FbaI (BclI) restriction enzyme

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	An aliquot (5 ml) of PCR product was digested with 8 U of FbaI (TaKaRa; Otsu, Shiga, Japan) and separated on a 3% agarose gel.

Protocol tips
An aliquot (5 ml) of PCR product was digested with 8 U of FbaI (TaKaRa; Otsu, Shiga, Japan) and separated on a 3% agarose gel.

Publication protocol

"We extracted genomic DNA from peripheral blood leukocytes of 5 ml whole blood using standard phenol/chloroform protocols. DNA samples were diluted to 8 ng/μl and placed in 96 well plates; each 96 well plate contained 94 samples and two controls containing water with no DNA. Three htSNPs (A-3348C, C-2128T, and C-1888T) were then selected for genotyping in the case−control population using PCR direct sequencing or PCR-RFLP analysis.

The polymorphism A-3348C was genotyped by PCR direct sequencing. The primers 5′-TGCCAGGTGCTCTCCATATTT-3′ and 5′-TTTCATTAACTGGCTGCACAAA-3′ were used for amplifying and sequencing the target region. PCR conditions were identical to those for SNP discovery,30,31 except for an annealing temperature of 56.5°C.

For the C-1888T polymorphism, an amplification using forward primer 5′-AGGTGAGACAGTTAAGCTATTTGAT-3′ and reverse primer 5′-AGGGGCCAAGAGATGAGACT-3′ was performed. An FbaI recognition site was introduced by a one base mismatch (underlined) in the forward primer. PCR conditions were identical to those for SNPs discovery30,31 except for an annealing temperature of 54.8°C and a total reaction volume of 25 μl. The reaction yielded a 192 bp amplicon. An aliquot (5 μl) of PCR product was digested with 8 U of FbaI (TaKaRa; Otsu, Shiga, Japan) and separated on a 3% agarose gel. The presence of the 1888T allele creates an FbaI restriction site; digested amplicons from 1888T homozygotes appear as a 170 bp and a 22 bp band, homozygotes for the 1888C allele appear as a 192 bp band, and heterozygotes have all three of these bands."

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