Melting temperature of surface-tethered DNA
2010 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 406, no 1, 34-40 p.Article in journal (Refereed) Published
A method for the accurate determination of the melting temperature (Tm) of surface-immobilized DNAduplexes that exploits the fluorescence-quenching properties of gold is reported. A thiolated singlestrandedDNA probe is chemisorbed onto a gold surface and then hybridized to a fluorophore-labeledcomplementary sequence. On formation of the duplex, the fluorescence of the label is effectivelyquenched by the gold surface. As the temperature is increased and the duplex denatures, the fluorophorelabel moves away from the gold surface and the fluorescence signal is again observed. The increase influorescence is measured as the temperature is ramped, and using first-derivative plots, the Tm is determined.To demonstrate the approach, the Tm of the cystic fibrosis DF508 mutation was determined inthree different phases: in solution, in suspension immobilized on gold nanoparticles, and immobilizedon gold film-coated substrate. The technique was further applied to optimize conditions for differentiationbetween a surface-immobilized DF508 mutant probe and a mutant/wild-type target exploitingincreasing stringency in varying salt and formamide concentrations. The approach has application inoptimization of assay conditions for biosensors that use gold substrates as well as in melting curveanalysis.
Place, publisher, year, edition, pages
Elsevier, 2010. Vol. 406, no 1, 34-40 p.
Melting temperature, Surface-immobilized DNA, Fluorescence quenching, Gold nanoparticles
IdentifiersURN: urn:nbn:se:liu:diva-92076DOI: 10.1016/j.ab.2010.06.049OAI: oai:DiVA.org:liu-92076DiVA: diva2:620079