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

我们开发了利用等位基因特异性扩增（ASA）引物进行单位点DNA多态性PCR分型的方法。Km （kappa marker）基因分型采用半嵌套PCR方法，采用ASA引物对人免疫球蛋白kappa轻链常数区Km**1、Km**1、Km** 2和Km**3基因中3个常见等位基因之间的碱基置换进行分型。该方法不仅可以测定淋巴细胞DNA中的Km基因型，还可以测定血液、血迹、唾液污渍和发根中的Km基因型。115名日本人的等位基因频率分别为0.739 Km**3和0.261 Km**1,2.2。IgA2基因分型采用巢式PCR方法，采用ASA引物对A2m**1和A2m**2等位基因3'侧区碱基替换进行分型。通过这种方法，可以从100 pg淋巴细胞DNA中分型IgA2基因。318例日本人IgA2**1和IgA2**2等位基因频率分别为0.561和0.439。该基因型可在全血、血迹、唾液污渍及各种器官和组织中检测到。HLA DRB1分型采用半巢式PCR和RFLP分析检测HLA DRB1位点的遗传多态性。该方法可从10pg淋巴细胞DNA中进行DRB1分型，并可在全血和血迹中进行分型。Hp基因分型采用三对ASA引物对Hp**1S、Hp**1F和Hp**2FS三种常见Hp等位基因核苷酸序列变化进行PCR分型。通过这种方法，可以从200 pg淋巴细胞DNA中分型Hp。Hp基因型也可在全血、血迹、唾液污渍和发根中检测到。α -2巨球蛋白基因分型由于难以设计合适的ASA引物，我们尚未实现α -2巨球蛋白基因分型。

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.