Polymerase chain reaction (PCR) conditions for genotyping of interleukin (IL)-1B, IL-10, and tumour necrosis factor α (TNF-A) markers
| Polymorphism | Primers | Temperature, time, and cycles for PCR; polymerase; PCR method; and definitions of the allele |
|---|---|---|
| RFLP, restriction fragment length polymorphism; CTPP, confronting two pair primers. | ||
| IL-1B | F 5′-CTG CAT ACC GTA TGT TCT CTG CC R 5′-GGA ATC TTC CCA CTT ACA GAT GG | 5 min at 94°C, 30 cycles of 30 s at 94°C, 59°C, and 72°C, and 5 min at 72°C; AmpliTaq Gold; PCR-RFLP (Eco811); C allele: 194 bp, T allele: 109 bp and 85 bp |
| C to T at −511 | ||
| IL-10 | F1 5′-TTT AGA CTC CAG CCA CAG AAG R1 5′-GCA AAC TGA GGC ACA GAG ATA F2 5′-CCC TTG TAC AGG TGA TGT AAC R2 5′-TAC TTT CCA GAG ACT GGC TTC | 5 min at 94°C, 30 cycles of 1 min at 94°C, 57°C, and 72°C, and 5 min at 72°C; AmpliTaq Gold; PCR-CTPP; C allele: 407 bp and 275 bp, T allele: 407 bp and 177 bp |
| T to C at −819 | ||
| TNF-A | F1 5′-AAG GCT CTG AAA GCC AGC TG R1 5′-CCA GAC CCT GAC TTT TCC TTC A F2 5′-GAA GCA AAG GAG AAG CTG AGA AGA C R2 5′-CTT CCA TAG CCC TGG ACA TTC T | 10 min at 95°C, 30 cycles of 1 min at 95°C, 66°C, and 72°C, and 5 min at 72°C; AmpliTaq Gold; PCR-CTPP; C allele: 444 bp and 174 bp, T allele: 444 bp and 316 bp |
| T to C at −1031 | ||