应用离子对反相高效液相色谱法检测微卫星DNA不稳定

目的　建立一种敏感、稳定、高通量的微卫星 DNA不稳定检测技术。方法　应用离子对反相高效液相色谱法在 2 0名正常人、115例散发性大肠癌患者中检测 5个微卫星位点。正常人取外周静脉血DNA,重复 PCR反应 (标准化参照 )。癌症患者取其癌组织及同源正常组织 DNA,配对分析 PCR产物。结果　 115例大肠癌样本中 17例 (14 .8% )存在微卫星 DNA高度不稳定 ,2 3例 (2 0 .0 % )存在微卫星 DNA低度不稳定。低龄和高龄大肠癌患者中微卫星不稳定 (microsatellite instability,MSI)检出率高于中位年龄患者(P<0 .0 5 ) ;低分化大肠癌患者中 MSI检出率高于中、高分化大肠癌患者 (P<0 .0 5 )。结论　离子对反相高效液相色谱法可用于检测 MSI,并具有敏感、稳定、高通量等特点。

The detection of microsatellite instability by ion-pair reversed-phase high performance liquid chromatography

ObjectiveTo set up a sensitive and stable technique which has high throughout to detect the instability of microsatellite DNA. Methods Genomic DNA extracted from the cancer tissues and their normal tissues were subjected to microsatellite instability(MSI) analysis on five of DNA markers in 115 sporadic colorectal cancers by means of PCR and ion-pair reversed-phase high performance liquid chromatography. Genomic DNA extracted from lymphocytes in blood of 20 normal persons were analysed and used as the standard control. The detection of microsatellite instability by ion-pair reversed-phase high performance liquid chromatography$$$$LIU Xiao-rong 1,2 , WANG Ya-ping 1, ZHANG Jun-ni 1, LI Jin-tian 1, ZHANG Xiao-mei 1, ZHU Ming 1, SHAN Xiang-nian 2. 1(Department of Molecular Biology, Jiangsu Institute of Cancer Research, Nanjing, Jiangsu, 210009 P.R.China.; 2(Department of Genetics, Medical School, Southeast University, Nanjing, Jiangsu, 210009 P.R.China) Abstract ObjectiveTo set up a sensitive and stable technique which has high throughout to detect the instability of microsatellite DNA. Methods Genomic DNA extracted from the cancer tissues and their normal tissues were subjected to microsatellite instability(MSI) analysis on five of DNA markers in 115 sporadic colorectal cancers by means of PCR and ion-pair reversed-phase high performance liquid chromatography. Genomic DNA extracted from lymphocytes in blood of 20 normal persons were analysed and used as the standard control. [Results Seventeen (14.8%) MSI-H and 23(20.0%) MSI-L were found in 115 sporadic colorectal cancers. The rates of MSI in the young patients and old patients were much higher than that in the middle-age patients ( P <0.05). And the rate of MSI in low differentiation group was also much higher than that in high or middle differentiation groups ( P <0.05). Conclusion The method the authors developed is a sensitive and accurate technique to detect MSI and has a high throughput.