ABSTRACT
Objective To develop an effective real time PCR method for genotyping mitochondrial aldehyde dehydrogenase-2(ALDH2)Glu504Lys polymorphism based on the hydrolysis probes.Methods The Mg~(2+)and probe concentration were optimized,the precision and sensitivity were also checked.The genotypings by this method were confirmed by the direct sequencing of amplified PCR products.Results The optimized Mg~(2+)and probe concentration were 2.5 mmol/L and 1.0 ?mol/L,respectively.The inter- group(n=20)and intra-group(n=20)CVs of Ct were 1.38% and 1.48 %,respectively.The method could detect human DNA in the range of 5.0?10~2-5.0 ?10~6 pg per 25 ?l reaction.The results from 150 individuals by this genotyping method are in full concordance with that by direct PCR products sequencing.Conclusion The combined merits of reliability,flexibility and simplicity should make this method suitable for routine clinical testing and cost-efficient large-scale genotyping.