HLA-DQB1 typing using DNA microarray.

我在2002年尝试基于HLA序列数据库设计一种寡核苷酸微阵列来鉴定HLA- dqb1等位基因。所得结果如下：1。材料采用苯酚/氯仿法从志愿者血液中提取dna。HLA-DQB1等位基因先前分别用PCR-SSP法和直接测序法分型。引物设计了扩增HLA-DQB1等位基因Exon2和Exon3的引物。为Exon2设计了1对正引物和2对反义引物，为Exon3设计了1对引物。这些引物在5'端标记生物素，混合后进行多重PCR。引物混合物经琼脂糖凝胶电泳扩增Exon2和Exon3，检测两种PCR产物。引物混合物用于DNA芯片分析。捕获DNA（寡核苷酸探针）设计了序列特异性的24条捕获Exon2和2条捕获Exon3来识别等位基因。将这些捕获物（寡核苷酸探针）定位在玻璃上制备DNA微阵列。采用该DNA芯片进行HIA-DQB1分型试验。利用DNA芯片从血液中分型HLA-DQB1。采用DNA微阵列固定化26个寡核苷酸探针进行HLA-DQB1分型。方法如下所示。用生物素标记的引物混合物扩增目标DNA，扩增HLA-DQB1等位基因的Exon2和Exon3。PCR产物变性并与捕获的dna杂交。与链亲和素-生物素标记过氧化物酶溶液偶联后，用过氧化物酶底物试剂盒TMB显色。等位基因类型由颜色发育模式决定。检测到等位基因特异型，但也有假阳性反应。显示这些假阳性反应的探针应该重新设计。

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.