Detection of Variation in Highly Polymorphic Mhc Genes by Denaturing Gradient Gel Electrophoresis Using the DCode System ( e re-running of less than 2% ...)

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

e re-running of less than 2% of all fish analyzed. This rapid rate of analysis of nucleotide sequence variation makes it possible to quickly accumulate large databases, and to use DGGE for applied, real-time fisheries issues such as stock identification in mixed stock fisheries, escapement enumeration within river systems, and the identification of escaped farmed fish, to name a few.

The DGGE methodology described herein can be used on any region of DNA with nucleotide sequence variation. Best results are obtained with fragments of 300500 bp, but larger fragments can be analyzed by first digesting them with restriction endonucleases (Sheffield 1989). In DGGE, the detection of length variation is often superseded by nucleotide variation, but DGGE could be an invaluable tool for detecting homoplasy in microsatellites with similar migration on acrylamide gels. The use of DGGE virtually eliminates the need to sequence large numbers of clones to examine sequence variability. Direct sequencing of fragments cut out of gradient gels can further expedite the process. Finally, by careful use of standard networks of known alleles, DGGE is a highly sensitive and expeditious technique for detecting nucleotide sequence variation in large-scale population surveys.

References
1. Brown, J. M., Leebens-Mack, J. H., Thompson, J. N., Pellmyr, O. and Harrison, R. G., Phylogeography and host association in a pollinating seed parasite Greya politella (Lepidoptera: Prodoxidae), Mol. Ecol., 6, 215224 (1997).

2. Fischer, S. G. and Lerman, L. S., DNA fragments differing by single basepair substitutions are separated in denaturing gradient gels: Correspondence with melting theory, Proc
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( e re-running of less than 2% ...)Detection of Variation in Highly Polymorphic Mhc Genes by Denaturing Gradient Gel Electrophoresis Using the DCode System