( Michael Hepburn and Glen...)A Multiple Mutation Model System as a Test Development and Training Tool for Denaturing Gradient Gel Electrophoresis

Michael Hepburn and Glenn A. Miller, Ph.D., Molecular Profiling Laboratory, Genzyme, Framingham, MA

Introduction
The search for sequence variations in genomic DNA is important in the study of genes that play a role in the development of cancer and a variety of other single and multiple gene disorders. A wide variety of different methods to detect DNA sequence variations have been developed (Dianzani, 1993). One of these methods, denaturing gradient gel electrophoresis (DGGE), has been shown to be a sensitive, reproducible, reliable technology for use in many research environments (Myers, 1985a; 1985b; 1987).

DGGE involves electrophoresis of double stranded DNA fragments through a polyacrylamide gel containing a linear gradient of denaturant. As the fragment migrates through the denaturant gradient the strands begin to melt. The melting profile of a given DNA duplex is predominantly determined by its base sequence with greater GC content resulting in higher melting temperatures. The ability of DGGE to detect sequence alterations is based on the differential melting characteristics of homoduplex DNA versus heteroduplex DNA. As heteroduplex DNA migrates through the denaturant gradient the areas of non-homology melt at a lower temperature than the comparable homoduplex region. This results in an area of decreased mobility within the fragment, retarding its progress through the gel. This reduction in mobility results in a separation of homoduplex from heteroduplex fragments thereby identifying a region of sequence alteration.

The melting characteristics of a double stranded DNA fragment can be predicted a priori, allowing one to predetermine
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