Establishment of Cloning and Sequencing Method for High-Resolution Human Leukocyte Antigen-B Genotype Assay
10.11895/j.issn.0253-3820.140494
- VernacularTitle:基于克隆测序技术的人类白细胞抗原基因高分辨率分型方法的建立
- Author:
Xiaoqing XING
;
Yanan CHU
;
Zheng XIANG
;
Qinxin SONG
;
Guohua ZHOU
- Publication Type:Journal Article
- Keywords:
Human leukocyte antigen-B gene;
TA clone;
Sanger Sequencing;
High-resolution genotyping
- From:
Chinese Journal of Analytical Chemistry
2014;(11):1574-1579
- CountryChina
- Language:Chinese
-
Abstract:
Ahigh-resolutionmethodforhumanleukocyteantigen-B(HLA-B)genotypingwasestablished based on the optimized polymerase chain reaction, cloning and sequencing technology. The exon2 and exon3 of HLA-B gene were amplified with primers based on the HLA-B gene sequence. These produced heterozygous alleles were effectively cloned into plasmid DNA based on the principle of plasmid incompatibility, and were followed by bacterial culture. Then Sanger sequencing was carried out and after analyzing the result by software ClustalX2 and IMTG/HLA database comparison, the HLA-B genotype of the samples was achieved. Seven clinical samples were detected, and the results were consistent with those of PCR-SBT genotyping method. The method was cost-effective, high-resolution and it did not require technical software. The use of universal primers simplified the cumbersome design and optimization process of specific primers.
