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DNA meter: Energy tunable, quantitative hybridization assay
Article first published online: 25 MAR 2013
Copyright © 2013 Wiley Periodicals, Inc.
Volume 99, Issue 6, pages 408–417, June 2013
How to Cite
Braunlin, W., Völker, J., Plum, G. E. and Breslauer, K. J. (2013), DNA meter: Energy tunable, quantitative hybridization assay. Biopolymers, 99: 408–417. doi: 10.1002/bip.22213
- Issue published online: 25 MAR 2013
- Article first published online: 25 MAR 2013
- Accepted manuscript online: 7 FEB 2013 09:09AM EST
- Manuscript Accepted: 15 JAN 2013
- Manuscript Received: 14 JAN 2013
- NIH. Grant Numbers: GM23509, GM34469, CA47995
- NSF. Grant Number: CBET-1033788
- NIH. Grant Number: AI074089
- novel hybridization assay;
- DNA meter;
- DNA arrays
We describe a novel hybridization assay that employs a unique class of energy tunable, bulge loop-containing competitor strands (C*) that hybridize to a probe strand (P). Such initial "pre-binding" of a probe strand modulates its effective "availability" for hybridizing to a target site (T). More generally, the assay described here is based on competitive binding equilibria for a common probe strand (P) between such tunable competitor strands (C*) and a target strand (T).
We demonstrate that loop variable, energy tunable families of C*P complexes exhibit enhanced discrimination between targets and mismatched targets, thereby reducing false positives/negatives. We refer to a C*P complex between a C* competitor single strand and the probe strand as a “tuning fork,” since the C* strand exhibits branch points (forks) at the duplex-bulge interfaces within the complex. By varying the loop to create families of such “tuning forks,” one can construct C*P “energy ladders” capable of resolving small differences within the target that may be of biological/functional consequence. The methodology further allows quantification of target strand concentrations, a determination heretofore not readily available by conventional hybridization assays. The dual ability of this tunable assay to discriminate and quantitate targets provides the basis for developing a technology we refer to as a “DNA Meter.” Here we present data that establish proof-of-principle for an in solution version of such a DNA Meter. We envision future applications of this tunable assay that incorporate surface bound/spatially resolved DNA arrays to yield enhanced discrimination and sensitivity. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 408–417, 2013.