Establishment of Cloning and Sequencing Method for High-Resolution HLA-B Genotype Assay

A high-resolution method for genotyping of human leukocyte antigen-B gene (HLA-B) based on the optimized polymerase chain reaction (PCR) cloning and sequencing procedure was established. The exon 2 and exon 3 of HLA-B gene were amplified by PCR with primers according to the HLA-B gene sequence. The produced heterozygous alleles were effectively cloned into plasmid DNA based on the principle of plasmid incompatibility, followed by bacterial culture. Then Sanger sequencing was carried out and after analyzing the result by software ClustalX2 and BLAST comparison in IMTG/HLA database, the genotyping of HLA-B was achieved. Seven clinical samples were detected by the proposed method, and the results were consistent with those of PCR-SBT genotyping method. The method had the advantages of cost-effectiveness and high resolution, with no requirement of specialized technical software. The use of universal primers simplified the cumbersome design and optimization process of specific primers in traditional methods.

Graphical abstractA high-resolution method for HLA-B genotyping was established using cloning and sequencing technology. HLA-B gene were amplified by PCR and the produced heterozygous alleles were cloned into plasmid DNA, followed by bacterial culture and Sanger sequencing.Figure optionsDownload full-size imageDownload as PowerPoint slide