HLA‐DRB sequencing‐based typing: an improved protocol which shows complete DRB exon 2 sequences and includes exon 3 of HLA‐DRB4/5

Abstract

Several human leukocyte antigen (HLA)-DRB protocols for sequencing-based typing have been described. In general, the DRB1 amplification is performed using group-specific amplification primers (GSAPs) located in HVR I or intron 1. Only some protocols include amplification of DRB3, DRB4, and DRB5. However, prior knowledge obtained by alternative methods such as PCR-SSP is preferred for some protocols and a large amount of DNA is often required for adequate typing of HLA-DRB1. We describe a new protocol that uses GSAPs located in the introns, includes also sequencing of exon 3 to enable high resolution. This protocol allows DRB typing without prior knowledge of the HLA type.

Anti–IFN-γ autoantibodies in adults with disseminated nontuberculous mycobacterial infections are associated with HLA-DRB1*16:02 and HLA-DQB1*05:02 and the reactivation of latent varicella-zoster virus infection

“…Briefly, the second exons of DRB1 and DQB1 were amplified by group-specific primers located in the flanking introns. 25,26 The same PCR conditions were the same as those used for HLA-A and HLA-B except that the 90-second PCR extension times were reduced to 45 seconds.…”

How much of the predisposition to Hashimoto’s thyroiditis can be explained based on previously reported associations?

“…120 ng DNA were used in each 25 µl reaction. Group-specific primers and PCR conditions were used according to van Dijk et al [ 21 ]. Products were visualized electrophoretically, and the remaining reaction mixture purified with NucleoSpin 96 PCR Clean-up (Macherey–Nagel).…”

“…The products were eluted with 70 µl nuclease-free water (Ambion), dried for 2 h at 75 °C, and pellets were resuspended in 11 µl nuclease-free water. The 10 µl sequencing PCR mixture was performed with BigDye Terminator v3.1 (ThermoFisher Applied Biosystems) and 5× sequencing buffer, 0.7 µl of the appropriate 10 µM sequencing primer (according to [ 21 ]), and 5.3 µl of the purified PCR product. The sequencing conditions were in agreement with the Manufacturer’s recommendations, with an annealing temperature of 55 °C.…”

Identification of nine novel HLA‐DRB1 alleles, HLA‐DRB1*04:91, DRB1*07:18, DRB1*11:01:12, DRB1*12:02:05, DRB1*12:22, DRB1*12:23, DRB1*13:100, DRB1*15:45, and DRB1*15:46 by polymerase chain reaction–sequence‐based typing

“…However, it turned out that there were novel mutations not yet reported in the most recent HLA nucleotide database (version 2.28 of the IMGT/HLA database, release, April 2010). To determine the mutated alleles, allele-specific sequencing at the second exon of HLA-DRB1 (exon 2) was performed using a group-specific primer according to van Dijk et al (2). The sequencing was conducted in forward and reverse directions by a 3730XL DNA Analyzer (Applied Biosystems, Foster City, CA) and BigDye ® Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems).…”