70 0bp。为进一步大批量筛选、克隆胃癌特异性表达的基因奠定了基础。
OBJECTIVE: To construct a human gastric cancer suppression subtractive cDNA library and lay the foundation for further mass screening and cloning of gastric cancer-specific genes. METHODS: Poly (A) + RNA was isolated from gastric cancer tissues and adjacent normal tissues. After reverse transcriptase, two inter-tissue differentially expressed genes were constructed by two rounds of hybridization and two inhibition PCR using suppression subtractive hybridization (SSH) CDNA subtractive library. RESULTS AND CONCLUSION: A total of 80 0 clones were selected for PCR amplification to detect whether there are inserted fragments. The results showed that 750 clones had inserted fragments ranging in size from 50 to 70 0 bp. In order to further mass screening, cloning gastric cancer-specific expression of the gene laid the foundation.