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

Development of HLA typing from forensic samples using molecular biological techniques.

使用分子生物学技术对法医样本进行 HLA 分型。

基本信息

批准号：
02557033
负责人：
OTA Masao
金额：
$ 5.95万
依托单位：
Department of Legal Medicine, Shinshu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02557033/
关键词：
HLA-DNA typing PCR-RFLP method genotype restriction enzyme HLA-class II antigen HLA-Class I antigen personal identification paternity testing HLA-DNAタイピング PCR-RFLP法 PCR-SSO法 HLA-クラスII抗原 HLA-クラスI抗原 HLAーDNAタイピングオリゴヌクレチオド PCRーS

项目摘要

1. In this study, we have developed the polymerase chain-reaction fragment polymorphism(PCR-RFLP)method based on digestion of PCRamplified DNAs with allele specific enzymes as a reliable, convenient and practical HLA class II DNA typing technique in stead of hybridization with multiple sequence specific oligonucleotide probes(PCR-SSO). Especially, the modified PCR-RFLP method incorporating informative enzymes, which have a single recognition site in some alleles but none in other alleles in the amplified regions, is simpler and more sensitive because genotypes can be defined mainly just by checking whether the amplified DNAs are digested or not. This modified one makes reading of the generated RFLP band patterns much easier, and thus all of the class II alleles(DRB1, DRB3, DRB5, DQA1, DQB1, DPAL and DPBL)can be clearly defined both for homozygotes and heterozygotes except three pairs of the alleles(DRB1*1103 and DRB1*1104, DQB1*0602 and DQB1*0603, and DRB5*0201 and DRB5*0202)using 29 restriction enzymes.2. We have applied this PCR-RFLP method to investigation of forensic works, HLA-disease association and transplantation matching.we could define HLA-class II genotype of DNA samples extracted from hairs, a small volume of whole blood, fresh or old dental pulp tissues at autopsy.In HLA-disease association study, we could explain the susceptibility gene to autoimmune hepatitis in the Japanese patients. The basic amino acid at position 13, which is present only on the DR2 and DR4Bl molecules(Arg on DR2 and His on DR4), contributed to the susceptibility to this disease.In bone marrow transplantation, we could explain that the DP disparity played an important role developing severe acute graft-versus-host disease.

1.本研究以等位基因特异性酶切聚合酶链反应片段多态性（PCR-RFLP）技术代替多序列特异性寡核苷酸探针杂交（PCR-SSO）技术，建立了一种可靠、简便、实用的HLA Ⅱ类DNA分型方法。特别是，引入信息酶的改进的PCR-RFLP方法更简单和更灵敏，因为主要通过检查扩增的DNA是否被消化就可以确定基因型，所述信息酶在扩增区域的某些等位基因中具有单一识别位点，而在其它等位基因中没有。这种修改的方法使得阅读产生的RFLP带型更容易，因此所有的II类等位基因DRB 1、DRB 3、DRB 5、DQA 1、DQB 1、DPAL和DPBL的等位基因除3对等位基因外，均能清楚地区分纯合子和杂合子（DRB 1 *1103和DRB 1 *1104，DQB 1 *0602和DQB 1 *0603，以及DRB 5 *0201和DRB 5 *0202）。本研究应用于法医学、HLA-疾病相关性和移植配型等方面的研究，可确定头发、少量全血、新鲜或陈旧牙髓组织的DNA样本的HLA-Ⅱ类基因型，在HLA-疾病相关性研究中，可解释日本患者自身免疫性肝炎的易感基因。DR 2和DR 4 B1分子上的13位碱性氨基酸（DR 2上的Arg和DR 4上的His）是引起急性移植物抗宿主病的重要原因，在骨髓移植中，DP差异可能是导致严重急性移植物抗宿主病的重要原因。