High-throughput sequence-based typing strategy for HLA-DRB1 based on real-time polymerase chain reaction Martin Danzer a, * Helene Polin a , Johannes Pröll a , Katja Hofer a , Ingrid Fae b , Gottfried F. Fischer b , Christian Gabriel a a Red Cross Transfusion Service of Upper Austria, Linz, Austria b Department for Blood Group Serology, University of Vienna, Vienna, Austria Received 31 July 2007; received in revised form 28 September 2007; accepted 5 October 2007 Summary DNA sequencing is the gold standard for high-resolution genotyping of the highly polymorphic human leukocyte antigen (HLA) loci. In the case of hematopoietic stem cell trans- plantation, four-digit typing of HLA class I and II genes is indicated. We developed a group- specific, real-time polymerase chain reaction (PCR) strategy for amplification of DRB1 applying TaqMan chemistry on different real-time machines. For evaluation of genotyping accuracy and ambiguity resolution, 115 well-characterized samples were adapted. Separate analysis of each allele was possible in 100 samples (87%). Of the samples, 13% (n = 15) showed amplification in one of the eight group-specific PCR mixes: one sample according to the group DRB1*15, 16, along with 14 samples according to the group DRB1*03, *11, *13, *14. Further testing of the 14 (12.2%) samples associated with group DRB1*03, *11, *13, *14 was performed with specific intron prim- ers. In conclusion this typing scheme generates results within 1 day, thereby making sequencing for different requirements attractive. © 2007 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. KEYWORDS HLA-DRB1; SBT; Real-time PCR; TaqMan Introduction DNA sequencing is the gold standard for high-resolution genotyping of the highly polymorphic human leukocyte an- tigens (HLA). The importance of HLA allele matching in bone marrow transplantation is well established, with the conse- quence that four-digit typing is indeed indicated for HLA class I and II genes to achieve the best clinical outcomes [1]. The advancement of sequence-based typing (SBT) at the two- and four-digit levels, together with the rapidly growing number of alleles, renders sequencing methods superior to polymerase chain reaction (PCR)–sequence-specific oligonu- cleotide hybridization (SSO) [2] or PCR–sequence-specific primer (SSP) [3], both limited in their analytical power by the use of recognition motifs that do not cover the whole sequence. Normally high-resolution HLA genotyping requires haplotype separation for the reduction of ambiguities caused by the cis/trans difficulty and can be achieved by group-spe- cific PCR [4] and other methods [5]. During the last decade * Corresponding author. Fax: 43-70-777000-430. E-mail address: martin.danzer@blutz.o.redcross.or.at (M. Danzer). Human Immunology (2007) 68, 915–917 0198-8859/$ -see front matter © 2007 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2007.10.005