N-ras基因DNA损伤检测方法的建立

Establishing a Method to Detect the DNA Damage of N-ras Gene

目的建立应用依赖随机化末端连接物PCR(RDPCR)技术检测N-ras基因DNA损伤的试验方法。方法采用单引物PCR技术制备N-ras基因外显子1单链探针,酶切构建DNA损伤模型,再经RDPCR扩增后与探针杂交显色。结果单引物PCR技术制备单链探针获得成功,目的基因在相应位置出现了清晰的杂交条带。结论RDPCR技术可定位检测到该酶切DNA损伤位点,表明该方法已成功建立,将有利于其在化学致癌作用机制研究及肿瘤预防领域的应用。

Objective Applying RDPCR to detect the DNA damage of N-ras gene in human. Methods Single-primer PCR was used to prepare single strand （ss） probes of extron 1 of N-ras gene in human. The genomic DNA was digested completely by restriction endonuclease, then amplified by RDPCR and detected by Southern hybridization with the probe. Results The ss probes were successfully prepared by single-primer PCR. The hybridized bands were clearly seen in the expected migration positions. Conclusion The result shows that the method to detect the damaged position of N-ras gene has been established, which would be helpful to further studies on chemical carcinogenesis and on the prevention of tumor.