摘要
目的 建立毛细管电泳 (CE)联合激光诱导荧光检测 (LIF)技术 ,分析单链构象多态性 (SSCP) ,检测人结直肠癌hMLH1基因点突变的方法。方法 PCR扩增 4 2例散发性结直肠癌患者与 2 0例健康志愿者外周血基因组DNAhMLH1基因 12外显子 ,采用CE LIF进行SSCP分析 ,对异常片段进行测序鉴定 ;研究不同筛分介质 (线性聚丙烯酰胺LPA)浓度、分离温度与分离电压对CE行为的影响。结果 4 2例患者中有 4例 (9.5 2 % )突变 ,CE测序证实为T115 1A的杂合子点突变 ;2 0例健康志愿者未发现突变。较高浓度的LPA(4 %～ 6 % ) ,2 0℃分离温度与 9kV分离电压有助于单链DNA峰分离。结论 调整适宜的LPA浓度、分离温度与分离电压可提高CE分析SSCP的效率 ,应用CE LIF技术SSCP分析检测hMLH1基因点突变快速、高效、重复性好 。
Objective To develop a single-strand conformation polymorphism (SSCP) method for detection of point mutation of hMLH1 gene in colorectal cancer by employing laser-induced fluorescence capillary electrophoresis (LIF-CE).Methods The exon 12 of hMLH1 genomic DNA in peripheral blood from 42 cases of sporadically colorectal cancer patients and 20 healthy individuals was amplified by polymerase chain reaction (PCR).SSCP of the PCR products was analyzed by LIF-CE.The abnormal samples were further confirmed by CE-sequencing.The effects of matrix concentration (linear polyacrylamide LPA),running temperature and running voltage on CE performance were also studied.Results A heterozygosis of mutation were found in four of 42 patients by SSCP analysis employing LIF-CE.CE-sequencing of these samples revealed a T1151A point mutation.No mutation was found in the 20 healthy individuals.The increased LPA concentration (4-6%),lower running temperature (20℃) and higher running voltage(9 kV) may beneficial for the separation of single-stranded DNA fragments.Conclusion Appropriate LPA concentration,running temperature and running voltage may improve CE performance in SSCP analysis.SSCP analysis by LIF-CE is a rapid,efficient and reproductive method for detection of the T1151A mutation in hMLH1 gene in a large number of clinical samples.
出处
《临床检验杂志》
CAS
CSCD
北大核心
2005年第1期25-27,共3页
Chinese Journal of Clinical Laboratory Science
