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High resolution HLA-DRB1 typing by the combination of group-specific amplification and RFLP
150
Abstracts
B-7.4 #183
HIGH RESOLUTIONHLA-DRBI TYPING BY THE COMBINATION OF GROUP-SPECIFIC AMPLIFICATION AND RFLP. S Mitsunaga, T Oguehi, ~ T Akaza, K Tadokoro and T Juji, Department of Research, Japanese Red Cross Central Blood Center, Tokyo, Japan. We developed a new reliable method for high-resolution DRBI typing using the combination of group-specific amplification and RFLP. Group-specific amplification was carried out under the same PCR conditions for all groups using seven different primer pairs divided into four tubes: I) DR1 and DR10; 2) DR2, DR7 and DR9; 3) DR3, DR5, DR6 and DR8; and 4) DR4. Computer a n a l y s i s on c u t t i n g s i t e s by v a r i o u s r e s t r i c t i o n enzymes was c a r r i e d out on 59 DRB1 a l l e l e s . I t was shown t h a t 4 a l l e l e s o f t h e DR1/10 g r o u p c o u l d be d i s t i n g u i s h e d w i t h 2 e n z y m e s , HphI and S a u 9 6 I ; 9 a l l e l e s o f t h e DR2/7/9 g r o u p w i t h 8 enzymes HphI, R s a I and S a u 9 6 I ; 33 a l l e l e s of the DR3/5/6/8 group w i t h 7 enzymes, BsoPI, B s r B I , C a c 8 I , HhaI, HphI, R s a I and XmnI; and 13 a l l e l e s o f the DR4 group i n c l u d i n g DRB1*1410 w i t h 5 enzymes, BsoFI, HhaI, HphI, MnlI and R s a I . DRBl*1501 and 1 5 0 3 , DRB1*1402 and 1409, and some a l l e l e s possessing s i l e n t m u t a t i o n s c o u l d n o t be d i s t i n g u i s h e d w i t h t h e s e or o t h e r r e s t r i c t i o n e n d o n u c l e a s e s . DRB1 a l l e l e s o f 100 J a p a n e s e human samples were d e t e r m i n e d by t h i s method and compared w i t h the s e r o l o g i c a l r e s u l t s . No d i s c r e p a n c y was o b s e r v e d between t h e two s e t s o f r e s u l t s . Also t h e c a l c u l a t e d DRB1 a l l e l e f r e q u e n c i e s showed good a g r e e m e n t w i t h t h o s e a n a l y z e d a t the llth International Histocompatibility Workshop. Therefore, this method i s u s a b l e as a r e l i a b l e h i g h - r e s o l u t i o n DRB1 t y p i n g method.
B-7.4 #184
AUTOMATED SEQUENCING-BASED TYPING OF PCR-AMPLIFIED DRB REGION GENES. M McGinnis 1, M Conrad 1 , A Bouwens 2, M Tilanus 2, and M Kronick I . 1perkin Elmer, Applied Biosystems Division, Foster City, CA. 2Diagnostic DNA Lab, Academic Hospital, Utrecht, The Netherlands. Direct DNA sequencing can provide high resolution typing of the DRB genes. We herein report a method which incorporates solid-phase automated fluorescent sequencing of PCR amplification products and computer-based data analysis. Because of the complexity of the DRB region, allele group-specific amplifications based on the first hypervariable region are used to generate specific sequencing templates. The allele groups analyzed are DR1, DR2, DR4, DR3/11/13/14, and DRS/12 as well as the DRB3 and DRB5 genes. In each amplification, one primer is labeled with biotin and the other primer has a 5' -21M13 universal sequencing primer recognition site. The locations of the biotin and -21M13 site determine the orientation of the sequence analysis (i.e. 5' or 3'). Among the advantages of this design are: i) a means of attaching sequencing templates to streptavidin-coated magnetic beads; ii) the use of common primers for sequencing all amplification products, regardless of allele group or sequencing orientation; and iii) group-specific amplification simplifies data analysis by minimizing the number of alleles present in any one sequencing template and thereby reducing the number of ambiguous heterozygous combinations. Following identification of heterozygous positions, the analysis software provides allele assignments as well as a means of evaluating the quality of the sequencing result. Allele assignment involves comparative sequence alignments against a library of known DRB alleles. The quality assessment is based on an analysis of errors at invariant and polymorphic positions. In addition to assigning currently recognized alleles, this strategy is also capable of identifying new sequence variants.
Abstracts
B-7.4 #183
HIGH RESOLUTIONHLA-DRBI TYPING BY THE COMBINATION OF GROUP-SPECIFIC AMPLIFICATION AND RFLP. S Mitsunaga, T Oguehi, ~ T Akaza, K Tadokoro and T Juji, Department of Research, Japanese Red Cross Central Blood Center, Tokyo, Japan. We developed a new reliable method for high-resolution DRBI typing using the combination of group-specific amplification and RFLP. Group-specific amplification was carried out under the same PCR conditions for all groups using seven different primer pairs divided into four tubes: I) DR1 and DR10; 2) DR2, DR7 and DR9; 3) DR3, DR5, DR6 and DR8; and 4) DR4. Computer a n a l y s i s on c u t t i n g s i t e s by v a r i o u s r e s t r i c t i o n enzymes was c a r r i e d out on 59 DRB1 a l l e l e s . I t was shown t h a t 4 a l l e l e s o f t h e DR1/10 g r o u p c o u l d be d i s t i n g u i s h e d w i t h 2 e n z y m e s , HphI and S a u 9 6 I ; 9 a l l e l e s o f t h e DR2/7/9 g r o u p w i t h 8 enzymes HphI, R s a I and S a u 9 6 I ; 33 a l l e l e s of the DR3/5/6/8 group w i t h 7 enzymes, BsoPI, B s r B I , C a c 8 I , HhaI, HphI, R s a I and XmnI; and 13 a l l e l e s o f the DR4 group i n c l u d i n g DRB1*1410 w i t h 5 enzymes, BsoFI, HhaI, HphI, MnlI and R s a I . DRBl*1501 and 1 5 0 3 , DRB1*1402 and 1409, and some a l l e l e s possessing s i l e n t m u t a t i o n s c o u l d n o t be d i s t i n g u i s h e d w i t h t h e s e or o t h e r r e s t r i c t i o n e n d o n u c l e a s e s . DRB1 a l l e l e s o f 100 J a p a n e s e human samples were d e t e r m i n e d by t h i s method and compared w i t h the s e r o l o g i c a l r e s u l t s . No d i s c r e p a n c y was o b s e r v e d between t h e two s e t s o f r e s u l t s . Also t h e c a l c u l a t e d DRB1 a l l e l e f r e q u e n c i e s showed good a g r e e m e n t w i t h t h o s e a n a l y z e d a t the llth International Histocompatibility Workshop. Therefore, this method i s u s a b l e as a r e l i a b l e h i g h - r e s o l u t i o n DRB1 t y p i n g method.
B-7.4 #184
AUTOMATED SEQUENCING-BASED TYPING OF PCR-AMPLIFIED DRB REGION GENES. M McGinnis 1, M Conrad 1 , A Bouwens 2, M Tilanus 2, and M Kronick I . 1perkin Elmer, Applied Biosystems Division, Foster City, CA. 2Diagnostic DNA Lab, Academic Hospital, Utrecht, The Netherlands. Direct DNA sequencing can provide high resolution typing of the DRB genes. We herein report a method which incorporates solid-phase automated fluorescent sequencing of PCR amplification products and computer-based data analysis. Because of the complexity of the DRB region, allele group-specific amplifications based on the first hypervariable region are used to generate specific sequencing templates. The allele groups analyzed are DR1, DR2, DR4, DR3/11/13/14, and DRS/12 as well as the DRB3 and DRB5 genes. In each amplification, one primer is labeled with biotin and the other primer has a 5' -21M13 universal sequencing primer recognition site. The locations of the biotin and -21M13 site determine the orientation of the sequence analysis (i.e. 5' or 3'). Among the advantages of this design are: i) a means of attaching sequencing templates to streptavidin-coated magnetic beads; ii) the use of common primers for sequencing all amplification products, regardless of allele group or sequencing orientation; and iii) group-specific amplification simplifies data analysis by minimizing the number of alleles present in any one sequencing template and thereby reducing the number of ambiguous heterozygous combinations. Following identification of heterozygous positions, the analysis software provides allele assignments as well as a means of evaluating the quality of the sequencing result. Allele assignment involves comparative sequence alignments against a library of known DRB alleles. The quality assessment is based on an analysis of errors at invariant and polymorphic positions. In addition to assigning currently recognized alleles, this strategy is also capable of identifying new sequence variants.