Detection of the NQO1 C609T polymorphism by a simple one step Tri-primer Amplification Refractory Mutation System-PCR method

Abstract

NAD(P)H:quinone oxidoreductase 1 (NQO1) is an enzyme with antioxidant functions. It catalyzes the two-electron reduction of quinones and nitrogen-oxides, reducing the formation of reactive oxygen species by decreasing one-electron reductions. A common polymorphism in the NQO1 gene involves the substitution of cytosine with thymine (609 C→T), giving rise to a proline to serine substitution, which decreases NQO1 enzymatic activity. The C609T polymorphism was shown to be important in a broad range of biomedical fields, especially cancer research. In this paper, we describe a new method for the detection of C609T based on the Amplification Refractory Mutation System – Polymerase Chain Reaction method (ARMS-PCR). Two specific forward primers differing in length and in the 3′ base, and one common reverse primer were combined in a single PCR reaction. Genotype adscription was based on the amplification of either one or both of two specific amplicons (75 bp for the C allele and 90 bp for the T allele) as shown following agarose gel electrophoresis. The method was validated using a PCR-RFLP method. The proposed method was used to characterize the genotype distribution of the C609T polymorphism in a sample population from Aleppo, Syria, where 2.8% of the sample population were found to be homozygous for the T allele, and 78.8% were found to be homozygous for the C allele.