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Kinetic and thermodynamic characterization of single-mismatch discrimination using single-molecule imaging

Gunnarsson, Anders LU ; Jönsson, Peter LU ; Zhdanov, Vladimir P and Höök, Fredrik LU (2009) In Nucleic Acids Research 37(14). p.99-99
Abstract

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... (More)

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.

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author
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Base Pair Mismatch, DNA, Kinetics, Liposomes, Microscopy, Fluorescence, Thermodynamics, Journal Article, Research Support, Non-U.S. Gov't
in
Nucleic Acids Research
volume
37
issue
14
pages
99 - 99
publisher
Oxford University Press
external identifiers
  • scopus:69049098982
ISSN
1362-4962
DOI
10.1093/nar/gkp487
language
English
LU publication?
no
id
879d7eca-c2ca-43bc-b3ed-83456611e87c
date added to LUP
2018-01-26 10:52:53
date last changed
2018-05-29 12:01:11
@article{879d7eca-c2ca-43bc-b3ed-83456611e87c,
  abstract     = {<p>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) &lt; 1 nM, k(off) &lt; 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.</p>},
  author       = {Gunnarsson, Anders and Jönsson, Peter and Zhdanov, Vladimir P and Höök, Fredrik},
  issn         = {1362-4962},
  keyword      = {Base Pair Mismatch,DNA,Kinetics,Liposomes,Microscopy, Fluorescence,Thermodynamics,Journal Article,Research Support, Non-U.S. Gov't},
  language     = {eng},
  number       = {14},
  pages        = {99--99},
  publisher    = {Oxford University Press},
  series       = {Nucleic Acids Research},
  title        = {Kinetic and thermodynamic characterization of single-mismatch discrimination using single-molecule imaging},
  url          = {http://dx.doi.org/10.1093/nar/gkp487},
  volume       = {37},
  year         = {2009},
}