Genotyping of Alcohol Dehydrogenase Gene by Pyrosequencing Coupled with Improved Linear_after_the_Exponential Polymerase Chain Reaction Using Human Whole Blood as Starting Material
10.11895/j.issn.0253_3820.140395
- VernacularTitle: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
- Author:
Zheng XIANG
;
Yunlong LIU
;
Xiaoqing XING
;
Yanan CHU
;
Qinxin SONG
;
Guohua ZHOU
- Publication Type:Journal Article
- Keywords:
Whole blood_polymerase chain reaction;
Linear_after_the_exponential_polymerase chain reaction;
Pyrosequencing;
Gene polymorphism;
Ethanolic metabolism
- From:
Chinese Journal of Analytical Chemistry
2015;(1):55-62
- CountryChina
- Language:Chinese
-
Abstract:
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.
