Kinetic and thermodynamic characterization of single-mismatch discrimination using single-molecule imaging
Artikel i vetenskaplig tidskrift, 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 (K-d < 1 nM, k(off) < 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 degrees 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.

hybridization

dissociation

sequence

density oligonucleotide arrays

base-pairs

surface-plasmon resonance

dna

nanoparticles

microarrays

nucleotide polymorphisms

nucleic-acids

Författare

Anders Gunnarsson

Chalmers, Teknisk fysik, Biologisk fysik

Peter Jönsson

Chalmers, Teknisk fysik, Biologisk fysik

Vladimir Zhdanov

Chalmers, Teknisk fysik, Biologisk fysik

Fredrik Höök

Chalmers, Teknisk fysik, Biologisk fysik

Nucleic Acids Research

0305-1048 (ISSN) 1362-4962 (eISSN)

Vol. 37 14 e99 (art no)-

Ämneskategorier

Biofysik

DOI

10.1093/nar/gkp487