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Development of an automated machine for HLA DNA typing

开发 HLA DNA 分型自动化机器

基本信息

批准号：
07557179
负责人：
INOKO Hidetoshi
金额：
$ 4.99万
依托单位：
Tokai University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557179/
关键词：
HLA DNA typing PCR-RFLP method fluorescently labeling dye-amidide automated DNA sequencer polymorphism DQA1 gene PCR-RFLP 自動化蛍光標識プライマー制限酵素 GENESCAN ジェノタイパ-

项目摘要

We have previously developed the PCR-RFLP method which is sensitive and reliable in HLA class II genotyping. In this study, to develop an automated typing machine, we analyzed for fluorescent labeling of primers and optimized the condition for scanning of gel patterns of PCR products using an automated DNA sequencer. Dye-amidide was more reliable and stable fluorescent than aminolink-2 for labeling of the 5'-end of DQA1 and DQB1 primers. The PCR-RFLP method was performed using DQA1 primers that was fluorescently labeled with dye-amidide, and informative restriction enzymes. Using Genescan^<TM>672 and Genotyper^<TM>software kits, all DQA1 genotypes of four HLA homozygous typing cells (HTC) and fourteen heterologous healthy individuals were easily defined from the band patterns and automatical fragment size assignment. These typing results were completely identical to those by the conventional PCR-RFLP method. Each fragment size can be determined precisely even if the quantity of PCR products is only 1 ng. Therefore, this new PCR-RFLP method is highly sensitive and reliable in HLA class II genotyping. Discrimination between specific and non-specific fragments in the PCR products was relatively easy for the precision of size and quantitative amount assignment. Electrophoretic data can be also displayd as a multi color digitized gel pattern using several dyes labeling. Therefore, this system is highly efficient for application to a lot of samples.

我们以前开发的PCR-RFLP方法是敏感和可靠的HLA II类基因分型。在这项研究中，开发一个自动分型机，我们分析了荧光标记的引物和优化的条件下扫描的凝胶模式的PCR产物使用自动DNA测序仪。DQA 1和DQB 1引物的5 '末端标记用染料酰胺法比氨基链2法更可靠、更稳定。PCR-RFLP方法使用DQA 1引物进行，DQA 1引物用染料-酰胺和信息限制性内切酶进行荧光标记。应用Genescan <TM>672和Genotyper<TM>软件包，从条带型和自动片段大小分配中很容易地确定了4个HLA纯合分型细胞（HTC）和14个异源健康个体的所有DQA 1基因型。这些分型结果与传统的PCR-RFLP方法完全一致。即使PCR产物的量仅为1 ng，也可以精确地确定每个片段的大小。因此，PCR-RFLP方法在HLA Ⅱ类基因分型中具有较高的敏感性和可靠性。PCR产物中特异性片段和非特异性片段的区分相对容易，以获得精确的大小和定量分配。电泳数据也可以显示为使用几种染料标记的多色数字化凝胶图案。因此，该系统对于大量样品的应用是高效的。