Psoralen-modified oligonucleotide primers improve detection of mutations by denaturing gradient gel electrophoresis and provide an alternative to GC-clamping.
Denaturing gradient gel electrophoresis (DGGE), a mutation-scanning procedure separating DNA fragments differing by as little as a single base change, is widely used in studies of genomic nucleotide sequence variability. The efficiency of the technique is greatly enhanced by attaching, through polymerase chain reaction (PCR) incorporation, a long GC-tail to the test DNA sequence which, as a result, becomes analysable throughout. As synthesis of GC-rich specific PCR primers is costly and time-consuming, we attempted to clamp the DNA fragment using a psoralen derivative (ChemiClamp) that promotes photo-induced cross-linking at one end. We found that this procedure provides an attractive alternative to GC-clamp in DGGE (and temperature gradient gel electrophoresis) and should prove useful in both research and diagnostic laboratories.
Costes
B
B
INSERM U91, Hôpital Henri Mondor, Créteil, France.
Girodon
E
E
Ghanem
N
N
Chassignol
M
M
Thuong
N T
NT
Dupret
D
D
Goossens
M
M
D003432:Cross-Linking Reagents; D003550:Cystic Fibrosis / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004252:DNA Mutational Analysis / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005069:Evaluation Studies as Topic; D005091:Exons; D011564:Furocoumarins; D005914:Globins / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic
C505032:CFTR protein, human; D003432:Cross-Linking Reagents; D011564:Furocoumarins; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; C081486:5-(hexyloxy)psoralen; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005914:Globins