全血改进线性指数聚合酶链式反应用于焦磷酸测序检测基因多态性

Genotyping of Alcohol Dehydrogenase Gene by Pyrosequencing Coupled with Improved Linear-after-the-Exponential Polymerase Chain Reaction Using Human Whole Blood as Starting Material

焦磷酸测序是目前基因多态性检测的主要方法之一,但是其前期的样本制备工作较为繁琐,限制了其在临床检测中的应用。为了简化焦磷酸测序的流程,本研究根据不对称PCR原理,改进了线性指数聚合酶链式反应(LATE-PCR)的引物设计方法,增加过量引物的长度和浓度,并结合全血直接扩增技术,建立了基于普通r Taq聚合酶和高p H缓冲液(Hp H Buffer)的全血改进LATE-PCR(Improved LATE-PCR,im LATE-PCR)方法。考察了方法的最优扩增体系、血液抗凝剂对其影响以及全血模板量。采用单管、一步法直接扩增出单链测序模板,成功地对24例临床血样的乙醇脱氢酶基因多态性进行了检测,检测结果可用于指导临床个体化用药。24例样本的基因型分别为ADH1B位点AA纯合6例、AG杂合14例、GG纯合4例;ADH1C位点GG纯合20例、AG杂合4例、AA纯合0例。

Pyrosequencing is one of the important genetic polymorphism detection methods currently,but the complicated pretreatment procedure limits its application in clinical test. To simplify the whole process of pyrosequencing,on the basis of the linear-after-the-exponential-polymerase chain reaction（ LATE-PCR）,we improved the primer design method of LATE-PCR,increased the length and the concentration of the excess primer,applied direct amplification technology with whole blood,and established a whole blood-imLATE-PCR method based on common r Taq polymerase and ＂HpH Buffer＂（ High pH buffer）. The amplification system was optimized,and the influences of blood anticoagulant and the amount of whole blood template were investigated. The single stranded template for the pyrosequencing was obtained by PCR amplification using a single tube in one-step process,and the alcohol dehydrogenase gene polymorphisms of 24 clinical blood samples were then detected successfully. The results could be used to guide clinical individualized medication.The genotypes of ADH1 B locus of 24 samples were 6 cases of AA homozygote,14 cases of AG heterozygote,and 4 cases of GG homozygote. The genotypes of ADH1 C were 20 cases of GG homozygote,4 cases of AG heterozygote,and no cases of AA homozygote.