Article

DNA conformation on surfaces measured by flourescence self-interference

Center for Advanced Biotechnology, Boston University, Boston, MA 02215, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 03/2006; 103(8):2623-8. DOI: 10.1073/pnas.0511214103
Source: PubMed

ABSTRACT

The conformation of DNA molecules tethered to the surface of a microarray may significantly affect the efficiency of hybridization. Although a number of methods have been applied to determine the structure of the DNA layer, they are not very sensitive to variations in the shape of DNA molecules. Here we describe the application of an interferometric technique called spectral self-interference fluorescence microscopy to the precise measurement of the average location of a fluorescent label in a DNA layer relative to the surface and thus determine specific information on the conformation of the surface-bound DNA molecules. Using spectral self-interference fluorescence microscopy, we have estimated the shape of coiled single-stranded DNA, the average tilt of double-stranded DNA of different lengths, and the amount of hybridization. The data provide important proofs of concept for the capabilities of novel optical surface analytical methods of the molecular disposition of DNA on surfaces. The determination of DNA conformations on surfaces and hybridization behavior provide information required to move DNA interfacial applications forward and thus impact emerging clinical and biotechnological fields.

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    • "The results showed that hybridization with the direct method was unspecific, obtaining sequences belonging to the Paracoccus sp., but non-homologous to the phaC gene. The mixed methods turned out to be the better strategy, allowing a better kinetic reaction between the probe and target-DNA, a better orientation of the probe, and avoidance of the steric hindrance that is commonly observed in immobilized probes to solid supports (Archer et al. 2010; Moiseev et al. 2006; Shchepinov et al. 1997). In addition, through the calculated values of nearestneighbor parameters, a higher affinity allows stringent washes to obtain phaC sequences. "

    Full-text · Dataset · Jun 2013
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    • "The results showed that hybridization with the direct method was unspecific, obtaining sequences belonging to the Paracoccus sp., but non-homologous to the phaC gene. The mixed methods turned out to be the better strategy, allowing a better kinetic reaction between the probe and target-DNA, a better orientation of the probe, and avoidance of the steric hindrance that is commonly observed in immobilized probes to solid supports (Archer et al. 2010; Moiseev et al. 2006; Shchepinov et al. 1997). In addition, through the calculated values of nearestneighbor parameters, a higher affinity allows stringent washes to obtain phaC sequences. "
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