HLA‐DQB1 sequencing‐based typing using newly identified conserved nucleotide sequences in introns 1 and 2

Abstract

Sequencing-based typing (SBT) human leukocyte antigen (HLA) class I and II genes should examine entire exon sequences where polymorphisms lie. Primers for the amplification of complete exons therefore anneal in introns and their design relies on accurate intron sequences being available. We decided to develop a SBT method for HLA-DQB1 using amplification primers which anneal in introns 1 and 2, yet the amount of intron sequence data previously available in databases was sparse. Therefore, we undertook a systematic sequencing of introns 1 and 2 using DNA from cell lines homozygous for DQB1. This study confirmed an earlier report that the non-coding regions of this gene are the most polymorphic seen in the human genome. Intron sequences within an allele group were largely identical, the exceptions being DQB1*0301 differing from other DQB1*03 allele groups and DQB1*0601 differing from all other DQB1*06 alleles. A retroviral Alu element, related to the AluYa5a2 subfamily, was identified uniquely inserted in intron 2 of DQB1*02 alleles. For the typing approach, six amplification primers were designed based on conserved allele group sequences covering all of the HLA DQB antigens, and two sequencing primers were also designed which anneal in intron 2. This method has proved to be very robust and has been used as part of a referral DNA sequencing service for a number of years.

Immunogenetic Markers Definition in Latvian Patients with Lyme Borreliosis and Lyme Neuroborreliosis

“…HLA-DR genotyping by polymerase chain reaction (PCR) with sequence-specific primers (PCR–SSP) for DRB1*01 to DRB1*10 was performed [28,29,30]. The reaction mixture (15 µL) included 1.0 µL DNA, 1.5 µL PCR buffer (50 mM KCl, 1.5 mM MgCl 2 , 10 mM Tris-HCl (pH 8.3)), 0.6 µL dNTPs (25 mmol/L), 1.0 µL specific primers (0.2 mmol/L), and 0.5 U of the Taq DNA polymerase (Promega, Madison, WI, USA).…”

“…The reaction mixture was subjected to 45 amplification cycles, each consisting of denaturation cycle at 94 °C (60 s), annealing cycles at 67 °C (40 s), and extension cycles at 65 °C (10 s). PCR products were visualized by agarose-gel electrophoresis [29,30]. After addition of 2 M loading buffer, the PCR reaction mixtures were loaded in agarose gels pre-stained with ethidium bromide (0.5 µL/mL gel).…”

“…After addition of 2 M loading buffer, the PCR reaction mixtures were loaded in agarose gels pre-stained with ethidium bromide (0.5 µL/mL gel). Gels were run for 15 min at 10 V/cm gel in 0.5 mM TBE (0.89 M Tris, 0.89 M boric acid and 0.02 M EDTA in aqueous solution) buffer and then were examined under ultraviolet (UV) illumination and recorded [29,30]. …”

Group-Specific Amplification of cDNA From DRB1 Genes. Complete Coding Sequences of Partially Defined Alleles and Identification of the New Alleles DRB1*040602, DRB1*111102, DRB1*080103, and DRB1*0113

“…The PCR profile was initial TaqGold enzyme (Applied Biosystems, Foster City, CA) activation for 10 minutes at 96°C, and then 15 cycles of 20 seconds at 96°C, 30 seconds at 62°C, and 30 seconds at 72°C, and 20 cycles of 20 seconds at 96°C, 30 seconds at 60°C, and 30 seconds at 72°C. For DQA1 and DQB1, SBT procedures have been already reported [8,9], and they were used with minor modifications.…”

Novel alleles at the HLA‐DRB1 and ‐DQB1 loci

“… a Exon 2 of the HLA‐DRB1 and exons 2 and 3 of HLA‐DQB1 alleles were characterized by DNA sequencing. In some cases, the HLA‐DRB1/DQB1 alleles were amplified separately from genomic DNA with group‐specific HLA‐DRB1/DQB1 primers and identified using sequence‐based typing as previously described (2, 3). The amplified HLA‐DRB1 and ‐DQB1 alleles in these samples were sequenced using an ABI Prism Big Dye™ Terminator Cycle Sequencing Ready Reaction kit and a 3730 xl sequencer (Applied Biosystems, Foster City, CA).…”