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DR4 high resolution typing by direct genomic sequencing
Abstracts
129
B-7.4 #245
HIGH RESOLUTION DQBI* TYPING BY PCR-RFLP. D.P.S.Sengar, R.(~01dstein. Ottawa General Hospital and University of Ottawa, Ottawa, Ontario. DQBI* pairs, 0602 and 0603; 0302 and 03032; and some other newly described alleles (0606, 0304) still need to be resolved by PCR-RFLP. We have attempted to resolve all 19 DQBI* alleles by PCR-RFLP. The second exon of DQBI* was amplified by PCR using two primer pairs (SDQ-01 and QB202; SDQ-01 or YS-01 and QB204) to obtain gpl (DQ5, 6) and gp2 (DQ2,3,4) specific products respectively. While 5 enzymes had either none or one site, all others had more than one site for a specific amplified product. All 11 gpl alleles (0501-0504, 0601-0606) and their heterozygotes could be resolved using 9 enzymes (ApaI, BssHII, HaelI, NciI, Sau96I, HaelII, HhaI, Fnu4HI and RsaI). RsaI was required to resolve allele 0602 (109, 81 bp) from 0603 (84, 81 bp) and Fnu4HI was required to resolve allele 0605 (154, 115 bp) from 0606 (154, 60 bp). BsaHI would resolve the silent allelic pair, 05031 (152, 61, 47 bp) from 05032 (152, 66, 61 bp). All 8 gp2 alleles (0201, 0301, 0302, 03031, 03032, 0304, 0401 and 0402) and their heterozygotes could be resolved using 6 enzymes (FokI, BglI, SacI, BsaHI, HpalI and Sau96I). Sau96I would resolve 0301 (101 bp) from 03032 and 0302 (161 bp). The latter pair with otherwise similar RFLP pattern could be resolved by BsaHI, 0302 (152, 123 bp) from 03032 (152, 104 bp). We have not yet verified a few of the patterns. However, the predicted patterns suggest that all 19 DQBI* alleles and their heterozygotes could be resolved by PCRRFLP. Thus, PCR-RFLP remains an effective means of high resolution HLA-DQ typing.
B-7.4 #246
DR4 HIGH RESOLUTION TYPING BY DIRECT GENOMIC SEQUENCING. Anne Bouwens, Willem Verduijn, Erik Rozemuller, Leone Versluis, Marcel Tilanus, Ieke Schreuder. Diagnostic DNA Laboratory. Academic Hospital Utrecht and the Dept. Immunohaematology and Blood Bank, University Hospital Leiden, The Netherlands. In HLA-DR4 haplotypes, two DRB genes, DRB1 and DRB4 are usually expressed. HLA-DRB1 alleles, which are associated with the HLA-DR4 serological determinant have been particularly studied since they are functionally different in the mixed lymphocyte culture (MLC). These DR4 variants show extensive polymorphism within the second exon of DRB1. In the previous workshop, twelve DRB1DR4 alleles have been characterized with DNA based techniques. We developed a fluorescent sequence based typing system to perform DR4 high resolution typing. In this study we tested a panel of 42 samples which are typed for DR4 by serology or SSO (Leiden). All DNA samples are amplified in the polymerase chain reaction with a 5' DRB1DR4 specific intron primer and a 3'biotinylated DRB intron primer. The generated PCR products are purified using streptavidin coated magnetic beads. Sequencing is performed with a DR4 specific fluorescent sequencing primer which is complementary to the shared first hypervariable region of all DR4 alleles. With this approach we are able to genotype most possible DR4 allelic combinations. For a few rare DR4 homozygous genotypes, in which two combinations of DR4 sequence based typed alleles cannot be distinguished, we use a second sequence reaction with a group specific sequence primer. With this DR4 high resolution sequence based typing method we are able to perform reliable genotyping of DR4.
129
B-7.4 #245
HIGH RESOLUTION DQBI* TYPING BY PCR-RFLP. D.P.S.Sengar, R.(~01dstein. Ottawa General Hospital and University of Ottawa, Ottawa, Ontario. DQBI* pairs, 0602 and 0603; 0302 and 03032; and some other newly described alleles (0606, 0304) still need to be resolved by PCR-RFLP. We have attempted to resolve all 19 DQBI* alleles by PCR-RFLP. The second exon of DQBI* was amplified by PCR using two primer pairs (SDQ-01 and QB202; SDQ-01 or YS-01 and QB204) to obtain gpl (DQ5, 6) and gp2 (DQ2,3,4) specific products respectively. While 5 enzymes had either none or one site, all others had more than one site for a specific amplified product. All 11 gpl alleles (0501-0504, 0601-0606) and their heterozygotes could be resolved using 9 enzymes (ApaI, BssHII, HaelI, NciI, Sau96I, HaelII, HhaI, Fnu4HI and RsaI). RsaI was required to resolve allele 0602 (109, 81 bp) from 0603 (84, 81 bp) and Fnu4HI was required to resolve allele 0605 (154, 115 bp) from 0606 (154, 60 bp). BsaHI would resolve the silent allelic pair, 05031 (152, 61, 47 bp) from 05032 (152, 66, 61 bp). All 8 gp2 alleles (0201, 0301, 0302, 03031, 03032, 0304, 0401 and 0402) and their heterozygotes could be resolved using 6 enzymes (FokI, BglI, SacI, BsaHI, HpalI and Sau96I). Sau96I would resolve 0301 (101 bp) from 03032 and 0302 (161 bp). The latter pair with otherwise similar RFLP pattern could be resolved by BsaHI, 0302 (152, 123 bp) from 03032 (152, 104 bp). We have not yet verified a few of the patterns. However, the predicted patterns suggest that all 19 DQBI* alleles and their heterozygotes could be resolved by PCRRFLP. Thus, PCR-RFLP remains an effective means of high resolution HLA-DQ typing.
B-7.4 #246
DR4 HIGH RESOLUTION TYPING BY DIRECT GENOMIC SEQUENCING. Anne Bouwens, Willem Verduijn, Erik Rozemuller, Leone Versluis, Marcel Tilanus, Ieke Schreuder. Diagnostic DNA Laboratory. Academic Hospital Utrecht and the Dept. Immunohaematology and Blood Bank, University Hospital Leiden, The Netherlands. In HLA-DR4 haplotypes, two DRB genes, DRB1 and DRB4 are usually expressed. HLA-DRB1 alleles, which are associated with the HLA-DR4 serological determinant have been particularly studied since they are functionally different in the mixed lymphocyte culture (MLC). These DR4 variants show extensive polymorphism within the second exon of DRB1. In the previous workshop, twelve DRB1DR4 alleles have been characterized with DNA based techniques. We developed a fluorescent sequence based typing system to perform DR4 high resolution typing. In this study we tested a panel of 42 samples which are typed for DR4 by serology or SSO (Leiden). All DNA samples are amplified in the polymerase chain reaction with a 5' DRB1DR4 specific intron primer and a 3'biotinylated DRB intron primer. The generated PCR products are purified using streptavidin coated magnetic beads. Sequencing is performed with a DR4 specific fluorescent sequencing primer which is complementary to the shared first hypervariable region of all DR4 alleles. With this approach we are able to genotype most possible DR4 allelic combinations. For a few rare DR4 homozygous genotypes, in which two combinations of DR4 sequence based typed alleles cannot be distinguished, we use a second sequence reaction with a group specific sequence primer. With this DR4 high resolution sequence based typing method we are able to perform reliable genotyping of DR4.