应用焦磷酸微测序技术行HLA-DRB基因型分析

目的应用焦磷酸微测序技术进行HLA-DRB基因型分析.方法PCR扩增外显子2基因片段,经链亲和素包被磁珠纯化、制备单链DNA测序模板,应用焦磷酸微测序技术进行实时测序和HLA-DRB基因分型.结果焦磷酸微测序技术-次可判读40～80个碱基,采用3-4个微测序可以判读全部扩增区域的多态性位点,测序结果与HLA数据库基因序列比较,可准确进行HLA-DRB基因型分析.结论焦磷酸微测序技术应用于HLA-DRB基因型分析具有高分辨率、高通量和快速等优点,该方法可应用于器官及骨髓移植的供体/受体筛查.

Applying pyrosequencing to HLA-DRB genotype analysis

Objective] To apply pyrosequencing technology to HLA-DRB genotyping. Methods] The DNA fragments of HLA-DRB exon-2 were amplified by PCR reaction. The biotinylated PCR products were purified with streptavidin-eoated sepharose beads. After the single-strand DNA templates had been prepared, the DNA sequences were analyzed by pyrosequencing and the HLA-DRB genotypes were determined. Results] The reading length was obtained up to 40-80 bp within single pyrosequencing reaction in our hands. The most polymorphie residues could be identified using 3-4 sequencing primers and the genotypes could be determined by compared with HLA sequences in public data bases. Conclusions] Pyrosequeneing-based HLA-DRB genotyping method offered a number of advantages, such as rapid, high resolution and high throughput, it can be applied to donor/recipient selection for organ as well as bone marrow transplantation.