( L. A. Knapp 1 2 E. Le...)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. Watkins^{2, 3
1}Department 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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