6 mmol/l (NH4)2SO4 and 20.0 mmol/l MgCl2, pH 8.8. After initial denaturation for 3 min at 94°C, 39 cycles were performed for 1 min at 94°C (denaturation), for 1 min at 60°C (annealing) and for 1 min at 72°C (extension), followed by a final step for 5 min at 72°C. The

GSTM1 (215-bp), GSTT1 (480-bp) and β-globin (268-bp) amplified products were visualized by electrophoresis on ethidium-bromide-stained 3% agarose gel (Fig. 1). For deletions Selleckchem PF-01367338 of GSTM1 and GST1 no amplified products can be observed, whereas the β-globin specific fragment confirms the presence of amplifiable DNA in the reaction mixture. Figure 1 Detection of polymerase chain reaction (PCR) amplification of GSTT1 (480 bp fragment), β-globin (268-bp fragment) and GSTM1 (215-bp fragment) genes. Absence of the PCR product indicates the null genotype. Ethidium bromide-stained electrophoresed representative PCR products samples: 100 bp ladder (lane L); absence of null genotypes (lanes 3, 4, 9); GSTT1 -null allele (lanes

2, 5) and GSTM1 -null allele (lanes 1, 2, 5, 6, 7, 8, 10, 11). The GSTP1 Ile 105 Val substitution was detected using the PCR-RFLP approach as the substitution by guanine introduced restriction site that can be recognized by an endonuclease Alw26I. PCR reactions were performed in a total volume of 25 μl of solution containing 10 × PCR buffer (16.6 mmol/l (NH4)2SO4, 20.0 mmol/l MgCl2, pH 8.8, 1.2 μl DMSO, 1.2 μl DTT), 200 μmol/l deoxynucleoside triphosphates, 1 U of KU-57788 chemical structure Taq DNA polymerase, 100 ng of genomic DNA and 25 pmol of GSTP1 primers (forward 5′-GTA GTT TGC CCA AGG TCA AG-3′ and reverse 5′-AGC CAC CTG AGG GGT AAG-3′, GenBank accession no. NM_000852). The reaction started for 3 min at 94°C, followed by 5 cycles of PCR (cycle 1: 94°C for 15 s, 64°C

for 30 s, and 72°C for 1 min) during which the annealing temperature decreased by 1°C for each cycle. This step was followed by 30 cycles of denaturation (for 15 s at 94°C), annealing (for 30 s at 59°C), and extension (for 1 min at 72°C). A final polymerization step (for 5 min at 72°C) was carried out to complete the elongation process and yield a 442-bp fragment. A negative control (PCR without template) was included in each set of PCR second reactions. Each PCR product (10 μl) was digested for 4 hours with the restriction enzyme Alw26I (5 U) and electrophoresed on ethidium-bromide-stained 1.5% agarose gel. The presence of the Ile/Ile allele was detected by 329-, and 113-bp fragments, whereas the Val/Val allele was confirmed by 216-, and 113-bp fragments. The heterozygote Ile/Val allele was characterized by fragments consisting of 329, 216, and 113 bp (Fig. 2) [7]. Figure 2 Cleavage of 442 bp PCR products of GSTP1 gene by the Alw26I restriction endonuclease.