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首页> 外文期刊>Chromatographia >A Method for Detection of Point Mutation Combined by Mutagenically Separated PCR with High Performance Liquid Chromatography
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A Method for Detection of Point Mutation Combined by Mutagenically Separated PCR with High Performance Liquid Chromatography

机译：高效液相色谱突变分离PCR结合点突变检测方法

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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.
机译：通过将诱变分离的聚合酶链反应与高效液相色谱相结合，已经开发出一种检测已知点突变的方法。选择亚甲基四氢叶酸还原酶基因（MTHFR）的C677T突变作为模型样品，以评估该方法的可行性。系统优化了MS-PCR的退火温度和HPLC的梯度条件。在优化的条件下，清楚地区分了野生型，纯合突变体和杂合体（C677C，T677T，C677T）这三种基因型，数据与从毛细管电泳（CE）和变性HPLC（DHPLC）获得的数据相同。根据保留时间计算的该方法的相对标准偏差（RSD）为±0.13％（n = 7）。我们的初步结果表明，MS-PCR与HPLC相结合是一种用于已知点突变检测的简单，有效且高度可重复的技术，在大规模临床诊断中可能具有潜在的应用。

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