HLA‐DQB1 and ‐DQA1 typing by PCR amplification with sequence‐specific primers (PCR‐SSP) in 2 hours

Abstract

In the present study PCR primers were designed for detecting all phenotypically expressed DQB1 and DQA1 allelic variability, 19 and 10 alleles, respectively, by PCR amplification with sequence-specific primers (PCR-SSP). For DQB1 typing, each sample was amplified by a first set of 14 PCR primer pairs, followed in some cases by two to six additional PCR reactions. The first 14 primer pairs allowed the identification/separation of all but a few of the recently described DQB1 alleles: DQB1*0504, DQB1*0605, DQB1*0606 and DQB1*0607 would not be identified; DQB1*0603 and DQB1*0608; and DQB1*0301 and DQB1*0304, respectively, would not be distinguished. Therefore an additional set of eight DQB1 primer pairs was used for a complete DQB1 typing, including all homozygous and heterozygous combinations. For DQA1 typing, 12 PCR reactions were performed per sample, 10 for detecting variability within the second exon and two for identifying first exon polymorphism. All homozygous and heterzoygous combinations of DQA1 alleles could be resolved by these primer pairs. In addition, four primer mixes were designed for determining codon 57 of the HLA-DQB1 gene. Thirty cell lines and 120 individuals were investigated by the DQB1 and DQA1 PCR-SSP technique, as well as with the HLA-DQ beta 57 primers. The concordance between PCR-SSP typing and assigning DQB1 and DQA1 alleles from TaqI DRB-DQA-DQB RFLP analysis was 100%. The reproducibility was 100% in 30 samples investigated on two separate occasions. Amplification patterns, investigated in 15 nuclear families, segregated according to dominant Mendelian inheritance. DQB1 and DQA1 PCR-SSP typing can be performed in 2 hours, including DNA extraction, PCR amplification and post-amplification processing. The method is technically simple and the typings are easy to interpret. The cost for typing one individual is low and is independent of the number of samples analyzed simultaneously, i.e. the technique is well-suited for routine clinical use.

The shared CTLA4-ICOS risk locus in celiac disease, IgA deficiency and common variable immunodeficiency

“…46 The kits used in this study included the HLA-B low resolution (L21, M26, J82, N80, N02, R56, X13 and X82), the HLA-, DQ-, DR SSP Combi Tray (K88, R60, V95, M01 and M84) and the HLA DQB1*06 high resolution (L 46) from Olerup SSP AB, Saltsjöbaden, Sweden.…”

Killer Ig-like receptor (KIR) genotype and HLA ligand combinations in ulcerative colitis susceptibility

“…50,51 PCR methodology PCR reactions were composed of 5 ml 2Â Biomix (containing Biotaq DNA polymerase) (Bioline, UK), between 0.01 and 0.1 mg of DNA, primer combinations (final reaction concentrations of each primer are given in Table 2), and brought to a total volume of 10 ml with 0.2 mm filtered H 2 O (Sigma). The Final 1 Â concentrations of the Biomix components are as follows: 1 mM deoxynucleoside triphosphates, 16mM (NH 4 ) 2 SO 4 , 62.5 mM Tris-HCl (pH 8.8 at 251C), 0.01% Tween 20 and 2 mM MgCl 2 .…”

Treatment of Graft-Versus-Host Disease After Liver Transplantation with Basiliximab Followed by Bowel Resection

“…The donor's HLA typing was A24 (9), A11, B7, B64 (14), DR15 (2), DR7, and he was seronegative for CMV and toxoplasma. The recipient's HLA antigens were A2, B18, B51 (5), DR15 (2), DR17 (3).…”

“…HLA typing was performed by polymerase chain reaction (PCR) using sequence specific primers (SSP) for HLA-A, -B, -DRB and DQBI based on methods previously described (13)(14)(15)(16). HLA typing was carried out on genomic DNA extracted from recipient peripheral blood taken immediately before and after transplantation.…”