Genetic polymorphism analysis of GSTM1 and GST1

GSTM1 and the GSTT1 genetic polymorphism analyses were determined using multiplex polymerase chain reaction (PCR) method.²⁵ In brief, isolated DNA was amplified in a 50 µL reaction mixture containing 200 µM deoxynucleotide mix, 10x Standard Taq Reaction Buffer, 0.5 mM MgC1₂, 2.5 units Taq DNA polymerase and 50 pmol of each GSTM1 primer: 5’ GAA CTC CCT GAA AAG CTA AAG C and 5’ GTT GGG CTC AAA TAT ACG GTG G; and GSTT1 primers, 5’-TIC CTT ACT GGT CCT CAC ATC TC and 5’-TCA CCG GAT CAT GGC CAG CA. The CYP1A1 gene was co-amplified using the primers 5’-GAA CTG CCA CTT CAG CTG TCT and 5’-CAG CTG CAT TTG GAA GTG CTC as internal controls to prove successful PCR. The PCR conditions consisted of an initial melting temperature of 94°C (5 min) followed by 35 cycles of melting (94°C, 2 min), annealing (59°C 1 min) and extension (72°C 1 min) with a final extension step (72°C) of 10 min. The PCR products of GSTT1, GSTM1, and CYP1A1 genes were analyzed using 2% agarose gel electrophoresis. GSTM1 and GSTT1 genes were detected through the presence or absence of a band at 215 bp (GSTM1) and a band at 480 bp (GSTT1). A band at 312 bp corresponding to CYP1Al gene was always present and used as an internal control for PCR amplification.