Kinetic and thermodynamic characterization of single-mismatch discrimination using single-molecule imaging
(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
- Gunnarsson, Anders LU ; Jönsson, Peter LU ; Zhdanov, Vladimir P and Höök, Fredrik LU
- publishing date
- 2009-08
- 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
-
- pmid:19509313
- 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
- 2024-01-14 14:17:01
@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) < 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.</p>}}, author = {{Gunnarsson, Anders and Jönsson, Peter and Zhdanov, Vladimir P and Höök, Fredrik}}, issn = {{1362-4962}}, keywords = {{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}}, doi = {{10.1093/nar/gkp487}}, volume = {{37}}, year = {{2009}}, }