title: Psoralen-modified oligonucleotide primers improve detection of mutations by denaturing gradient gel electrophoresis and provide an alternative to GC-clamping.
abstract: 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.
authors.affiliation: INSERM U91, Hôpital Henri Mondor, Créteil, France.
authors.forename: B
authors.initials: B
authors.lastname: Costes
authors.forename: E
authors.initials: E
authors.lastname: Girodon
authors.forename: N
authors.initials: N
authors.lastname: Ghanem
authors.forename: M
authors.initials: M
authors.lastname: Chassignol
authors.forename: N T
authors.initials: NT
authors.lastname: Thuong
authors.forename: D
authors.initials: D
authors.lastname: Dupret
authors.forename: M
authors.initials: M
authors.lastname: Goossens
mesh_terms: 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
chemical_list: 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