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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。
OBJECTIVE: To establish a technology platform for rapid screening of minor mutations of hMLH1 and hMSH2 based on denaturing high performance liquid chromatography (DHPLC). Methods: Primers for exon3 of hMLH1 and hMSH2 were designed by PCR. Minimal mutations of hMLH1 and hMSH2 in 26 hereditary nonpolyposis colorectal cancer (HNPCC) pedigrees were detected by DHPLC. The results of direct DNA sequencing were compared. Results: The exons and splice regions of hMLH1 and hMSH2 exon were all amplified well. All the known mutations were detected by DHPLC, and the positive and negative mutations were detected by DHPLC Sensitivity and specificity were both 100%. The splicing region of the corresponding exons in amplicons 12A, hMLH1 and amplicons 2, 3, 7, and 5 of hMSH2 spanned two temperatures and differed greatly (2.2-8.5 ° C ): Compared with DNA direct sequencing, DHPLC has the advantages of fast, high efficiency, low labor intensity, low cost, small human error, high sensitivity and specificity. Conclusion: The mutation screening platform based on DH-PLC can effectively screen small mutations of hMLH1 and hMSH2 with high cost-efficiency ratio.