CAJ | 학술논문

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rTaq聚合酶和高pH 缓冲液( HpH Buffer)的全血改进LATE_PCR( Improved LATE_PCR, imLATE_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。
초린산측서시목전기인다태성검측적주요방법지일,단시기전기적양본제비공작교위번쇄,한제료기재림상검측중적응용。위료간화초린산측서적류정,본연구근거불대칭PCR원리,개진료선성지수취합매련식반응( LATE_PCR)적인물설계방법,증가과량인물적장도화농도,병결합전혈직접확증기술,건립료기우보통rTaq취합매화고pH 완충액( HpH Buffer)적전혈개진LATE_PCR( Improved LATE_PCR, imLATE_PCR)방법。고찰료방법적최우확증체계、혈액항응제대기영향이급전혈모판량。채용단관、일보법직접확증출단련측서모판,성공지대24례림상혈양적을순탈경매기인다태성진행료검측,검측결과가용우지도림상개체화용약。24례양본적기인형분별위ADH1B위점AA순합6례、AG잡합14례、GG순합4례； ADH1C위점GG순합20례、AG잡합4례、AA순합0례。
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 rTaq 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 ADH1B locus of 24 samples were 6 cases of AA homozygote, 14 cases of AG heterozygote, and 4 cases of GG homozygote. The genotypes of ADH1C were 20 cases of GG homozygote, 4 cases of AG heterozygote, and no cases of AA homozygote.