Single tube genotyping ofGSTM1,GSTT1andTP53polymorphisms by multiplex PCR

Abstract

Glutathione S-transferases (GST) are enzymes involved in the conjugation of a number of human carcinogens, while p53 tumour suppressor gene is the most frequently mutated gene identified till now in human neoplasias. Typically, GSTM1 and GSTT1 genotyping are performed together, with several different protocol described and sometimes with the risk of misclassification due to "false negative", depending on the internal positive control employed. Here, we report a modification of the classical multiplex polymerase chain reaction (PCR) method, allowing the genotyping of GSTM1, GSTT1, together with a polymorphism within the intron 3 of TP53 tumour suppressor gene (a 16 base pairs (bp) duplication) in a single tube, with an appropriate internal positive control. To test the applicability of the method, the frequencies of the deleted alleles of GSTM1 and GSTT1 (null genotypes), and the 16 bp duplication of TP53 gene were assayed in a series of Caucasian DNA samples.