Development of an automated machine for HLA DNA typing using mass spectrometry

为了研制一台全自动HLA分型机，我们采用电喷雾质谱（electrospray ionization mass spectrometry，MS）技术对HLA-DRB 1基因扩增后的PCR产物进行了分子量测定，并优化了PCR产物的纯化条件。由于限制性内切酶消化和纯化后的PCR产物中存在杂质，阻碍了PCR-RFLP方法在MS中的应用。相比之下，用PCR-SSP方法的序列特异性引物组扩增的PCR产物允许测量其可靠的分子量。此外，针对大量序列特异性引物组修订了分型软件。然而，在一些杂合子样本中，即使我们使用修改后的软件也很难精确地分配等位基因。为了验证MS和PCR-SSP方法的结合，我们优化了PCR产物的大小，并最大限度地减少了质谱仪载玻片芯片的测量时间。PCR产物经简单纯化后，质谱可准确测定其分子量。MS和PCR-SSP方法的这种组合是一种简单而灵敏的HLA分型系统，因此可以适用于使用相对短长度的PCR产物，特别是其分子量小于50 kDa的产物，在杂合样品的分型中鉴定一个碱基对错配的应用。

In order to develop an automated HLA typing machine, we have estimated the molecular weight of PCR products after DNA amplification of the HLA DRB1 gene using electrospray ionization mass spectrometry (MS) and optimized the condition for purification of PCR products. The application to MS of the PCR-RFLP method was hindered due to impurities in PCR products after restriction enzyme digestion and purification. In contrast, PCR products amplified with sequence specific primer sets for the PCR-SSP method allowed measurement of their reliable molecular mass. Further, the typing software was revised for the numerous sequence specific primer sets. However, in several heterozygous samples, it is difficult to assign the alleles precisely even if we use the revised software. To validate the combination of the MS and PCR-SSP methods, we have optimized the size of PCR products and minimized the time for measurement by slide chips of mass spectrometer. The molecular mass of PCR products with a 90 bp-length can be determined precisely by MS after their simplified purification. This combination of the MS and PCR-SSP methods is a simple and sensitive HLA typing system, and so may be suited for application to identify one base pair mismatch in the typing of heterozygous samples using relatively short length PCR products, especially their molecular weight of less than 50 kDa.