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

Abstract

Abstract Pyrosequencing is one of principal methods to detect genetic polymorphism currently, but the complicated sample pretreatment procedure limits its application in clinical tests. In order to simplify the 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 by increasing the length and the concentration of the excess primer and using whole blood as template for direct amplification, and established an improved LATE-PCR (imLATE-PCR) method on the basis of common rTaq polymerase and high-pH buffer (HpH Buffer). The amplification conditions and the amount of whole blood template were optimized and the influence of blood anticoagulant was investigated. By amplifying the whole blood in a single tube with one-step process to get the sequencing template, alcohol dehydrogenase (ADH) gene polymorphisms of 24 clinical blood samples were successfully detected, and the results could served as a guide for individualized medication clinically. The genotypes of ADH1B locus of 24 samples are 6 cases of AA homozygote, 14 cases of AG heterozygote, and 4 cases of GG homozygote. The genotypes of ADH1C are 20 cases of GG homozygote, 4 cases of AG heterozygote, with no cases of AA homozygote.