Improvements in gel composition and electrophoretic conditions for broad-range mutation analysis by denaturing gradient gel electrophoresis.
Denaturing gradient gel electrophoresis (DGGE) is believed to be the most powerful pre-screening method for mutation detection currently available, being used mostly on an exon-by-exon basis. Broad-range DGGE for the analysis of multiple fragments or an entire gene is rarely applied. We and others have already shown that one or two DGGE conditions are usually sufficient to analyse an entire gene. Conditions, however, have never been profoundly tested and compared with alternative methods suggested in the literature. Trying to do so in this study, we found significant differences between the various gel systems. The optimal conditions we found for broad-range DGGE include 9% polyacrylamide for the gel, a denaturing gradient with a difference of 30-50% between the lowest and the highest concentration of denaturant, and electrophoresis in 0.5x TAE buffer at a voltage >100 V and <200 V.
Hayes
V M
VM
Department of Medical Genetics, University of Groningen, Antonius Deusinglaan 4, 9713 AW Groningen, The Netherlands.
Wu
Y
Y
Osinga
J
J
Mulder
I M
IM
van der Vlies
P
P
Elfferich
P
P
Buys
C H
CH
Hofstra
R M
RM
D000180:Acrylic Resins / Q000737:chemistry; D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D004563:Electrochemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005782:Gels; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction
D000180:Acrylic Resins; D004273:DNA, Neoplasm; D005782:Gels; C016680:polyacrylamide gels