Rapid simultaneous genotyping of polymorphisms in ADHIB and ALDH2 using high resolution melting assay
作者单位：Department of Medical GeneticsSchool of Basic Medical SciencesSouthern Medical University
会议名称：《第十二次全国医学遗传学学术会议》
会议日期：2013年
学科分类：0710[理学-生物学] 07[理学] 071007[理学-遗传学]
关 键 词：alcohol dehydrogenase aldehyde dehydrogenase genotyping high resolution melting assay
摘 要：Background:Alcohol dehydrogenase-1B(ADH1B) and aldehyde dehydrogenase-2 (ALDH2) are important enzymes in human alcohol metabolism.The genetic polymorphisms of ADH1B 143GA(ADH1B*1/ADH1B*2) and ALDH2 15100A(ALDH2*1/ALDH2*2) are responsible for serious alcohol related disorders.A rapid,accurate,sensitive and high-throughput assay for genotyping the two genes was developed,and the assay could be useful in the epidemiological studies and risk evaluation of alcohol-related diseases.Methods:In this study,we designed two amplicons with different melting temperature by adding GC-or AT-rich 5’ tails,and the two amplicons include ADH1B 143GA and ALDH2 1510GA respectively.A total of 200 genomic DNA samples from Chinese population were enrolled to evaluate the specificity of the assay.40 samples randomly selected from each of the genotype groups tested by HRM assay were validated by DNA sequencing.The assay reproducibility and detection limit was evaluated by replicate experiments and two-fold serial dilutions of DNA concentration,respectively.Results: All samples were unambiguously distinguished by HRM assay.There was 100%concordance between the results of the HRM assay and DNA sequencing.Meanwhile,the assay has highly reproducibility,and the coefficients of variation were in the range 0.022%to 0.062%.In addition, only 6.25ng of genomic DNA can be detected as well.Conclusion:The method we present is proved to be high-throughput and reliable,which is suitable for rapid simultaneous genotyping of all nine genotype groups of ADH1B and ALDH2 gene in a single closed tube with $0.5 in 2 hours.