The PCR for TNF-α-308G/A SNP was carried out in a total volume of 25 μL containing 100 ng–1 μg of genomic DNA, 0.7-1 U Taq DNA polymerase with lX Standard Taq reaction buffer (New England Biolabs, UK), 2.1 mM MgCl2; 0.28 mM deoxynucleotide triphosphate mix (New England Biolabs, UK); 0.56 μM forward and revere oligonucleotide primers (Integrated DNA Technologies, India) and nuclease-protease free water (Qiagen, Germany) added up to a final volume of 25 μL.
The PCR conditions used for the amplification of TNF-α region encompassing − 308G/A SNP were as follows; initial denaturation at 95 °C for 6 min followed by 35 cycles of denaturation at 95 °C for 45 s; annealing at 65 °C for 60 s and extension at 72 °C for 60 s followed by a single final extension step at 72 °C for 10 min. The oligonucleotide primers used for this amplification were 5′-GGAGGCAATAGGTTTTGAGGGC CAT-3′ (forward) and 5′-CTGTCT-CGGTTTCTTCTCCATGGCG-3′ (reverse). The underlined base at the 3′-end of the forward primer was incorporated to generate an NcoI restriction site in the polymorphic region under study (Wilson et al., 1992 (link)). The desired PCR product obtained for TNF-α-308G/A SNP was 195 bp in size (Fig. 1 ).
The PCR conditions used for the amplification of TNF-α region encompassing − 308G/A SNP were as follows; initial denaturation at 95 °C for 6 min followed by 35 cycles of denaturation at 95 °C for 45 s; annealing at 65 °C for 60 s and extension at 72 °C for 60 s followed by a single final extension step at 72 °C for 10 min. The oligonucleotide primers used for this amplification were 5′-GGAGGCAATAGGTTTTGAGGG