Ten milliliters of blood was obtained from each patient by venipuncture. Genomic DNA was extracted from leukocytes of the peripheral blood (Nucleon DNA Extraction kits; Tepnel Life Sciences, Manchester, UK). We performed genotyping for the IL-1 gene cluster in three polymorphic loci to determine the presence of single nucleotide. The IL-1α (−889C/T), IL-1β (−511C/T), and IL-1β (+3953C/T) variants were detected by specific polymerase chain reaction (PCR; using primers in
Table 1 ) with a DNA thermocycler (model 9700; Applied Biosystems, Foster City, CA).
PCR reactions were performed in 50-μL reaction volumes containing 10 mM Tris HCl (pH 8.9), 50 mM KCl, 1.5 mM MgCl2, 25 picomoles of each primer, 200 μM each dNTP, 50 to 100 ng of patient genomic DNA and 0.7 units of Taq thermostable DNA polymerase (Promega, Madison, WI). Cycling parameters were 3 minutes at 95°C, followed by 35 cycles of 30 seconds at 95°C, 30 seconds at the annealing temperature (Tm) of the primers (58–60°C), and 30 seconds at 72°C with a final 5-minute extension at 72°C. The PCR products for IL-1β (−511C/T) and IL-1β (+3953C/T) were then subject to restriction enzyme digestion, to determine the presence of polymorphisms.
The PCR products were incubated at the optimal temperature for enzyme digestion for 8 hours and then separated on 2% agarose gels. The separation of the DNA products were visualized by staining with ethidium bromide, and the size of the expected DNA products was compared against a DNA ladder (Hyperladder IV; Bioline, London, UK) which was run in an adjacent gel lane. Confirmation of polymorphisms at these two loci was performed by direct sequencing.
The IL-1α (−889) PCR products were not subject to restriction enzyme digest but were sequenced. The PCR products were purified on PCR columns (GFX purification column; GE Healthcare, Amersham, UK). Sequence variations were identified by automated bidirectional sequencing (BigDye terminator ver. 3.1; Applied Biosystems) on an automated DNA sequencer (Prism 3100; Applied Biosystems).