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.
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 conditions consisted of 30 rounds of 90 seconds denaturation at 94 C, 90 seconds annealing at 55 C and 90 seconds extension at 74 C.
Optimal conditions for separation of HLA-DRB alleles using DGGE were established on a perpendicular denaturing gradient as described by Myers and coworkers.3, 4 Briefly, 100 l of the GC-clamped PCR product was electrophoresed in a 12.6% acrylamide (37.5:1 acrylamide:bisacrylamide) gel with an increasing gradient from 0% to 80% denaturant (100% denaturant=7 M urea and 40% formamide). The perpendicular DGGE was run in 1x TAE buffer at 60 C constant temperature. Samples were electrophoresed for 3.5 hours at 300 constant volts. Following electrophoresis, the perpendicular gel was silver-stained according to a method described by Bassam et al.,5 and the optimum gradient conditions were identified.
To separate HLA-DRB alleles, 30 l of the GC-clamped PCR products were mixed with 15 l of loading buffer and electrophoresed 4065% parallel denaturing gradient gel according to the method described by Myers et al.3, 4 As described for perpendicular DGGE, the 12.6% acrylamide gel was electrophoresed in 1x TAE buffer for 3.5 hours at 300 constant volts and at 60 C, constant temperature. Individual bands on the parallel DGGE gels were visualised using SYBR-Green stain and UV illumination.
Results and Discussion
A scheme for identifying HLA-DRB alleles using DGGE is shown in Figure 1.
Analysis of PCR-amplified HLA-DRB alleles by perpendicular DGGE revealed a single-domain melting structure when the GC-clamp was positioned at the 3 side of the PCR product and suggested that the optimal denaturing gradient for allele separation ranged from 4065%. Thus, our PCR products were electrophoresed on a 4065% parallel denaturing gradient. Figures 2a and 2b demonstrate separation of HLA-DRB alleles on 4065% parallel denaturing gradient gels.
To validate our DGGE separation method, we first analyzed samples from two unrelated individuals, for which PCR-SSCP and cloning and sequencing data were available. DGGE separated 46 bands from each individual (Figure 2a). Subsequent analysis of the two remaining individuals revealed up to 5 bands per individual (Figure 2b). Interestingly, each individual had a unique DGGE banding pattern. Moreover, when DGGE was combined with direct sequencing, we were able to identify all HLA-DRB alleles previously described using PCR-SSCP or cloning and sequencing. A comparison of results from three different typing methods is shown in Table 1.
High-resolution typing of HLA-DRB alleles generally relies upon labor-intensive cloning and sequencing techniques or PCR-SSCP, however we rapidly and unambiguously identified 11 different class II HLA-DRB alleles in four unrelated individuals. Thus, separation of HLA-DRB alleles using DGGE followed by direct sequencing represents a significant improvement over traditional class II typing methods. Incorporation of a GC-clamped PCR primer theoretically allows separation of alleles that differ by a single nucleotide substitution. In the present study, denaturing gradient gel electrophoresis resulted in physical separation of HLA-DRB1, -DRB3 and -DRB4 alleles. Notably, our results suggest that DGGE could also be used for rapid screening of HLA-DR subtype-identical unrelated bone marrow donor/recipient pairs. Specifically, when two indivi duals share the same combination of HLA-DRB alleles, they should also share the same banding patterns on gradient gels. Thus, the DGGE approach can be used for both HLA-DRB typing and screening.
1. Sheffield, V. C., Cox, D. R., Lerman, L. S. and Myers, R. M., Proc. Natl. Acad. Sci. USA, 86, 232236 (1989).
2. Knapp, L. A., Lehmann, E., Hennes, L., Eberle, M. E., Watkins, D. I., Tissue Antigens, 50, 170177 (1997).
3. Myers, R. M. and Maniatis, T., Cold Spring Harbor Symp. Quant. Biol., 51, 275284 (1986).
4. Myers, R. M., Sheffield, V. C. and Cox, D. R., In: (K. Davies, ed.) Genome Analysis: A Practical Approach, Oxford: IRL press, pp. 95139 (1988).
5. Bassam, B. J., Caetano-Anolles, G. and Gresshoff, P. M., Analytical Biochem., 196, 8083 (1991).
* The Polymerase Chain Reaction (PCR) process is covered by patents owned by Hoffmann-LaRoche. Use of the PCR process requires a license.
SYBR-Green is a trademark of Molecular Probes.
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