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Development of methods for typing of single locus DNA polymorphisms, Km, A2m, HLA DRB1 and alpha2-macroglobulin genotypes

开发单基因座 DNA 多态性、Km、A2m、HLA DRB1 和 α2-巨球蛋白基因型分型方法

基本信息

批准号：
06454247
负责人：
ISHIZU Hideo
金额：
$ 2.62万
依托单位：
Okayama University Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

We have developed methods for genotyping single locus DNA polymorphisms by PCR using allele-specific amplification (ASA) primers.1.Km genotypingKm (kappa marker) genotyping was performed by semi-nested PCR using ASA primers designed for discriminating base substitutions between three common alleles in a gene of the human immunoglobulin kappa light chain constant region, Km**1, Km**1,2 and Km**3, which manifest Km allotypic specificity. By this method, Km genotypes were determined in not only lymphocyte DNA but also blood, bloodstains, saliva stains and hair roots. The estimated allele frequency in 115 Japanese was 0.739 for Km**3 and 0.261 for Km**1,2.2.IgA2 genotypingIgA2 genotyping was performed by nested PCR using ASA primers which were designed for discriminating base substitutions in the 3'-flanking region of alleles, A2m**1 and A2m**2, which manifest A2m serum types. By this method, IgA2 genotyping was possible from 100 pg of lymphocyte DNA.The estimated allele frequency in 318 Japanese was 0.561 for IgA2**1 and 0.439 for IgA2**2. This genotype could be detected in whole blood, bloodstains, saliva stains, and various organs and tissues.3.HLA DRB1 typingThe genetic polymorphism of the HLA DRB1 locus was examined by semi-nested PCR followed by RFLP analysis. DRB1 typing was possible from 10 pg of lymphocyte DNA by this method and the type could be determined in whole blood and bloodstains.4.Hp genotypingHp genotyping was performed by PCR using three pairs of ASA primers which were designed for discriminating changes in the nucleotide sequences among three common Hp alleles, Hp**1S,Hp**1F and Hp**2FS.By this method, Hp genotyping was possible from 200 pg of lymphocyte DNA.Hp genotypes could also be detected from whole blood, bloodstains, salliva stains and hair roots.5.Alpha-2-macroglobulin genotypingWe have not yet achieved alpha-2-macroglobulin genotyping because of the difficulty to design suitable ASA primers.

我们已经开发了使用等位基因特异性扩增（阿萨）引物通过PCR对单位点DNA多态性进行基因分型的方法。使用阿萨引物通过半巢式PCR进行（κ标记）基因分型，所述ASA引物被设计用于区分人免疫球蛋白κ轻链恒定区基因中的三个常见等位基因Km**1、Km** 1，2和Km**3之间的碱基取代，其表现Km同种异型特异性。用这种方法不仅可以检测淋巴细胞DNA中的Km基因型，还可以检测血液、血痕、唾液和发根中的Km基因型。115名日本人Km**3等位基因频率为0.739，Km** 1、2.2等位基因频率为0.261。IgA 2基因分型采用巢式PCR方法，用阿萨引物区分A2 m血清型等位基因A2 m **1和A2 m **2的3 '侧翼区碱基替换。用这种方法可以从100 pg的淋巴细胞DNA中进行IgA 2基因分型，在318名日本人中估算的等位基因频率为IgA 2 **1为0.561，IgA 2 **2为0.439。HLA-DRB 1基因分型采用半巢式PCR结合RFLP技术检测HLA-DRB 1基因座的遗传多态性。4. Hp基因分型用3对阿萨引物进行PCR分型，以区分Hp** 1 S、Hp**1F和Hp** 2FS三种常见等位基因的核苷酸序列变化，用此方法进行Hp基因分型。Hp基因分型可从200 pg淋巴细胞DNA中检出，也可从全血、血痕、唾液斑和发根中检出。5. α-2-巨球蛋白基因分型由于阿萨引物设计困难，尚未实现α-2-巨球蛋白基因分型。