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


Darby, R.A.J., Sollogoub, M., McKeen, M., Brown, Lynda J., Risitano, A., Brown, N., Barton, C., Brown, Tom and Fox, K.R. (2002) High throughput measurement of duplex, triplex and quadruplex melting curves using molecular beacons and a LightCycler. Nucleic Acids Research, 30, (9), e39-[9pp]. (doi:10.1093/nar/30.9.e39). (PMID:11972354).

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Description/Abstract

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.

Item Type: Article
ISSNs: 0305-1048 (print)
1362-4962 (electronic)
Keywords: resonance energy-transfer, double-stranded dna, g-quartet formation, 2', -aminoethoxy-modified oligonucleotides, dual recognition, scorpionprimers, promoter region, helix formation, nucleic-acids, fluorescence
Subjects: Q Science > QD Chemistry
Q Science > QH Natural history > QH426 Genetics
Divisions: University Structure - Pre August 2011 > School of Biological Sciences
University Structure - Pre August 2011 > School of Chemistry
ePrint ID: 24241
Date Deposited: 29 Mar 2006
Last Modified: 27 Mar 2014 18:13
Contact Email Address: k.r.fox@soton.ac.uk
URI: http://eprints.soton.ac.uk/id/eprint/24241

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