HLA-DPA1和-DPB1基因第2、第3外显子同步测序分型的研究

鲍自谦; 蔡思齐; 钟艳平; 陈瑞; 甄建新; 邓志辉

doi:10.13201/j.issn.1004-2806-b.2018.12.011

HLA-DPA1和-DPB1基因第2、第3外显子同步测序分型的研究

- 1. 深圳市血液中心(广东深圳, 518035);
- 2. 深圳市宝安区妇幼保健院
基金项目:
深圳市科技计划项目(No:JCYJ20160427144249175)

详细信息

通讯作者: 邓志辉,E-mail:zhihui_deng@sina.cn

中图分类号: R331.1

收稿日期: 2018-09-16

Study on simultaneous sequence-based typing at exons 2 and 3 of HLA-DPA1 and-DPB1 genes

- 1. Shenzhen Blood Center, Shenzhen, 518035, China;
- 2. Shenzhen Baoan District Maternal and Child Health Care Hospital

More Information

Corresponding author: DENG Zhihui, E-mail: zhihui_deng@sina.cn

Received Date: 16 September 2018

摘要

摘要: 目的:建立可靠的HLA-DPA1和 -DPB1基因第2、第3外显子同步测序分型的方法。方法:采用一对位点特异性引物扩增HLA-DPA1基因的第2外显子、第2内含子、第3外显子及其两翼的部分内含子序列,采用2对位点特异性引物扩增HLA-DPB1目的基因片段:第1对引物扩增HLA-DPB1基因第2外显子及两翼的部分内含子序列,第2对引物扩增HLA-DPB1基因第3外显子及两翼的部分内含子序列。PCR产物经核酸外切酶和碱性磷酸酶纯化,用本文的测序引物分别对HLA-DPA1和 -DPB1第2、第3外显子进行双向测序。纯化的测序反应产物,经ABI 3730测序仪电泳,用Assign 4.7.1分析软件判定基因型。结果:HLA-DPA1及 -DPB1基因的PCR扩增获得了清晰的目的条带,对其第2、第3外显子进行测序,所获的序列中无背景杂峰。用本文的方法对随机的96人份造血干细胞志愿捐献者标本进行HLA-DPB1测序分型,与采用SeCore^TM DPB1商品化测序分型试剂盒平行对照检测的结果相一致。结论:本文的方法在群体遗传学及疾病关联研究等领域具有广泛的使用价值。
- 人类白细胞抗原 /
- HLA-DPA1 /
- HLA-DPB1 /
- 测序分型
Abstract: Objective: To establish a reliable assay for simultaneous sequence-based typing(SBT)at exons 2 and 3 of HLA-DPA1 and -DPB1 genes.Method:One pair of HLA-DPA1 specific PCR primers was utilized to amplify the target sequence encompassing the entire exon 2,intron 2,exon 3 and partial flanking intronic sequences of HLA-DPA1.Two pairs of HLA-DPB1 specific PCR primers,namely,the first PCR primer pair specially amplified exon 2 and its partial flanking intronic sequences,the second PCR primer pair specially amplified exon 3 and its partial flanking intronic sequences of HLA-DPB1.PCR amplicons purified by exonuclease I and Alkaline phosphatase were then subjected to sequencing reaction using the in-house sequencing primers at exons 2 and 3 of HLA-DPA1 and -DPB1 genes in both directions.The purified products of sequencing reactions were run electrophoresis on the ABI 3730 DNA sequencer and the genotype was determined using the Assign 4.7.1 SBT software.Result:The specific target sequences of HLA-DPA1 and -DPB1 could be obtained in the PCR procedure.No background and noise was observed in the obtained sequences.96 randomly-selected samples from stem cell voluntary donors subjected to HLA-DPA1and -DPB1 genotyping using the present SBT method,the genotyping results were completely in accordance with the results typed by the SeCore^TM HLA-DPB1 commercial kit.Conclusion:Our results implied that the established simultaneous HLA-DPA1 and -DPB1 SBT method showed a broad application in the further genetic population and HLA-DP associated disease studies.
- human leukocyte antigens /
- HLA-DPA1 /
- HLA-DPB1 /
- sequence-based typing

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