A quadruplex tetra-primer ARMS-PCR method for the simultaneous detection of TP53 Arg72Pro, IVS3 16bp Del/Ins and IVS6+62A>G, and NQO1 C609T polymorphisms.
The apoptotic pathway has been shown to be crucial in the development of cancers in addition to a variety of neurodegenerative disorders. The tumor suppressor gene (TP53) encodes p53, the central protein in the apoptotic pathway. The NAD(P)H:quinone oxidoreductase 1, which is encoded by the NQO1 gene and, plays a direct role in apoptosis in addition to its recently discovered role as a regulator for p53. Three most commonly studied polymorphisms that were shown to affect the biochemical functions of p53 protein are the exon 4 Arg72pro, Intron 3 16bp Del/Ins, and Intron 6 A>G polymorphisms. The exon 6 C609T polymorphism was shown to significantly affect NQO1 enzymatic activity. The currently used methods for the separate detection of the four polymorphisms are either slow and laborious or extremely expensive. In this paper, a new highly optimized method for the simultaneous detection of the four polymorphisms is described. The proposed method utilizes 13 primers in a single PCR reaction to detect the four polymorphisms simultaneously based on the principle of tetra-primer ARMS-PCR (also known as PCR-CTPP). The proposed method offers extremely fast, economical, and simple detection. The proposed method was successfully applied to a sample of the Syrian population (n=144), where we found a unique distribution for TP53 polymorphisms that differed from the major ethnic groups. The proposed method is the first to simultaneously detect four polymorphisms including 3 SNPs in a single PCR reaction based on tetra-primer ARMS-PCR or PCR-CTPP, and can serve as an invaluable tool for the investigation of TP53 haplotypes and the combined effects of the TP53 and NQO1 genes with respect to apoptosis and susceptibility for various types of cancers and neurodegenerative disorders.
University of Aleppo, Department of Analytical Chemistry, Faculty of Pharmacy, Aleppo, Syria. BassamL7@yahoo.co.uk
Bassam
B
Lajin
Amal
A
Alachkar
Amir
A
Alhaj Sakur
D001120:Arginine / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers; D016158:Genes, p53; D006801:Humans; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D011392:Proline / Q000235:genetics*; D013593:Syria
D017931:DNA Primers; D001120:Arginine; D011392:Proline; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human