OBJECTIVETo develop a method for separating the human leukocyte antigen (HLA)-A, -B and -C haploid using biotinylated probes and streptavidin magnetic beads in order to solve ambiguous HLA genotyping results.

METHODSBased on sequence information of HLA alleles from the IMGT/HLA database, the 5-biotinylated probes were designed. The probe was mixed and extended with corresponding genomic DNA, and incubated with streptavidin magnetic beads, which could form a streptavidin magnetic beads-biotin-probe DNA complex. The unique DNA haploid binding to corresponding probe was isolated after washes and elution. The separated haploid genomic DNA was used as template for HLA-A, -B and -C loci amplification and sequencing analysis.

RESULTSAmong the 12 HLA-A probes, 19 HLA-B probes and 13 HLA-C probes, DNA sequencing has confirmed that 9 HLA-A probes, 9 HLA-B probes and 5 HLA-C probes could successfully separate the haploid from genomic DNA samples.

CONCLUSIONThe developed method for HLA-A, -B and -C haploid separation is reliable, which can solve certain ambiguity and improve the accuracy of HLA genotyping.

Genotype ; HLA-A Antigens ; genetics ; HLA-B Antigens ; genetics ; HLA-C Antigens ; genetics ; Haploidy ; Humans ; Molecular Probe Techniques ; instrumentation ; Polymerase Chain Reaction ; instrumentation ; methods ; Streptavidin ; chemistry