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（ASA）引物通过PCR通过PCR进行基因分型的方法。1.KM基因型（KAPPA标记）基因分型通过半巢PCR使用ASA使用，使用用于区分鉴别基础基础替代品之间的ASA PRCR进行了半巢PCR在人类免疫球蛋白Kappa轻链常数区域的基因中，km ** 1，km ** 1,2和km ** 3，表现为km同型特异性。通过这种方法，不仅在淋巴细胞DNA中确定了KM基因型，还确定了血迹，血迹，唾液染色和发根。 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等位基因，A2M ** 1和A2M ** 2，表现为A2M血清类型。通过这种方法，IgA2基因分型可以从100 pg的淋巴细胞DNA中进行。318日语的估计等位基因频率为IGA2 ** 1的0.561，而IGA2 ** 2的估计等位基因频率为0.561。可以在全血，血迹，唾液染色以及各种器官和组织中检测到该基因型。3.HLADRB1键入HLA DRB1基因座的遗传多态性，通过半纽扣PCR检查，然后进行RFLP分析。通过这种方法，可以从10 pg的淋巴细胞DNA中键入DRB1，并且可以在全血和血迹中确定类型。4.HPGenotypingHP基因分型通过PCR使用三对ASA引物进行，这些ASA引物设计用于鉴别核苷酸序列变化的变化在三个常见的HP等位基因中，HP ** 1S，HP ** 1F和HP ** 2fs.通过这种方法，也可以从200 pg的淋巴细胞DNA.HP基因型中从全血，血迹，salliva salliva stains中检测到HP基因分型。 5.Alpha-2-macroglobolin Genotypingwe尚未实现Alpha-2巨球蛋白基因分型，因为很难设计合适的ASA底漆，因此尚未实现α-2-摩克洛蛋白基因分型。