High throughput measurement of duplex, triplex and quadruplex melting curves using molecular beacons and a LightCycler.
We have used oligonucleotides containing molecular beacons to determine melting profiles for intramolecular DNA duplexes, triplexes and quadruplexes (tetraplexes). The synthetic oligonucleotides used in these studies contain a fluorophore (fluorescein) and quencher (methyl red) attached either to deoxyribose or to the 5 position of dU. In the folded DNA structures the fluorophore and quencher are in close proximity and the fluorescence is quenched. When the structures melt, the fluorophore and quencher are separated and there is a large increase in fluorescence. These experiments were performed in a Roche LightCycler; this requires small amounts of material (typically 4 pmol oligonucleotide) and can perform 32 melting profiles in parallel. We have used this technique to compare the stability of triplexes containing different base analogues and to confirm the selectivity of a triplex-binding ligand for triplex, rather than duplex, DNA. We have also compared the melting of inter- and intramolecular quadruplexes.
Darby
Richard A J
RA
Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK.
Sollogoub
Matthieu
M
McKeen
Catherine
C
Brown
Lynda
L
Risitano
Antonina
A
Brown
Nicholas
N
Barton
Christopher
C
Brown
Tom
T
Fox
Keith R
KR
D001391:Azo Compounds / Q000737:chemistry; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D019793:Fluorescein / Q000737:chemistry*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D054856:G-Quadruplexes; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*
D001391:Azo Compounds; D005456:Fluorescent Dyes; D008024:Ligands; D009838:Oligodeoxyribonucleotides; C051802:triplex DNA; C008492:methyl red; D004247:DNA; D019793:Fluorescein