Table 2.
Details of the PCR protocol for genotyping polymorphisms.
| Genotype | Content of the PCR reaction | PCR conditions | PCR amplified product |
|---|---|---|---|
| MDR1 (C3435T) | 1 U Taq DNA polymerase, 2.5 μl of 10X Buffer with 25 mM MgCl2, 0.5 μl of 10 mM dNTPs, 10 pmoles of primers and 100 ng of genomic DNA | Initial denaturation at 95 °C for 5 min followed by 30 cycles of denaturation at 95 °C for 45 s, annealing at 62 °C for 30 s and extension at 72 °C for 30 s followed by final extension at 72 °C for 5 min. | 197 base pair (bp) |
| CYP2C19*2 and CYP2C19*3 polymorphisms by multiplex PCR | The 50 μl reaction mixture contained 2 U Taq DNA polymerase, 5 μl 10X Buffer with 25 mM MgCl2, 1 μl 10 mM dNTPs and 15 pmoles of each specific primers and 100 ng of genomic DNA | The PCR amplification conditions consisted 3 steps – an initial denaturation step at 95 °C for 5 min followed by 30 cycles of denaturation at 95 °C for 45 s, annealing at 58 °C for 50 s and extension at 72 °C for 1 min followed by final extension at 72 °C for 3 min | 321 bp of CYP2C19*2 (G681A) and 271 bp of CYP2C19*3 (G636A) |
| CYP2C19*17 polymorphism by nested PCR |
First PCR The first 25 μl reaction mixture contained 1 U Taq DNA polymerase, 2.5 μl of 10X buffer with 25 mM MgCl2, 0.5 μl of 10 mM dNTPs and 10 pmoles of each specific primers and 100 ng of genomic DNA Second PCR The next 50 μl reaction mixture contained 1U Taq DNA polymerase, 5 μl of 10X Buffer with 25 mM MgCl2, 0.5 μl of 10 mM dNTPs and 10 pmoles of each specific primers. For each sample to be genotyped 1 μl of earlier amplified PCR product was added later. |
An initial denaturation step at 95 °C for 5 min followed by 30 cycles of denaturation 95 °C for 45 s, annealing at 62 °C for 30 s and denaturation at 72 °C for 30 s followed by final extension at 72 °C for 5 min. Second PCR Initial denaturation step at 95 °C for 5 min followed by 30 cycles of denaturation at 95 °C for 45 s, annealing at 62 °C for 30 s and extension at 72 °C for 30 s followed by final extension at 72 °C for 3 min |
473bp 143bp |
|
P2Y12 (i-T744C) |
2 U Taq DNA polymerase (Fermentas - MBI), 2.5 μl 10X Buffer with 25 mM MgCl2, 0.5 μl of 10 mM dNTPs and 10 pmoles of each specific primers and 100 ng of genomic DNA. | Initial denaturation step at 95 °C for 5 min followed by 30 cycles of denaturation at 95 °C for 45 s, annealing at 60 °C for 2 min and extension at 72 °C for 2 min followed by final extension at 72 °C for 5 min | 220bp |