( DGGE gels of sufficient qual...)Detection of Variation in Highly Polymorphic Mhc Genes by Denaturing Gradient Gel Electrophoresis Using the DCode System

DGGE gels of sufficient quality to score reliably over time. The purity and age of the acrylamide and acrylamide denaturant solutions, the maximum voltage, and the overall run time all strongly affect the resolution and migration of fragments. Optimally, for our analysis DGGE was the most reliable when run slowly (6080 V) overnight (<17 hours). However, to maximize the throughput of the DCode system, we conduct the A1 analysis on daytime runs (at 120 V max) and analyze other loci on overnight runs. We use a single brand of acrylamide for analysis of each gene locus, and run a time series on new batches of acrylamide for slight run-time adjustments. Acrylamide over 6 months old and acrylamide denaturant solutions over 7 days old can be problematic and are avoided. In general, run-times for A1 are decreased by 5 minutes per day after the acrylamide solution is made. Furthermore, in order to accurately reproduce the run-time conditions from the time-series to the population gels, population gels were pre-run for 1.5 hr prior to loading. A more refined time series (from 5 to 6 hr) would decrease the necessary pre-run time.

Discussion
DGGE has been utilized for population analysis in only a limited number of studies (Brown et al. 1997; Norman et al. 1994), most merely to identify haplotypes among a few individuals for sequencing. Our study offers the first example of the potential use of DGGE for large scale population surveys of highly polymorphic DNA loci. In our laboratory, each technician runs 160 fish per day on DGGE (8 gels) using two DCode systems (80 fish each for daytime (A1 locus) and nighttime (A2locus) runs). Ambiguous scoring of shifting alleles necessitates th
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