|Budget Amount *help
¥4,100,000 (Direct Cost : ¥4,100,000)
Fiscal Year 1995 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1994 : ¥3,100,000 (Direct Cost : ¥3,100,000)
We have developed methods for genotyping single locus DNA polymorphisms by PCR using allele-specific amplification (ASA) primers.
Km (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.
IgA2 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 typing
The 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.
Hp 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.
We have not yet achieved alpha-2-macroglobulin genotyping because of the difficulty to design suitable ASA primers.