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Nucleic Acids Research Advance Access originally published online on May 31, 2008
Nucleic Acids Research 2008 36(12):e74; doi:10.1093/nar/gkn301
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Nucleic Acids Research, 2008, Vol. 36, No. 12 e74
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Improved assay-dependent searching of nucleic acid sequence databases

Jason D. Gans* and Murray Wolinsky

Biosciences Division, Los Alamos National Laboratory, Los Alamos, NM, USA

*To whom correspondence should be addressed. Tel: +1 505 667 3770; Fax: +1 505 665 2564; Email: jgans{at}lanl.gov

Correspondence may also be addressed to Murray Wolinsky. Tel: +1 505 665 0952; Fax: +1 505 665 2564; Email: murray{at}lanl.gov

Received April 9, 2008. Revised April 9, 2008. Accepted April 30, 2008.

Nucleic acid-based biochemical assays are crucial to modern biology. Key applications, such as detection of bacterial, viral and fungal pathogens, require detailed knowledge of assay sensitivity and specificity to obtain reliable results. Improved methods to predict assay performance are needed for exploiting the exponentially growing amount of DNA sequence data and for reducing the experimental effort required to develop robust detection assays. Toward this goal, we present an algorithm for the calculation of sequence similarity based on DNA thermodynamics. In our approach, search queries consist of one to three oligonucleotide sequences representing either a hybridization probe, a pair of Padlock probes or a pair of PCR primers with an optional TaqManTM probe (i.e. in silico or ‘virtual’ PCR). Matches are reported if the query and target satisfy both the thermodynamics of the assay (binding at a specified hybridization temperature and/or change in free energy) and the relevant biological constraints (assay sequences binding to the correct target duplex strands in the required orientations). The sensitivity and specificity of our method is evaluated by comparing predicted to known sequence tagged sites in the human genome. Free energy is shown to be a more sensitive and specific match criterion than hybridization temperature.


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