Probing DNA surface attachment and local environment using single molecule spectroscopy

Osborne, M A, Barnes, C L, Balasubramanian, S and Klenerman, D (2001) Probing DNA surface attachment and local environment using single molecule spectroscopy. Journal of Physical Chemistry B, 105 (15). pp. 3120-3126. ISSN 1089-5647

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Abstract

We have employed single molecule fluorescence spectroscopy, using a total internal reflection geometry and wide-angle detection, to study the attachment of singly fluorescently labeled DNA to a silica surface by either a streptavidin-biotin of a covalent linkage. In both cases the DNA is highly monodispersed with no evidence for aggregation. The covalent coupling gave higher signal-to-noise than the streptavidin-biotin Linkage and was therefore studied in more detail. Two components in the photobleaching times, corresponding to different states of the tetramethyl rhodamine probe, were observed: a short and long component with populations in the ratio 6.7:1. Only rarely was interconversion between these two states detected during the 30-s observation time of the experiment. Hybridization experiments using a complementary strand of DNA labeled with a different fluorophore gave a low level of colocalized fluorescence, indicating a significant fraction of the surface attached DNA was not available for hybridization. These results are consistent with the surface attached DNA spending significant time collapsed on the surface.

Item Type: Article
Additional Information: First measurement of mono-dispersity in random arrays of immobilized single-molecules as a step towards massively high density single-molecule DNA arrays. Experimental design, construction and imaging performed by MAO and principle author.
Schools and Departments: School of Life Sciences > Chemistry
Depositing User: Mark Osborne
Date Deposited: 06 Feb 2012 20:44
Last Modified: 28 Mar 2012 11:01
URI: http://sro.sussex.ac.uk/id/eprint/27843
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