变性高效液相色谱法筛检hMLH1和hMSH2微小突变技术

目的:建立基于变性高效液相色谱法(DHPLC)的快速筛检错配修复基因hMLH1和hMSH2微小突变的技术平台。方法:自行设计PCR扩增hMLH1和hMSH2各外显子的引物,应用DHPLC检测26个遗传性非息肉病性结直肠癌(HNPCC)家系的先证者hMLH1和hMSH2种系微小突变,并与先前进行的DNA直接测序结果相比较。结果:hMLH1与hMSH2各外显子的PCR扩增引物,均能很好地扩增出相应的外显子及剪接区;DHPLC检出了所有已知突变,突变阳性筛检与阴性筛检的灵敏度和特异性均为100%;hMLH1的扩增子12A和hMSH2的扩增子2、3、7、5中相应外显子的剪接区跨越2个温度,而且相差较大(2.2-8.5℃):与DNA直接测序相比较,DHPLC具有快速、高效、低劳动强度、费用低、人为误差小、灵敏度和特异性高等优点。结论:基于DH- PLC的突变筛检平台,能够有效地筛检hMLH1和hMSH2微小突变,并具有较高的费用效率比。

Screening of Minor Mutation of hMLH 1 and hMSH2 Based on Denatured HPLC

Objective:To establish a method based on denatured high performance liquid chromatography(DHPLC)and quickly detect minor mutation of mismatch repair gene hMLH1 and hMSH2.Methods:The primers to amplify all exons of hMLH1 and hMSH2 were designed by ourselves.Polymerase chain reaction(PCR)and DHPLC were deployed to screen minor mutation of hMLH1 and hMSH2.The results were compared with previous results of DNA sequencing to screen mutation of these genes.Results:All primers of hMLH1 and hMSH2 amplified corresponding exons and splicing regions of these genes very well.DHPLC detected all known mutations of these genes and sensitivity and specificity of screening mutation and nonmutation were all 100 %.Two temperatures were used to screen the mutation in some exons and splicing regions of hMLH1 and hMSH2 such as amplicon 12A of hMLH1 and amplicon 2,3,5 and 7 ofhMSH2.In contrast with DNA sequencing,DHPLC was quicker,highly efficient,with low work intensity,low-cost,low personal error,high sensitivity and specificity.Conclusion:The platform based on DHPLC can efficiently screen minor mutation of hMLH1 and hMSH2,and has high cost efficiency.