Development of a method for the separation of HLA-A, -B and -C haploid using biotinylated probe and streptavidin magnetic beads.
- Author:
Sudan TAO
1
;
Yanmin HE
;
Wei ZHANG
;
Wei WANG
;
Ji HE
;
Famin ZHU
;
Hangjun LYU
Author Information
- Publication Type:Journal Article
- MeSH: 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
- From: Chinese Journal of Medical Genetics 2014;31(3):383-387
- CountryChina
- Language:Chinese
-
Abstract:
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.
