Rapid, accurate genotyping of alcohol dehydrogenase-1B and aldehyde dehydrogenase-2 based on the use of denaturing HPLC.
The genotypes of alcohol dehydrogenase-1B (ADH1B) and aldehyde dehydrogenase-2 (ALDH2) are related to alcohol dependence and some human disorders. Rapid, accurate genotyping methodologies for specific polymorphisms of these two genes are needed for molecular screening and testing of alcohol-related problems in populations. A polymerase chain reaction (PCR) composed of two separate amplicons was designed to generate sequences containing the polymorphic site of interest in the ADH1B and ALDH2 genes. The PCR amplicons for each sample were subjected to denaturing high-performance liquid chromatography (DHPLC), analysis performed under partially denaturing conditions as determined by profiling the mixture of a unique homozygous control and a tested sample amplicon. A total of 150 genomic DNA samples were tested to validate this assay by blind analysis. Direct DNA sequencing was performed on samples randomly selected from each of the genotype groups detected by DHPLC profiling. The results indicate 100% concordance between the sequencing analysis and the DHPLC detection. The method we present provides a reliable and fast genotyping procedure for molecular screening of alcohol-related problems.
Li
Liang
L
Department of Medical Genetics, Southern Medical University (formerly: First Military Medical University), Guangdong, PR China.
Mo
Qiu-Hua
QH
Xu
Xiang-Min
XM
D000426:Alcohol Dehydrogenase / Q000235:genetics*; D000444:Aldehyde Dehydrogenase / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019411:Clinical Laboratory Techniques; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005820:Genetic Testing / Q000379:methods; D023281:Genomics; D005838:Genotype; D006720:Homozygote; D006801:Humans; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D004247:DNA; D000426:Alcohol Dehydrogenase; D000444:Aldehyde Dehydrogenase