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Nucleic Acids Research, 2002, Vol. 30, No. 9 e39
© 2002 Oxford University Press

High throughput measurement of duplex, triplex and quadruplex melting curves using molecular beacons and a LightCycler

Richard A. J. Darby, Matthieu Sollogoub1, Catherine McKeen2, Lynda Brown2, Antonina Risitano, Nicholas Brown, Christopher Barton, Tom Brown1 and Keith R. Fox*

Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK, 1Department of Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, UK and 2Oswel Research Products Ltd, Biological and Medical Sciences Building, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK

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.

* To whom correspondence should be addressed. Tel: +44 23 8059 4374; Fax: +44 23 8059 4459; Email: k.r.fox{at}soton.ac.uk Present addresses:Richard A. J. Darby, Life and Health Sciences, Pharmaceutical Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UKMatthieu Sollogoub, Département de Chimie, Ecole Normale Supérieure, 24 rue Lhomond, 75231 Paris Cédex 05, France


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