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Anne-Lise Brresen-Dale, Sigrid Lystad, and Anita Langerd, Department of Genetics, Institute for Cancer Research, The Norwegian Radium Hospital, N-0310 Oslo, Norway.
Introduction
Temporal temperature gradient electrophoresis (TTGE) exploits the principles
upon which denaturing gradient gel electrophoresis (DGGE) and constant
denaturing gel electrophoresis (CDGE) are based. TTGE combines some of
the advantages of these techniques, and eliminates some of the problems.
The focusing of bands obtained by DGGE seems to be retained, and in TTGE
there is no need for a chemical denaturing gradient which is the advantage
in CDGE. The problem of determining the exact running time and denaturing
conditions is eliminated, and since the denaturing conditions in TTGE
span a wider range, several fragments with different melting behavior
can be analyzed on the same gel. A new electrophoretic instrument from
Bio-Rad Laboratories called the DCode universal mutation detection system
has the ability to perform TTGE.
Improvements of DGGE by CDGE
DGGE, which is based on the discontinuous phenomenon of strand dissociation,
allows the resolution of DNA fragments differing by as little as a single
nucleotide substitution.1 In mutation analysis using parallel DGGE, field
strength, temperature, and run time must be strictly controlled to achieve
reproducible results. DGGE is complicated by the difficulties of choosing
the exact running time and gel conditions to achieve the optimal separation.
By running a DGGE gel too long, an achieved separation decreases, and
may even be lost. Mutations present in a homo- or hemizygote state, where
heteroduplexes are not formed, will be
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