摘要
目的 建立一种以多重 PCR-高效液相色谱 (high performance liquid chromatography,HPL C)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 ML H1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 ML H1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- HPL C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。
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.
出处
《中华医学遗传学杂志》
CAS
CSCD
2003年第6期517-521,共5页
Chinese Journal of Medical Genetics
基金
江苏省卫生厅重大研究项目基金(H9805
H200207)
江苏省卫生厅重点人才基金(RC2002070)~~
关键词
高效液相色谱
基因错配
基因修复
基因大片段缺失
多重聚合酶链反应
大肠癌
multiplex polymerase chain reaction
high performance liquid chromatography
mismatch repair genes
mutation
deletion
colorectal cancer
