Cf96 real time pcr system

Genomic DNA for MICA genotyping was obtained from WCB using QIAamp® DNA blood minikit (Qiagen, GmbH, Hilden, Germany). The SNP rs1051792 (G/A) causing substitution of Val (G) for Met (A) at position 129 of the MICA gene was genotyped using a TaqMan® assay (Applied Biosystems, Foster City, CA, USA). PCR amplification conditions were as recommended by the manufacturer and reactions contained the forward primer 5′-GCTCTTCCTCTCCCAAAACCT-3′ and reverse primer 5′-CGTTCATGGCCAAGGTCTGA-3′ and the two allele-specific dye labeled probes FAM-5′-AATGGACAGTGCCCC-3′ and VIC-5′-AATGGACAATGCCCC-3′. Results were obtained and interpreted using the CF96 real-time PCR system (Biorad, Hercules, CA, USA). Confirmatory typing was performed using DNA extracted from WCB and polymerase chain reaction (PCR) amplification and sequencing of exon 3 of the MICA gene. Results were used to determine the presence of “A,” “G” or both encoding Met or Val at residue 129 (ATG or GTG, respectively). Exons 2–6 of the MICA gene were amplified as previously described (21 (link)) using a forward primer located in intron 1 (5′- CACCTGTGATTTCCTCTTCCCCAGAGC-3′) and reverse primer in the 3′ untranslated region (5′-CTAACAATTTGCAGCMTCCAACAAC-3′). Cycle sequencing was performed using standard protocols and exon 3 forward sequencing primer (5′- CCCTGGGCTGAGTTCCTC-3′) and reverse sequencing primer (5′- ATAGCACAGGGAGGGTTT-3′).