HLA-DQB1 typing using DNA microarray.

使用 DNA 微阵列进行 HLA-DQB1 分型。

• 批准号: 15590572
• 负责人: SATO Yayoi
• 金额: $ 2.24万
• 依托单位: Chiba University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590572/
• 关键词: DNA microarray; HLA-DQB1; human genome; Forensic medicine; biotin-streptavidin; PCR; primer; HLA-DQB1; クローン

I tried to design an oligonucleotide microarray to identify HLA-DQB1 alleles based on the HLA sequence data base in 2002 year. The result was obtained described as below.1.MaterialsDNA was extracted from blood of volunteers using phenol/chloroform method. HLA-DQB1 alleles were previously typed by PCR-SSP method and direct sequence method.2.PrimersThe primers to amplify HLA-DQB1 alleles in Exon2 and Exon3 were designed. One sense primer and two antisense primers were designed for Exon2 and one primer pair was designed for Exon3. These primers were labeled with Biotin at the site of 5' end and mixed for multiplex PCR. Two PCR products were detected using the primer mixture to amplify Exon2 and Exon3 by the agarose gel electrophoresis. The primer mixture was used for the DNA microarray analysis.3.Capture DNA (oligonucleotide probe)Sequence-specific 24 captures for Exon2 and 2 captures for Exon3 to identify the alleles were designed. DNA microarray was prepared by spotting these captures (oligonucleotide probes) on the glass. HIA-DQB1 typing was tried by this DNA microarray.4.HLA-DQB1 typing from blood using DNA microarray.HLA-DQB1 typing was tried using DNA microarray immobilized 26 oligonucleotide probes. The method was such as below. The target DNA was amplified with primer mixture labeled with biotin to amplify Exon2 and Exon3 of HLA-DQB1 alleles. PCR products were denatured and hybridized with capture DNAs. After conjugating with streptavidin-biotin labeled peroxidase solution, color development was performed with peroxidase substrate kit TMB. The allele type was determined by the pattern of color development. The allele specific pattern was detected, but some false positive reaction was also obtained. The probes to show these false positive reactions should be redesigned.

我于2002年在HLA基因数据库的基础上，尝试设计了一种HLA-DQB 1基因芯片。实验结果如下：1.材料采用酚/氯仿法从志愿者血液中提取DNA。2.引物设计扩增HLA-DQB 1基因外显子2和外显子3的引物。针对外显子2设计了一对正义引物和两对反义引物，针对外显子3设计了一对引物。将这些引物在5'端用生物素标记，混合用于多重PCR。使用引物混合物扩增外显子2和外显子3，通过琼脂糖凝胶电泳检测两个PCR产物。3.捕获DNA（寡核苷酸探针）设计了24个外显子2的序列特异性捕获和2个外显子3的序列特异性捕获来鉴定等位基因。通过在玻璃上点样这些捕获物（寡核苷酸探针）来制备DNA微阵列。4.应用DNA微阵列技术对血液样本进行HLA-DQB 1分型应用固定26个寡核苷酸探针的DNA微阵列技术进行HLA-DQB 1分型。方法如下。用生物素标记的引物混合物扩增目的DNA，以扩增HLA-DQB 1等位基因的外显子2和外显子3。将PCR产物变性并与捕获DNA杂交。与链霉亲和素-生物素标记的过氧化物酶溶液缀合后，用过氧化物酶底物试剂盒TMB进行显色。等位基因类型由显色模式决定。检测到等位基因特异性模式，但也有假阳性反应。应该重新设计显示这些假阳性反应的探针。