Nucleic Acids Research Advance Access published online on June 9, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp487
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Methods Online |
Kinetic and thermodynamic characterization of single-mismatch discrimination using single-molecule imaging
1Department of Applied Physics, Chalmers University of Technology, SE-41296 Gothenburg, Sweden and 2Boreskov Institute of Catalysis, Russian Academy of Sciences, Novosibirsk, 630090, Russia
*To whom correspondence should be addressed. Tel: +46 31 772 6130; Fax: +46 31 772 3134; Email: fredrik.hook{at}chalmers.se
Received April 2, 2009. Revised May 18, 2009. Accepted May 19, 2009.
A single-molecule detection setup based on total internal reflection fluorescence (TIRF) microscopy has been used to investigate association and dissociation kinetics of unlabeled 30mer DNA strands. Single-molecule sensitivity was accomplished by letting unlabeled DNA target strands mediate the binding of DNA-modified and fluorescently labeled liposomes to a DNA-modified surface. The liposomes, acting as signal enhancer elements, enabled the number of binding events as well as the residence time for high affinity binders (Kd < 1 nM, koff < 0.01 s–1) to be collected under equilibrium conditions at low pM concentrations. The mismatch discrimination obtained from the residence time data was shown to be concentration and temperature independent in intervals of 1–100 pM and 23–46°C, respectively. This suggests the method as a robust means for detection of point mutations at low target concentrations in, for example, single nucleotide polymorphism (SNP) analysis.