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

Kristi M. Miller, Tobi J. Ming and Ruth E. Withler, Pacific Biological Station, Department of Fisheries and Oceans, Nanaimo, B.C. V9R 5K6, Canada


Introduction

We describe a rapid and sensitive method for the detection of nucleotide sequence variation which can be used for largescale screening of population markers. Denaturing gradient gel electrophoresis (DGGE) detects sequence variants of amplified fragments by the differences in their melting behavior (Fischer and Lerman 1983). By adding a GC-clamp to one of the primers, nearly all single base substitutions can be detected (Sheffield et al. 1989). Although DGGE has primarily been utilized for the detection of single base mutations in disease studies, it offers great potential for use in population analysis of genetic markers with higher levels of sequence variation. Herein we describe our DGGE analysis of a Mhc class I locus in salmon.

The methodology described was developed to identify the number and distribution of Mhc class I A1 (exon 2) alleles among chinook salmon (Oncorhynchus tshawytscha) populations (Miller et al. 1997, Miller and Withler 1997, 1998). DGGE detects 25 of the 28 identified A1 sequences, with a nucleotide sequence variation between them of 116 nt plus a 2 codon indel. By creating a unique network of control alleles, 22 of these alleles can be rapidly and accurately resolved by a single gel run condition. This technique has been used in surveys of over 20,000 salmon, representing two Mhc markers (class I A1 and A2) in two salmon species (chinook and coho salmon). A single person in our laboratory now analyses 160 salmon per day with DGGE using two DCode systems.
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