A method for detection of point mutation combined by mutagenically separated PCR with high performance liquid chromatography

被引：1

作者：

Fang, NH ^{[1
]}

Lin, L ^{[1
]}

Ren, JC ^{[1
]}

Wu, D ^{[1
]}

机构：

[1] Shanghai Jiao Tong Univ, Dept Chem, Shanghai 200240, Peoples R China

来源：

CHROMATOGRAPHIA | 2005年 / 62卷 / 1-2期

基金：

中国国家自然科学基金;

关键词：

column liquid chromatography; mutagenically separated polymerase chain reaction; mutation detection; C677T methylenetetrahydrofolate reductase gene;

D O I：

10.1365/s10337-005-0572-1

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

A method for detecting a known point mutation has been developed by combining mutagenically separated polymerase chain reaction with high performance liquid chromatography. C677T mutation from methylenetetrahydrofolate reductase gene (MTHFR) was chosen as model samples to assess the feasibility of this method. The annealing temperature for MS-PCR and gradient conditions for HPLC were systematically optimized. Under the optimized conditions, three genotypes of wild type, homozygous mutant and heterozygote (C677C, T677T, C677T) were clearly distinguished, and the data are identical to those obtained from capillary electrophoresis (CE) and from denaturing HPLC (DHPLC). The relative standard deviation (RSD) of this method calculated on the basis of retention times is 0.13% (n=7). Our preliminary results demonstrate that MS-PCR combined with HPLC is a simple, effective and highly reproducible technique for known point mutation detection, and may have potential applications in large-scale clinical diagnosis.

引用

页码：99 / 102

页数：4

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