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Separation of HLA-DRB Alleles by Denaturing Gradient Gel Electrophoresis using the DCode System

L. A. Knapp,1, 2 E. Lehmann,2 L. Hennes,3 M. E. Eberle,2 and D. I. Watkins2, 3
1Department of Biological Anthropology, University of Cambridge,U.K.
2Wisconsin Regional Primate Research Center, University of Wisconsin,
3University of Wisconsin Histocompatibility Laboratory, University of Wisconsin Hospital and Clinics, Madison, Wisconsin, U.S.A.


Introduction
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 separation of HLA-DRB alleles, following PCR amplification of the highly variable second exon of HLA-DRB alleles, using denaturing gradient gel electrophoresis (DGGE). When separation of HLA-DRB alleles is followed by direct sequencing, this technique provides a reliable, specific and relatively rapid way of identifying all HLA-DRB alleles for high-resolution tissue typing.


Materials and Methods
Genomic DNA was extracted from peripheral blood or B-cell lines from four unrelated individuals. Two samples had been typed previously for HLA-DRB alleles using both PCR-SSCP and cloning and sequencing and two samples had been characterized using cloning and sequencing techniques. Thirty to forty nanograms of genomic DNA was amplified in 50 l of 1x PCR* buffer (pH 8.5), 1.5 mM MgCl2, 2.5 mM of each of the four deoxyribonucleotide triphosphates (dGTP, dATP, dTTP and dCTP), 25 picomoles of each of the forward and GC-clamped1 reverse primers2 and 1 U of Taq polymerase. Cycling
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