High-throughput sequence-based typing strategy for HLA-DRB1 based on real-time polymerase chain reaction.
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 transplantation, 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 primers. In conclusion this typing scheme generates results within 1 day, thereby making sequencing for different requirements attractive.
Danzer
Martin
M
Red Cross Transfusion Service of Upper Austria, Linz, Austria. martin.danzer@blutz.o.redcross.or.at
Polin
Helene
H
Pröll
Johannes
J
Hofer
Katja
K
Fae
Ingrid
I
Fischer
Gottfried F
GF
Gabriel
Christian
C
D000483:Alleles; D001483:Base Sequence; D006684:HLA-DR Antigens / Q000235:genetics*; D059811:HLA-DRB1 Chains; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D006684:HLA-DR Antigens; D059811:HLA-DRB1 Chains