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目的 检测肿瘤发生发展过程中DNA和染色体的不稳定性及筛选与新的癌基因或抑癌基因相关的分子标志。方法 用 9个随机引物对 5种肿瘤共 12 8个样本进行RAPD分析 ,检测其DNA和染色体的不稳定性。不稳定性的扩增带被切割回收纯化 ,克隆 ,然后被标记为探针作Southern和Northern印迹杂交分析 ,再进一步测序分析。结果 在所有的癌组织标本中胃癌样本 5号和 3号显示出最高的遗传变异。这 5种癌组织中遗传不稳定性的平均检出率分别从 4 2 2 %到 4 9 4 %。每个随机引物的检测能力有极显著差异 ,分别从 2 7%到 6 8%不等。其中引物2最高达 6 8% ,引物 8最低 ,为 2 7%。图 2中的B带为一个单拷贝片段经RFLP分析后发现其在胃癌和结肠癌中是缺失突变 ,测序后 ,经查询是一个新的序列并被GeneBank收录 (登录号AF15 10 0 5 )。进一步研究显示它可能是一个新抑癌基因的调控元件部分 ,含有一个顺式作用的“CACA”启动子 ,一个“GATA”家族序列的增强子和一个转录起始密码子。结论 RAPD与其他方法结合使用能检测肿瘤发生过程中DNA和染色体的不稳定性 ,并筛选与新的癌基因或抑癌基因相关的分子标志。
Objective To detect DNA and chromosomal instability during tumorigenesis and to screen molecular markers associated with novel oncogenes or tumor suppressor genes. Methods A total of 12 8 samples of 5 kinds of tumors were analyzed by RAPD using 9 random primers to detect the DNA and chromosomal instability. Unstable amplification bands were cut and recovered for purification, cloned, and then labeled as probes for Northern and Southern blot analysis followed by further sequencing analysis. Results Gastric cancer samples Nos. 5 and 3 showed the highest genetic variation in all of the cancerous tissue specimens. The average detection rate of genetic instability in these five kinds of cancer tissues was from 42.2% to 494%. The detection of each random primer has a very significant difference, ranging from 27% to 68%. Primer 2 up to 68%, primer 8 lowest, 27%. The B-band in Fig. 2 is a single-copy fragment that was found to be absent in gastric and colon cancers by RFLP analysis. After sequencing, a new sequence was obtained and registered on GeneBank (accession number AF15 10 05). Further studies have shown that it may be part of a regulatory element of a novel tumor suppressor gene that contains a cis-acting “CACA” promoter, an enhancer of the “GATA” family of sequences and a transcription initiation codon. Conclusion The combination of RAPD and other methods can detect DNA and chromosomal instability during tumorigenesis and screen molecular markers associated with novel oncogenes or tumor suppressor genes.