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HLA-B typing by separation of alleles followed by direct automated sequencing
Abstracts
115
P624 AN ANALYSIS
OF MIGRATION ANOMALIES OBSERVED IN MIXED·BASE CLASS I DNA SEQUENCE FRAGMENTS. LeslieJohnston-Dow and Mel Kronick. AppliedBiosystems Divisonof PerkinElmer,FosterCity,CA Seven! methods havebeen recently ck:scribed for !he determination of !he sequeeces '"" resulting alleleassignment of HLA samples for bolh Class I and Class It has been observed in !he d3Ia from the Class n methods that an occasional polymorphic sample will give rise to a sequence laddeo" that exhibits differing mobilities at mixed-ba'le positions. Recently, SBT methods have been descnred for the Class I genes that involveIbe simoltaneoos sequencing of bothallelesin these higbly polymorphic genes. It was CXjJeCIod that sequence mobility anomalies would also be seen in Class I data sincea similarsequencing stralegy wasused. However. while !he polymorphism in !he Class n genes tend to cluster in dofined bypervariable regions, the Class I gene polymorphisms tendnot 10 occurin clusters, It has been suggested by os and other> that thislackof clustering and !he higherdegreeof polymorphism in the Class I genesmight resultin an increasein !he appareot mobility differences relative10 what is observed in the Class n genes, Indeed, in certain Class I mixed base samples we have oblelved situations in whichthe mobility anomalies are so markedas to appear 10 resultfroman inserted
P625
A SEQUENCE DATA EVALUATION METHOD FOR SEQUENCING BASED HLA CLASS I SUBTYPING
Bjorkesten Lennart, Pharmacia Biotech, Uppsala, Sweden
n.
A data evaluation method is presented for sequencing based lILA class I subtyping. Compared to class II genes, the class I genes generally require a much larger sequence region for the non-ambigous subtyping. This implies the need for including more than one sequencing fragment in the typing procedure. The method presented incorporates lILA typing based on multiple sequencing fragments. The method also includes confidence calculations based on consensus information between overlapping sequence fragments and On data in the conserved regions
temperanne,
P626
P627
DIRECT SUBTYPING OF HLA·B and -A GENES BY AUTO· MATED SEQUENCING FOLLOWING REVERSE TRANSCRIPTION·peR
eDNA SEQUENCING BASED TYPING OF HLA-A ALLELES USING A ROBOTIC WORKSTATION
KnipperAJ, Enczmann J, Hakenberg P, KOgler G, Wernet P. Bone Marrow Donor Center (with Transplantation Immunology and Stem Cell Bank). Heinrich-Heine University, Dusseldorf,Germany. A peR approach for the amplification of distinct locus specific exons and the prevention of amplifying pseudogenes are two prerequisites of a molecuiar genetic typing strategy for HLA class I genes. A direct subtyping approach for the HLA-B and -Alocus on the basis of an mRNA template has been developed, here. RNAwas isolated from peripheral blood mononuclear cells using an RNA isolation kit. A subsequent reverse transcription resulted in the generation of eDNA, which was used as a template for the amplification of 734bp (-6) and 807bp (-A)eDNAfragment. For direct sequencing of the PCR products a solidphase single-strand separation was performed and three different internal sequencing primers were employed in separate reactions to produce overlapping sequencing iadders (L1-COR Model 4000 DNA Sequencer). The derived sequences were aligned against reference sequences. The analysis of 35 patient and unrelated marrow donor samples demonstrated the applicability of this approach for routine use. By incorporating the serological pretyping data and the achievable sequence information of this sUbtyping strategy (exon 2, 3, and part of exon 4) in a computer analysis only 53 out of 8001 (B-Iocus) and 4 out of 1711 (A-locus) heterozygous alleliccombinations were ambigious. Thus, the application of this automated sequencing-based typing approach revealed the highest possible typing resolutionof the extracellular regionof HLA-B and also -Alocus genes.
Fischer Gottfried F., Fae Ingrid, Petrasek Monika, Broer Edeltraud, Mayr WolfgangR. Department of Blood Group Serology, University of Vienna, Wiihringergiirtel 18-20, A-1090 Wien
P628 HLA-B TYPING BY SEPARATION OF ALLELES FOLLOWED BY DffiECT AUTOMATED SEQUENCING Mary Eberle. Leslie A. Knapp, Kris Aiyer". David I. Watkins Universityof Wisconsin. Madison, WI, USA *Pel-Freez Clinical Systems, Brown Deer, WI, USA
Due to the enormous allelic diversity of the HLA-B locus, it has been difficult to design an unambiguous method for molecular typing of alleles at this locus. Here we describe an alternativeapproach for the automated sequencing of HLA-B alleles in which each allele is sequeuced independently. HLA-B alleles are co-amplified during a locus-specific reverse transcription from RNA followed by peR. After amplification, alleles are separated using denaturing gradient gel electrophoresis which separates DNA fragments based on their sequence composition. Bands are excised and direetly sequenced. The sequences are then aligned to a database of published HLA-B allele sequences, and an allele assignment is made. With the physical separation and independentexamination of alleles, we avoid the problems associated with interpreting heterozygousdata, Using this method, we analyzed cells of various types from different sources. Samples included immortalized cell lines from the International HistocompatibilityWorkshop and aboriginal peoples. and blood samples from a panel of cells which were difficult to type using serological techniques. In all cases, an unambiguous typing was assigned to each sample.
Sequencing based typing can refine the resolution of serologic results when one specificity comprises several alleles. We consider three issues necessary to obtain robust and reliable sequencing results of lILA class I alleles: (i) both strands of the amplification product should be sequenced, (ii) at least exon 2 plus exon 3 of the gene should be analysed, (iii) possibly all heterozygous positions, no matter whether they are in exon 2 and/or exon 3 should be resolved by sequencing a group specific amplification product. For HLA-A typing these goals can be achieved by using eDNA transcribed with an lILA-A locus specific primer as template for generating of two overlapping PCR amplification fragments which span exon I to exon 4. In each offour amplification reactions one of the primers contains a universal sequencing primer binding site added to its 5' end allowing dye primer sequencing of amplification products in an automated workstation. This typing approach has been validated on 50 serologically typed individuals.
P629
A GENERAL APPROACH FOR SEQUENCING-BASED TYPING OF HLA-A Bini D, Johnston-Dow L, Pozzi S, Bengston A, Ferrara GB, Kronik M. Immunogenetics Lab. CBA/IST, Genova, Italy Perkin Elmer, ABI Division, Foster City, CA, USA Here we present a method for the sequence analysis of HLA-A. This technique is based upon PCR amplification and direct fluorescent sequencing of exon 2 and 3 of this gene followed by software analysis. A locus-specific PCR reaction using primers located in exon I and exon 5 supplies a 2 Kb DNA fragment which is used with no purification in a sequencing reaction. We adopted a fluorescently-Iabelled primer cycle sequencing chemistry using AmplitaqTM DNA Polymerase FS (Perkin-Elmer). 4 sequencing reactions are needed to get the information sufficient to determine the HLA type. The sequence data obtained after electrophoresis on an Applied Biosytems 377 DNA Sequencer is analyzed by a software which performs sequence comparison against allele library and provides HLA-A typing for either homozygous and heterozygous samples. This protocol allowed us to present the local DNA panel (120 samples) and the XII IHWC Saquence Based Typing HLA-A key reference panel (34 samples).
115
P624 AN ANALYSIS
OF MIGRATION ANOMALIES OBSERVED IN MIXED·BASE CLASS I DNA SEQUENCE FRAGMENTS. LeslieJohnston-Dow and Mel Kronick. AppliedBiosystems Divisonof PerkinElmer,FosterCity,CA Seven! methods havebeen recently ck:scribed for !he determination of !he sequeeces '"" resulting alleleassignment of HLA samples for bolh Class I and Class It has been observed in !he d3Ia from the Class n methods that an occasional polymorphic sample will give rise to a sequence laddeo" that exhibits differing mobilities at mixed-ba'le positions. Recently, SBT methods have been descnred for the Class I genes that involveIbe simoltaneoos sequencing of bothallelesin these higbly polymorphic genes. It was CXjJeCIod that sequence mobility anomalies would also be seen in Class I data sincea similarsequencing stralegy wasused. However. while !he polymorphism in !he Class n genes tend to cluster in dofined bypervariable regions, the Class I gene polymorphisms tendnot 10 occurin clusters, It has been suggested by os and other> that thislackof clustering and !he higherdegreeof polymorphism in the Class I genesmight resultin an increasein !he appareot mobility differences relative10 what is observed in the Class n genes, Indeed, in certain Class I mixed base samples we have oblelved situations in whichthe mobility anomalies are so markedas to appear 10 resultfroman inserted
P625
A SEQUENCE DATA EVALUATION METHOD FOR SEQUENCING BASED HLA CLASS I SUBTYPING
Bjorkesten Lennart, Pharmacia Biotech, Uppsala, Sweden
n.
A data evaluation method is presented for sequencing based lILA class I subtyping. Compared to class II genes, the class I genes generally require a much larger sequence region for the non-ambigous subtyping. This implies the need for including more than one sequencing fragment in the typing procedure. The method presented incorporates lILA typing based on multiple sequencing fragments. The method also includes confidence calculations based on consensus information between overlapping sequence fragments and On data in the conserved regions
temperanne,
P626
P627
DIRECT SUBTYPING OF HLA·B and -A GENES BY AUTO· MATED SEQUENCING FOLLOWING REVERSE TRANSCRIPTION·peR
eDNA SEQUENCING BASED TYPING OF HLA-A ALLELES USING A ROBOTIC WORKSTATION
KnipperAJ, Enczmann J, Hakenberg P, KOgler G, Wernet P. Bone Marrow Donor Center (with Transplantation Immunology and Stem Cell Bank). Heinrich-Heine University, Dusseldorf,Germany. A peR approach for the amplification of distinct locus specific exons and the prevention of amplifying pseudogenes are two prerequisites of a molecuiar genetic typing strategy for HLA class I genes. A direct subtyping approach for the HLA-B and -Alocus on the basis of an mRNA template has been developed, here. RNAwas isolated from peripheral blood mononuclear cells using an RNA isolation kit. A subsequent reverse transcription resulted in the generation of eDNA, which was used as a template for the amplification of 734bp (-6) and 807bp (-A)eDNAfragment. For direct sequencing of the PCR products a solidphase single-strand separation was performed and three different internal sequencing primers were employed in separate reactions to produce overlapping sequencing iadders (L1-COR Model 4000 DNA Sequencer). The derived sequences were aligned against reference sequences. The analysis of 35 patient and unrelated marrow donor samples demonstrated the applicability of this approach for routine use. By incorporating the serological pretyping data and the achievable sequence information of this sUbtyping strategy (exon 2, 3, and part of exon 4) in a computer analysis only 53 out of 8001 (B-Iocus) and 4 out of 1711 (A-locus) heterozygous alleliccombinations were ambigious. Thus, the application of this automated sequencing-based typing approach revealed the highest possible typing resolutionof the extracellular regionof HLA-B and also -Alocus genes.
Fischer Gottfried F., Fae Ingrid, Petrasek Monika, Broer Edeltraud, Mayr WolfgangR. Department of Blood Group Serology, University of Vienna, Wiihringergiirtel 18-20, A-1090 Wien
P628 HLA-B TYPING BY SEPARATION OF ALLELES FOLLOWED BY DffiECT AUTOMATED SEQUENCING Mary Eberle. Leslie A. Knapp, Kris Aiyer". David I. Watkins Universityof Wisconsin. Madison, WI, USA *Pel-Freez Clinical Systems, Brown Deer, WI, USA
Due to the enormous allelic diversity of the HLA-B locus, it has been difficult to design an unambiguous method for molecular typing of alleles at this locus. Here we describe an alternativeapproach for the automated sequencing of HLA-B alleles in which each allele is sequeuced independently. HLA-B alleles are co-amplified during a locus-specific reverse transcription from RNA followed by peR. After amplification, alleles are separated using denaturing gradient gel electrophoresis which separates DNA fragments based on their sequence composition. Bands are excised and direetly sequenced. The sequences are then aligned to a database of published HLA-B allele sequences, and an allele assignment is made. With the physical separation and independentexamination of alleles, we avoid the problems associated with interpreting heterozygousdata, Using this method, we analyzed cells of various types from different sources. Samples included immortalized cell lines from the International HistocompatibilityWorkshop and aboriginal peoples. and blood samples from a panel of cells which were difficult to type using serological techniques. In all cases, an unambiguous typing was assigned to each sample.
Sequencing based typing can refine the resolution of serologic results when one specificity comprises several alleles. We consider three issues necessary to obtain robust and reliable sequencing results of lILA class I alleles: (i) both strands of the amplification product should be sequenced, (ii) at least exon 2 plus exon 3 of the gene should be analysed, (iii) possibly all heterozygous positions, no matter whether they are in exon 2 and/or exon 3 should be resolved by sequencing a group specific amplification product. For HLA-A typing these goals can be achieved by using eDNA transcribed with an lILA-A locus specific primer as template for generating of two overlapping PCR amplification fragments which span exon I to exon 4. In each offour amplification reactions one of the primers contains a universal sequencing primer binding site added to its 5' end allowing dye primer sequencing of amplification products in an automated workstation. This typing approach has been validated on 50 serologically typed individuals.
P629
A GENERAL APPROACH FOR SEQUENCING-BASED TYPING OF HLA-A Bini D, Johnston-Dow L, Pozzi S, Bengston A, Ferrara GB, Kronik M. Immunogenetics Lab. CBA/IST, Genova, Italy Perkin Elmer, ABI Division, Foster City, CA, USA Here we present a method for the sequence analysis of HLA-A. This technique is based upon PCR amplification and direct fluorescent sequencing of exon 2 and 3 of this gene followed by software analysis. A locus-specific PCR reaction using primers located in exon I and exon 5 supplies a 2 Kb DNA fragment which is used with no purification in a sequencing reaction. We adopted a fluorescently-Iabelled primer cycle sequencing chemistry using AmplitaqTM DNA Polymerase FS (Perkin-Elmer). 4 sequencing reactions are needed to get the information sufficient to determine the HLA type. The sequence data obtained after electrophoresis on an Applied Biosytems 377 DNA Sequencer is analyzed by a software which performs sequence comparison against allele library and provides HLA-A typing for either homozygous and heterozygous samples. This protocol allowed us to present the local DNA panel (120 samples) and the XII IHWC Saquence Based Typing HLA-A key reference panel (34 samples).