Illustratmblood genomicprep mini spin kit

To further support the diagnosis of CD all serologically positive individuals from both groups (group I and group II) were genotyped for the presence of HLA predisposing alleles for CD. Briefly, genomic DNA was extracted from peripheral venous blood samples using the IllustraTMBlood genomicPrep Mini Spin Kit (GE Healthcare, Buckinghamshire, UK). HLA-DQA1*0501 (DQ2 α chain), HLA-DQB1*02 (DQ2 β chain), HLA-DQA1*0301 (DQ8 α chain) and DQB1*0302 (DQ8 β chain) genotyping was performed by polymerase chain reaction amplification using sequencespecific primers (PCR-SSP). For internal positive amplification control, each PCR reaction included a primer pair for a conserved region of the DRB1 gene. The amplified products were separated using 2% agarose gel, stained with ethidium bromide and then visualized under a ultraviolet (UV) trans illuminator.

Genomic DNA was extracted from whole blood using IllustraTM Blood GenomicPrep Mini Spin Kit (GE Healthcare) according to manufacturer’s instructions. SNPs selection was based on minimum allele frequency (MAF) of 10% and/or SNP consequence/function upon gene action. A total of five SNPs were selected distributed as follows PTPN22 + 1858G/A (rs2476601) at codon 620, CTLA-4 +49A/G (rs231775) within codon 17 in the first exon and MBL2 promoter region -550(H/L) (rs11003125), -221(X/Y) (rs7096206). Genotyping was performed using TaqMan SNP genotyping assays and Taqman Universal Master Mix (Applied Biosystems®, CA) according to manufacturer instructions. SNP assessment within MBL2 exon 1 (A/O) was performed using SYBR Green (Qiagen, Hilden, Germany) as previously described (Hladnik et al., 2002 (link)). All SYBR Green endpoint PCRs were performed, including all three possible genotypes as positive controls, in a Rotor-Gene 6000 TM apparatus (Corbett Research Mortlake, Sydney, Australia). Ten randomly chosen MBL2 genotyped samples were sequenced in order to double-check the Melting Temperature assay (MTA) results. We found 100% concordance between sequenced samples and the MTA results.