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

ng in different run times, subsets of the "known" alleles were combined into three standard sets to use as controls in the following way: The relative positions of alleles run under optimal conditions were determined from the time series analysis, and the fastest (latest melting) to slowest (earliest melting) alleles were alternately placed into each one of the 3 standard sets. Alleles that were known to shift relative positions at different denaturing concentrations (as identified in the time-series analysis) were placed in different standard sets. The A, B, and C lanes in the time-series are the actual standard sets used in the population gels.

On the population gels, allele standard sets were loaded in three adjacent lanes in the center and on each side of the gel, with 20 individuals from one or two populations run in the remaining lanes. Parallel DGGE gels were photographed with a CCD camera utilizing a 1024 x 1024 cm pixel array format and analyzed with standard gel documentation software. Standard allele sets were used to confirm that the electrophoretic conditions were sufficient to resolve all of the standard alleles, to monitor the relative positions of shifting alleles on the gel, and to form a network on which to score individual alleles. Alleles within the standard lanes were assigned "DGGE allele" numbers ranging from 50 to 400, approximately corresponding with their (logarithmic) positions on the gel. New alleles identified in the population survey with denaturing points that did not coincide with any of the known DGGE alleles were added to standard lanes as they were found.


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