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

ration distance through the denaturing gradient was strongly correlated with increasing overall GC content. However, the presence/absence of the 2-codon insert, and the GC content of the last 70 nt (distal to the GC-clamp) also had apparent influence on allelic migration. Prior to any allele dissociation, alleles separated according to size, with the alleles containing the two-codon insert migrating slower than those without the insert. However, once alleles began to dissociate, the insert was apparently less influential on allele migration than was the GC content, and some of the alleles that contained the insert and a high overall GC content migrated past alleles that did not contain the insert. In addition, because much of the gradient sampled variation in the low melt domain, GC content distal to the GC-clamp was more influential on the overall migration distances of the homoduplexes than GC content close to the clamp.

Alleles that shifted (exchanged relative positions in a ladder of migration distance) often did not share the presence/absence of the 2-codon insert and/or differed in their relative GC content in the low and the high melt domains. The most notoriously shifting allele (9; fourth band from the bottom of lane A in Figure 3) shifts position with four other alleles so readily it can actually shift across a single gel if the denaturing gradient is not completely even. Careful monitoring of the standard allele sets is necessary to accurately score this allele, and it is often used as a marker for gel to gel run uniformity.

To facilitate rapid and accurate scoring of alleles on population gels, including those that shift relative positions duri
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