【摘 要】
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Background: Alcohol dehydrogenase-1B (ADH1B) and aldehyde dehydrogenase-2 (ALDH2) are important enzymes in human alcohol metabolism.The genetic polymorphisms of ADH1B 143G>A (ADH1B*1/ADH1B*2) and ALDH
【机 构】
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Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University,Guangz
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Background: Alcohol dehydrogenase-1B (ADH1B) and aldehyde dehydrogenase-2 (ALDH2) are important enzymes in human alcohol metabolism.The genetic polymorphisms of ADH1B 143G>A (ADH1B*1/ADH1B*2) and ALDH2 1510G>A (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 143G>A and ALDH2 1510G>A 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.
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