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目的采用焦磷酸微测序技术,建立一种高通量、高分辨率的HLADRB基因型分析新方法,为临床应用提供依据。方法对HLADRB外显子2DNA序列进行同源性分析,设计PCR引物,提取健康人外周血淋巴细胞基因组DNA,PCR扩增外显子2基因片段,克隆构建含有外显子2的重组质粒pMD/DRB302023。扩增产物经链亲和素包被磁珠纯化、变性、分离制备单链DNA测序模板,采用PSQ96焦磷酸测序仪进行实时测序,分析判断HLADRB基因型。结果扩增并克隆了HLADRB外显子2,焦磷酸微测序结果与克隆质粒常规测序结果比较,测序正确,与HLA数据库基因序列比较,可准确进行HLADRB基因型分析。采用重组质粒pMD/DRB302023和pMD/DRB10405等比例混合模拟杂合子基因型,焦磷酸微测序分析获得预期结果。结论焦磷酸微测序技术应用于HLADRB基因型分析具有高分辨率和快速等优点,可有效分析杂合子基因型,该新方法有望应用于器官及骨髓移植的供体/受体筛查。
OBJECTIVE: To establish a high-throughput and high-resolution method for genotyping HLADRB using pyrosequencing technology and provide a basis for clinical application. Methods The homology analysis of exon 2 of HLADRB gene was carried out. The PCR primers were designed and the genomic DNA of peripheral blood lymphocytes was extracted from healthy volunteers. The exon 2 gene fragment was amplified by PCR. The recombinant plasmid pMD / DRB302023. The amplified product was purified by streptavidin-coated magnetic beads, denatured and separated to prepare a single-stranded DNA sequencing template. The real-time sequencing of the amplified product was performed by using a PSQ96 pyrosequencing sequencer to analyze and determine the HLADRB genotype. Results The exon 2 of HLADRB was amplified and cloned. The results of pyrosequencing were compared with the conventional sequencing results of cloned plasmids. Sequencing was correct. Compared with the HLA database, the HLADRB genotypes could be accurately analyzed. The mixed plasmids pMD / DRB302023 and pMD / DRB10405 were used to simulate heterozygous genotypes and pyrosequencing was used to obtain the expected results. CONCLUSION: Pyrosequencing with pyrosequencing can be used to genotype heterozygotes for HLADRB genotyping with high resolution and rapidity. The new method is expected to be used in donor / recipient screening for organ and bone marrow transplantation.