定量多重PCR-高效液相色谱分析错配修复基因大片段缺失

目的 建立一种以多重PCR-高效液相色谱(high performance liquid chromatography，HPLC)分析为基础的错配修复基因DNA大片段缺失检测技术。方法 设计合成35对引物，分8个多重PCR反应扩增MSH2和MLHl基因的35个外显子，PCR产物经高效液相色谱半定量分析，确定各外显子拷贝数。(1)双盲法分析阴性和阳性对照样本，完成方法学可靠性检验。(2)分析14例遗传性非息肉性大肠癌患者外周血细胞DNA和13例散发性大肠癌患者癌组织细胞DNA样本，筛查MSH2和．MLHl基因DNA大片段缺失。结果 (1)稳定检出阳性对照样本的DNA大片段缺失；(2)在筛查样本检出2例新的MSH2基因DNA大片段缺失，分别为MSH2基因外显子7遗传性缺失和MSH2基因外显子1～6体细胞性缺失。结论 多重PCR—HPLC分析系统可以作为突变基因分析系统的一个重要补充，在基因DNA大片段缺失检测中发挥作用。

Large deletion in mismatch repair genes uncovered by quantitative multiplex PCR-high performance liquid chromatography system

Objective Establishing a new method on the basis of multiplex PCR-high performance liquid chromatography(HPLC) for screening a large deletion in mismatch repair genes. Methods Thirty-five pairs of primers were used to amplify all 16 exons of MSH2 and all 19 exons of MLH1 gene in 8 multiplex PCR. The products of multiplex PCR were analysed for the large deletion with Double Strand DNA Analysis System of HPLC. Firstly, validation of the method was tested on positive and negative controls in blind analysis. Secondly, 14 blood cell DNA samples from hereditary nonpolyposis colorectal cancer (HNPCC) patients and 13 colorectal cancer (CRC) tissues DNA samples from sporadic CRC patients were checked with the new developed method. Results (1) the genomic deletions in all 4 of positive controls were identically uncovered with the new method; (2) a novel germline and a novel somatic large deletions were unveiled in 1/14 HNPCC patients and in 1/13 CRC tissues. Conclusion The method developed on multiplex PCR-HPLC is reliable for uncovering large genomic deletion in mismatch repair genes, and can be taken as a valuable addition to mutation screening system.