中国人常见HLA-DPB1不明确杂合子SSP分型解决方案

Resolution of HLA-DPB1 Indefinite Heterozygotes in China Using the Method of SSP

目的:人类染色体是二倍体,这使得以测序方法研究多态性区域时会遇到不明确杂合子,这个问题在HLA-DPB1的分型上尤为突出。试图寻找一个来解决HLA-DPB1分型中不明确杂合子比例高给分型带来的困难的方案。方法:对946例样品进行HLA-DPB1分型,其中不明确杂合子有353例,共30种,占总例数的37%。建立了一套SSP分型方法,设计上游引物6条,下游引物15条,每一个样品同时用两对特异性引物进行扩增,两对引物分别代表两种不同的杂合模式。再从30种不明确杂合子类型中各挑2个样品进行克隆测序,结果与SSP分型结果比较。结果:SSP分型方法采用同一套扩增体系与两套循环反应条件,只需一次PCR扩增,就可以方便快捷地实现不明确杂合子的分辨,其结果与克隆测序结果相符。结论:与以往的克隆测序、SSCP方法相比,本研究中SSP分型方法具有高效率、高通量、省时省力的特点。

Objective： HLA-DPB1 was one of most polymorphic genes in HLA class-Ⅱ, which was the most polymorphic gene complex in the whole human genome. When dlrect-sequencing of PCR product without chain separation method was applied to HLA-DPB1 typing, heterozygotes would always be produced mainly because of interallelic recombination, indicating that the heterozygosity could be composed of different allele pairs. So a project was searched to resolve heterozygotes in HLA-DPB1 typing. Methods： In the 946 samples collected for typing in our laboratory, 353 samples were found to be indefinite heterozygotes in HLA-DPB1, and could be classified into 30 types, which accounted for 37% of the total numbers. In order to make HLA-DPB1 typing work easier and quicker, a SSP typing system was developed in our laboratory, using six forward primers and fifteen reverse primers, to type out the exact genotype. The typing results were compared with the sequencing of clone. Results： Generally each sample needs two pairs of primers, that is two independent PCR reactions, to distinguish the exact genotype.The identification of indefinite heterozygotes could be quickly realized, only needing PCR amplification once, by SSP typing system. Conclusions ： Compared with other methods usually used in heterozygote typing, the SSP typing system is efficient,easy to operate, besides with high flux.