High-resolution HLA-DRB typing using denaturing gradient gel electrophoresis and direct sequencing

L. A. Knapp, E. Lehmann, L. Hennes, M. E. Eberle, D. I. Watkins

Research output: Contribution to journalArticle

37 Scopus citations

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 previo usly 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.

Original languageEnglish (US)
Pages (from-to)170-177
Number of pages8
JournalTissue Antigens
Volume50
Issue number2
DOIs
StatePublished - Jan 1 1997
Externally publishedYes

Keywords

  • DGGE
  • DNA
  • HLA-DRB
  • PCR
  • Sequencing
  • Typing

ASJC Scopus subject areas

  • Immunology
  • Cell Biology

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