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Detection of Variation in Highly Polymorphic Mhc Genes by Denaturing Gradient Gel Electrophoresis Using the DCode System

different denaturant concentrations. Virtually all of the alleles were differentiated by DGGE, with the exception of two sets of alleles with a single C-G transversion within 10 bp of the clamped primer and one set of alleles with a single A-T transversion at nt 237. The remaining 5 sets of alleles with single base differences at positions ranging from nt 85 to 282 (24 to 243 not including 5'-clamped primer) were differentiated, as well as all alleles with a greater number of differences. Thus, the range of denaturant gradient used and the addition of the GC-clamp facilitated sampling of sequence variation over virtually the entire A1 fragment.

As expected from the presence of two melting domains within the A1 sequence, time series analysis revealed a number of alleles which shifted relative positions on the gel when electrophoresed into different ranges of denaturant concentration. Moreover, some alleles that migrated differentially through the gel at low concentrations of denaturant became indistinguishable when electrophoresed in high denaturant concentrations (Figure 3). As a result, there was no single run-time that consistently separated all of the alleles. In the time series shown in Figure 3, the best separation of alleles was obtained at a run-time of 5.5 hr, which placed the DNA fragments between 48% and 53% denaturant, a range in which dissociation of the A1 low melting domain was completed and the high melting domain was initiated. At this run-time, 18 alleles were distinctive, and 5 sets of alleles migrated to similar positions and were consequently binned into 5 allelic groups.

For the homoduplex A1 allele fragments, increasing mig
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