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A high resolution HLA class I typing method with group specific amplification and novel SSO probe set
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Abstracts
7.4 #159
IDENTIFICATION OF ALLELES OF DRBI*04 GROUP USING BIOTINYLATED PRIMERS SPECIFIC FOR THE CODON 86 POLYMORPHISM. FJ Pellett, AB Begovich, CM Ng, JF Novotny, and JA Wade.Regional Histocompatibility Laboratory,The Toronto Hospital,Faculty of Medicine, University of Toronto, Canada and Roche Molecular Systems, Alameda, CA., Somerville, NJ. The hybridization to immobilized probes of amplified biotin labelled DNA, ie Reverse Line Blot (RLB) technique, allows for batching of samples and a turnaround time of less than 8 hours. Low resolution Class II HLA-DR molecular typing is routinely performed in our clinical lab using the AMPLICOR'" HLA DRB method on genomic DNA .We examined the feasibility of using the same immobilized probes for high level resolution typing using biotinylated specific primers. The 22 alleles of DRB I *04 can be divided into 2 groups - those with either glycine (0401,0405,0407-0409,0414,0416,0417,0419-0421) or valine (0402-0404,0406,0410-0413, 0415,0418,0422) at codon 86. A DRB I *04 group specific forward primer and reverse primers detecting the codon 86 polymorphism were used to amplify genomic DNA in a single cycle PCR program. Annealing temperatures of 60,62,63,64 and 65"C were assessed by gcl electrophoresis of PCR amplified product from genomic DNA extracted from well characterized homozygous and heterozygous DR4 positive and negative cells. None of the DR4 negative DNA amplified. 64°C was the optimal annealing temperature for DR4 positive DNA and subsequently was used in a modified RLB PCR cycling profile. Hybridization patterns of the biotin labelled amplified DNA to the 29 inunobilized probes could theoretically distinguish 13 of the 22 alleles of DRB I *04 with the unresolved groups being: 0401/0416/0421; 040310406; 0407/0420 and 0408/0419. Hybridization patterns after preferential amplification were as predicted . This preferential amplification can be extended to other antigen groups providing clinical HLA laboratories with a high throughput, fast turnaround technique using a common standardized panel of probes. RLB is easily amenable to automation (eg PCR setup, hybridization) and to computerization (eg analysis of probe patterns) to facilitate handling large numbers of samples with a minimum of errors.
7.4 #160
A HIGH RESOLUTION HLA CLASS I TYPING METHOD WITH GROUP SPECIFIC AMPLIFICATION AND NOVEL SSO PR08E SET. D Gallardo, R ArgClello, I Rojas, AM Little, H Avakian, JA Madrigal. Anthony Nolan Research Institute, London, U.K. Ambiguities in SSO typing are frequently the result of overlapping sequence homologies in heterozygous samples. This is a particularly serious impediment to high resolution analysis of HLA class I alleles. We have devised a SSP group amplification approach which yields allelic products which can be resolved by a set of 40 motif specific probes that are universal for HLA-A, -8 and-Cwo Group specific SSP mixes have been devised for each locus amplification. The group amplification products can be probed unambiguously by a set of probes which have been targeted at the recombinational motifs. 36 mixes - 9 HLA-A, 18 -8 and 9 for -Cw have been used to cover all known class I alleles. The PCR products were analysed in agarose gel electrophoreSis to determine the productive amplifications. The amplified segments from HLA-A, -8 and -Cw reactions were hybridised on the same nylon membrane and were probed simultaneously. Patterns produced by motif specific probes identified all 80 different class I alleles that were tested. This method is a fast, economical and high resolution technique for typing HLA class I genes at the allelic level.
Abstracts
7.4 #159
IDENTIFICATION OF ALLELES OF DRBI*04 GROUP USING BIOTINYLATED PRIMERS SPECIFIC FOR THE CODON 86 POLYMORPHISM. FJ Pellett, AB Begovich, CM Ng, JF Novotny, and JA Wade.Regional Histocompatibility Laboratory,The Toronto Hospital,Faculty of Medicine, University of Toronto, Canada and Roche Molecular Systems, Alameda, CA., Somerville, NJ. The hybridization to immobilized probes of amplified biotin labelled DNA, ie Reverse Line Blot (RLB) technique, allows for batching of samples and a turnaround time of less than 8 hours. Low resolution Class II HLA-DR molecular typing is routinely performed in our clinical lab using the AMPLICOR'" HLA DRB method on genomic DNA .We examined the feasibility of using the same immobilized probes for high level resolution typing using biotinylated specific primers. The 22 alleles of DRB I *04 can be divided into 2 groups - those with either glycine (0401,0405,0407-0409,0414,0416,0417,0419-0421) or valine (0402-0404,0406,0410-0413, 0415,0418,0422) at codon 86. A DRB I *04 group specific forward primer and reverse primers detecting the codon 86 polymorphism were used to amplify genomic DNA in a single cycle PCR program. Annealing temperatures of 60,62,63,64 and 65"C were assessed by gcl electrophoresis of PCR amplified product from genomic DNA extracted from well characterized homozygous and heterozygous DR4 positive and negative cells. None of the DR4 negative DNA amplified. 64°C was the optimal annealing temperature for DR4 positive DNA and subsequently was used in a modified RLB PCR cycling profile. Hybridization patterns of the biotin labelled amplified DNA to the 29 inunobilized probes could theoretically distinguish 13 of the 22 alleles of DRB I *04 with the unresolved groups being: 0401/0416/0421; 040310406; 0407/0420 and 0408/0419. Hybridization patterns after preferential amplification were as predicted . This preferential amplification can be extended to other antigen groups providing clinical HLA laboratories with a high throughput, fast turnaround technique using a common standardized panel of probes. RLB is easily amenable to automation (eg PCR setup, hybridization) and to computerization (eg analysis of probe patterns) to facilitate handling large numbers of samples with a minimum of errors.
7.4 #160
A HIGH RESOLUTION HLA CLASS I TYPING METHOD WITH GROUP SPECIFIC AMPLIFICATION AND NOVEL SSO PR08E SET. D Gallardo, R ArgClello, I Rojas, AM Little, H Avakian, JA Madrigal. Anthony Nolan Research Institute, London, U.K. Ambiguities in SSO typing are frequently the result of overlapping sequence homologies in heterozygous samples. This is a particularly serious impediment to high resolution analysis of HLA class I alleles. We have devised a SSP group amplification approach which yields allelic products which can be resolved by a set of 40 motif specific probes that are universal for HLA-A, -8 and-Cwo Group specific SSP mixes have been devised for each locus amplification. The group amplification products can be probed unambiguously by a set of probes which have been targeted at the recombinational motifs. 36 mixes - 9 HLA-A, 18 -8 and 9 for -Cw have been used to cover all known class I alleles. The PCR products were analysed in agarose gel electrophoreSis to determine the productive amplifications. The amplified segments from HLA-A, -8 and -Cw reactions were hybridised on the same nylon membrane and were probed simultaneously. Patterns produced by motif specific probes identified all 80 different class I alleles that were tested. This method is a fast, economical and high resolution technique for typing HLA class I genes at the allelic level.