DNA surface hybridization regimes
- *Department of Chemical Engineering, Columbia University, New York, NY 10027; and
- †Department of Chemical and Biological Engineering, Polytechnic University, Brooklyn, NY 11201
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Edited by Jacqueline K. Barton, California Institute of Technology, Pasadena, CA, and approved January 30, 2008 (received for review October 3, 2007)
Abstract
Surface hybridization reactions, in which sequence-specific recognition occurs between immobilized and solution nucleic acids, are routinely carried out to quantify and interpret genomic information. Although hybridization is fairly well understood in bulk solution, the greater complexity of an interfacial environment presents new challenges to a fundamental understanding, and hence application, of these assays. At a surface, molecular interactions are amplified by the two-dimensional nature of the immobilized layer, which focuses the nucleic acid charge and concentration to levels not encountered in solution, and which impacts the hybridization behavior in unique ways. This study finds that, at low ionic strengths, an electrostatic balance between the concentration of immobilized oligonucleotide charge and solution ionic strength governs the onset of hybridization. As ionic strength increases, the importance of electrostatics diminishes and the hybridization behavior becomes more complex. Suppression of hybridization affinity constants relative to solution values, and their weakened dependence on the concentration of DNA counterions, indicate that the immobilized strands form complexes that compete with hybridization to analyte strands. Moreover, an unusual regime is observed in which the surface coverage of immobilized oligonucleotides does not significantly influence the hybridization behavior, despite physical closeness and hence compulsory interactions between sites. These results are interpreted and summarized in a diagram of hybridization regimes that maps specific behaviors to experimental ranges of ionic strength and probe coverage.
Footnotes
- ‡To whom correspondence should be addressed. E-mail: rlevicky{at}poly.edu
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Author contributions: P.G. and R.L. designed research; P.G. performed research; P.G. and R.L. analyzed data; and P.G. and R.L. wrote the paper.
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The authors declare no conflict of interest.
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This article is a PNAS Direct Submission.
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This article contains supporting information online at www.pnas.org/cgi/content/full/0709416105/DC1.
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↵ § The precise threshold value of Π is expected to vary somewhat with base sequence, temperature, and other parameters that influence hybridization equilibrium.
- © 2008 by The National Academy of Sciences of the USA
