Methods and Results
Single locus Mhc class I A1 primers were used to amplify fragments of 301307 bp (including primers) (Miller et al. 1996). Because a large proportion of the sequence variation and the lower melting domain reside on the 3' end of the fragment, a GC-clamp was added to the 5' primer.
DGGE was performed on the DCode system. We determined initial DGGE conditions by electrophoresing three the amplified chinook salmon A1 chinook alleles (5, 9, and 10), differing from one another by 712 nt and a 2 codon indel on a perpendicular gradient gel (Figure 1). Dissociation of A1 alleles occurred over a 3654% gradient. The presence of three distinctive but overlapping curves indicated that all three A1 alleles melted at different concentrations of denaturant and could be differentiated on parallel DGGE gels, but might shift relative positions if run into different portions of the denaturant gradient. Further analysis of these alleles by WinMelt software again showed three distinctive curves, with two melting domains still apparent (despite the GC-clamp), and elucidated the crossover point between alleles 9 and 10 at nt position 207 (Figure 2). This crossover point directly coincided with the location of the 2 codon insert, present in allele 10, but absent in allele 9. Thus, it appeared that the 2 codon insert and the presence of two melting domains may cause shifting of some alleles on parallel gels.
Time-series analyses on 4065% (7.5% acrylamide) parallel
gradient gels was used to determine the run-time that
separated most of the 28 A1 alleles (homoduplexes only) and
to assess changes in the relative positions of alleles at