用生物素标记探针和链亲和素磁珠分离HLA-A、-B、-C位点单体型

Development of a method for the separation of HLA-A, -B and -C haploid using biotinylated probe and streptavidin magnetic beads

目的建立生物素标记探针和链亲和素磁珠分离人类白细胞抗原（humanleukocyteantigen，HLA）-A、-B、-C单体型的技术，以解决HLA分型过程中部分模棱两可的结果。方法根据IMGT／HLA数据库中HLA等位基因的序列信息，设计5’端生物素标记探针。将探针与104份标本基因组DNA混合杂交，然后加入链亲和素磁珠孵育，形成链亲和素磁珠一生物素探针一单链DNA复合物，通过洗涤和解析从而实施有效的分离。对分离出的单体型基因组DNA进行HLA-A、-B或-C位点扩增和测序分析。结果设计的12个HLA-A、19个HLA-B、13个HLA-C位点探针中，经测序证实分别有9个、9个、5个探针成功分离了单链DNA标本。结论建立的探针和磁珠分离HLAA、-B、-C单体型技术具有可行性，可以解决HLA测序分型中部分模棱两可结果，提高分型的准确性。

Objective To develop a method for separating the human leukocyte antigen （HLA）-A, - B and -C haploid using biotinylated probes and streptavidin magnetic beads in order to solve ambiguous HLA genotyping results. Methods Based on sequence information of HLA alleles from the IMGT/HLA database, the 5＇-biotinylated probes were designed. The probe was mixed and extended with corresponding genomic DNA, and incubated with streptavidin magnetic beads, which could form a streptavidin magnetic beads-biotin-probe-DNA complex. The unique DNA haploid binding to corresponding probe was isolated after washes and elution. The separated haploid genomic DNA was used as template for HLA-A, -B and -C loci amplification and sequencing analysis. Results Among the 12 HLA-A probes, 19 HLA-B probes and 13 HLA-C probes, DNA sequencing has confirmed that 9 HLA-A probes, 9 HLA-B probes and 5 HLA-C probes could successfully separate the haploid from genomic DNA samples. Conclusion The developed method for HLA-A, -B and -C haploid separation is reliable, which can solve certain ambiguity and improve the accuracy of HLA genotyping.