Double-gradient DGGE for optimized detection of DNA point mutations.
A novel technique is reported for screening point mutations is genomic DNA: double gradient, denaturing gradient gel electrophoresis (DG-DGGE). Unlike conventional DGGE, which exploits a single gradient of denaturing chemicals (typically urea and formamide) along the migration path to force the two hetero- and two homo-duplexes to partially unwind and separate, DG-DGGE superimposes a second (porous) gradient over the denaturing one. With the help of the sieving gradient, molecules such as the hetero-duplexes, which often produce curtains and smears instead of sharp zones, due to lack of a sharp melting transition, are re-compacted into remarkably narrow bands. Even homo-duplexes with minute melting temperature differences, giving a single band in DGGE, are resolved into two zones in DG-DGGE. The technique has been applied to the analysis of a number of point mutations in several exons of the cystic fibrosis transmembrane conductance regulator gene.
Cremonesi
L
L
I.R.C.C.S., H. San Raffaele, Milan, Italy.
Firpo
S
S
Ferrari
M
M
Righetti
P G
PG
Gelfi
C
C
D003550:Cystic Fibrosis / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D004586:Electrophoresis / Q000379:methods*; D005091:Exons; D006801:Humans; D009691:Nucleic Acid Denaturation; D017354:Point Mutation; D012967:Sodium Dodecyl Sulfate
C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D012967:Sodium Dodecyl Sulfate; D004247:DNA