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

开发使用质谱进行 HLA DNA 分型的自动化机器

基本信息

批准号：
10557252
负责人：
INOKO Hidetoshi
金额：
$ 7.81万
依托单位：
Tokai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10557252/
关键词：
HLA typing mass spectrometry automated analysis allele high-performance liquid chromatography 自動判定

项目摘要

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.

为了研制一台自动化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PCR产物的分子量进行了测定，并对PCR产物的纯化条件进行了优化。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，因此可能适合于使用相对较短长度的PCR产物（特别是其分子量小于50）在杂合样本分型中识别一个碱基对错配的应用。