权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Development of automated high-resolutoin HLA-DNA typing system using DNA tips

使用 DNA 吸头开发自动化高分辨率 HLA-DNA 分型系统

基本信息

批准号：
12559008
负责人：
INOKO Hidetoshi
金额：
$ 7.62万
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：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

In order to establish the automated high-resolution HLA typing system, we have developed the PCR-SSOP (polymerase chain reaction - sequence specific oligonucleotide probe) method using microarray technique.In this study, a total of 365 allelic polymorphisms of HLA-DRB genes were searched. A total of 20 sets of primer pairs were used for specific PCR amplification specific for HLA-DRB1, -DRB3, -DRB4 and -DRB5 genes, then hybridized with 124 oligonucleotide probes which were designed to be complimentary to the target olymorphic region of each primer pair. Each 5' primer employed in this study were labeled with fluorescence dye, which can be detected by the scanner of microarray system in the case of successful hybridization with the probe spotted on a glass tip.As a result, 14 sets of primer pairs and 103 of probes adopted to these focus on a Japanese population, were successfully applied to genotyping of 70 genomic DNAs extracted from 20 Japanese homozygous cells and 50 individual. Identity between genotypes of 20 homozygous DNAs typed by this method and those obtained by PCR-RFLP (restriction fragment length polymorphism) were 80%, likewise, 69% for 50 individuals. It will be necessary to modify probe design to increase a accuracy, however, it will become suitable for HLA allele typing if low-background can be attained to prevent false signals of heterozygous.

为了建立高分辨率的HLA分型系统，我们利用基因芯片技术建立了聚合酶链反应-序列特异性寡核苷酸探针（PCR-SSOP）方法，共检测了365个HLA-DRB基因的等位基因多态性。用20对引物对HLA-DRB 1、-DRB 3、-DRB 4和-DRB 5基因进行特异性PCR扩增，然后与124个寡核苷酸探针杂交，所述寡核苷酸探针被设计成与每个引物对的目标多态性区域互补。本研究中使用的每个5'引物都用荧光染料标记，在与点在玻璃尖上的探针成功杂交的情况下，可以通过微阵列系统的扫描仪检测。结果，14对引物对和103个探针用于这些针对日本人群，成功地应用于从20个日本纯合子细胞和50个个体中提取的70个基因组DNA的基因分型。用该方法对20个纯合DNA进行分型，其基因型与PCR-RFLP（限制性片段长度多态性）方法所得基因型的一致性为80%，50个个体的一致性为69%。有必要修改探针设计以提高准确性，然而，如果可以获得低背景以防止杂合的错误信号，则它将变得适合于HLA等位基因分型。