Validation of double gradient denaturing gradient gel electrophoresis through multigenic retrospective analysis.
Among established techniques for the identification of either known or new mutations, denaturing gradient gel electrophoresis (DGGE) is one of the most effective. However, conventional DGGE is affected by major drawbacks that limit its routine application: the different denaturant gradient ranges and migration times required for different DNA fragments. We developed a modified version of DGGE for high-throughput mutational analysis, double gradient DGGE (DG-DGGE), by superimposing a porous gradient over the denaturant gradient, which maintains the zone-sharpening effect even during lengthy analyses. Because of this innovation, DG-DGGE achieves the double goals of retaining full effectiveness in the detection of mutations while allowing identical run time conditions for all fragments analyzed. Here we use retrospective analysis of a large number of well-characterized mutations and polymorphisms, spanning all predicted melting domains and the whole genomic sequence of three different genes--the cystic fibrosis transmembrane conductance regulator (CFTR), the beta-globin, and the p53 genes--to demonstrate that DG-DGGE may be applied to the rapid scanning of any sequence variation.
Cremonesi
L
L
Instituto di Ricovero e Cura a Caraterre Scientifico, O San Raffaele, Unità di Genetica e Diagnostica Molecolare, Via Olgettina 60, 20132 Milan, Italy.cremonisi@laura.hsr.it
Carrera
P
P
Fumagalli
A
A
Lucchiari
S
S
Cardillo
E
E
Ferrari
M
M
Righetti
S C
SC
Zunino
F
F
Righetti
P G
PG
Gelfi
C
C
D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D004247:DNA / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D004586:Electrophoresis / Q000379:methods*; D005914:Globins / Q000235:genetics; D006801:Humans; D009154:Mutation; D009691:Nucleic Acid Denaturation; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
C505032:CFTR protein, human; D016159:Tumor Suppressor Protein p53; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005914:Globins; D004247:DNA