title: High-resolution HLA-DRB typing using denaturing gradient gel electrophoresis and direct sequencing.
abstract: High-resolution HLA-DRB typing is required for bone marrow transplantation between unrelated donors and recipients and also for identification of novel HLA-DRB alleles. Here we describe a method for the unambiguous identification of HLA-DRB alleles using the polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE) and direct sequencing. The highly variable second exon of all HLA-DRB1, -DRB3, -DRB4, -DRB5, -DRB6 and -DRB7 alleles was amplified using a single pair of generic DRB-specific primers and alleles were separated by DGGE. DNA was then reamplified from plugs removed from the gel and the sequences of these alleles were determined using fluorescent-based sequencing and allele-assignment software. The validity of this typing procedure was confirmed by identification of HLA-DRB alleles for 17 individuals previously characterized by PCR-SSP and/or cloning and sequencing techniques. We identified 34 different HLA-DRB alleles in these 17 unrelated individuals. Importantly, our analysis revealed HLA-DRB1 alleles which had not been identified using the PCR-SSP typing technique. Additionally, alleles from the HLA-DRB3, -DRB4 and -DRB5 loci were identified. Whereas traditional HLA-DRB typing methods provide limited information or require the use of multiple oligonucleotide primers or probes, our technique provides a reliable, specific and relatively rapid way of identifying all HLA-DRB alleles for high-resolution tissue typing.
authors.affiliation: Wisconsin Regional Primate Research Center, University of Wisconsin, Madison, USA.
authors.forename: L A
authors.initials: LA
authors.lastname: Knapp
authors.forename: E
authors.initials: E
authors.lastname: Lehmann
authors.forename: L
authors.initials: L
authors.lastname: Hennes
authors.forename: M E
authors.initials: ME
authors.lastname: Eberle
authors.forename: D I
authors.initials: DI
authors.lastname: Watkins
mesh_terms: D000483:Alleles; D004247:DNA / Q000032:analysis; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005091:Exons; D006684:HLA-DR Antigens / Q000032:analysis / Q000145:classification*; D006650:Histocompatibility Testing / Q000379:methods; D006801:Humans; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D017931:DNA Primers; D006684:HLA-DR Antigens; D004247:DNA