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Using the DCode System to Identify DNA Sequence Variation for Studies of Population Structure in Marine Organisms

alignment of oyster and fly 16S sequences, in order to construct a theoretical melting profile of the PCR product (Figure 1). The theoretical profile suggests a gradient range of 2040% denaturant.

After heteroduplex formation, pooled PCR products were loaded onto 6% acrylamide gels and run at constant temperature (60 C) for 45 h, followed by staining in ethidium bromide and visualization with a standard UV transilluminator.


Results
For the tautog, no previous genetic data were available, so the amount of genetic variation to be expected in the amplified fragments was unknown. Seventy-two individuals collected from the geographical range of the species (Rhode Island to Virginia) were examined. The LDH intron appeared to be virtually invariant, with only a single variant encountered among the 144 alleles screened. The cyt b fragment was likewise nearly monomorphic; 70 individuals possessed the common haplotype and two individuals were unique variants (singletons).

As an indirect test of the possibility that sequence variation in these fragments was going undetected with our protocols, we examined cyt b products from spot (Leiostomus xanthurus), a fish species shown by RFLP analysis to have high mtDNA sequence diversity (T. Lankford, University of Delaware, pers. comm.). Even without optimizing DGGE protocols, we detected four to six different haplotypes among the eight individuals examined, suggesting that the low diversity observed in the tautog was not due to poor resolution by DGGE.

For the oyster 16S product, more than 250 individuals were examined using heteroduplex DGGE. Initial screening showed that each geographical region tended to have a si
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