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

L. A. Knapp, E. Lehmann, L. Hennes, M. E. Eberle, David Watkins

Research output: Contribution to journalArticle

37 Citations (Scopus)

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
Pages (from-to)170-177
Number of pages8
JournalTissue Antigens
Volume50
Issue number2
StatePublished - Aug 19 1997
Externally publishedYes

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Dichlororibofuranosylbenzimidazole
Histocompatibility Testing
Denaturing Gradient Gel Electrophoresis
Alleles
HLA-DRB3 Chains
HLA-DRB1 Chains
HLA-DRB4 Chains
Polymerase Chain Reaction
Unrelated Donors
DNA Primers
Oligonucleotide Probes
Bone Marrow Transplantation
Organism Cloning
Exons
Software

Keywords

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

ASJC Scopus subject areas

  • Immunology
  • Cell Biology

Cite this

High-resolution HLA-DRB typing using denaturing gradient gel electrophoresis and direct sequencing. / Knapp, L. A.; Lehmann, E.; Hennes, L.; Eberle, M. E.; Watkins, David.

In: Tissue Antigens, Vol. 50, No. 2, 19.08.1997, p. 170-177.

Research output: Contribution to journalArticle

Knapp, LA, Lehmann, E, Hennes, L, Eberle, ME & Watkins, D 1997, 'High-resolution HLA-DRB typing using denaturing gradient gel electrophoresis and direct sequencing', Tissue Antigens, vol. 50, no. 2, pp. 170-177.
Knapp, L. A. ; Lehmann, E. ; Hennes, L. ; Eberle, M. E. ; Watkins, David. / High-resolution HLA-DRB typing using denaturing gradient gel electrophoresis and direct sequencing. In: Tissue Antigens. 1997 ; Vol. 50, No. 2. pp. 170-177.
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