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Nucleic Acids Research Advance Access published online on November 13, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm983
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© 2007 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

Using force spectroscopy analysis to improve the properties of the hairpin probe

Liu Yang1, Kemin Wang1,*, Weihong Tan1, Huimin Li2, Xiaohai Yang1, Changbei Ma1 and Hongxing Tang1

1State Key Laboratory of Chemo/Biosensing and Chemometrics, Biomedical Engineering Center, Engineering Research Center for Bio-Nanotechnology of Hunan Province, College of Chemistry and Chemical Engineering and 2College of Material Science and Engineering, Hunan University, Changsha 410082, P.R. China

*To whom correspondence should be addressed. Tel: (86)731- 8821566; Fax: (86)731- 8821566; Email: kmwang{at}hnu.cn

Received July 19, 2007. Revised October 19, 2007. Accepted October 19, 2007.

The sensitivity of hairpin-probe-based fluorescence resonance energy transfer (FRET) analysis was sequence-dependent in detecting single base mismatches with different positions and identities. In this paper, the relationship between the sequence-dependent effect and the discrimination sensitivity of a single base mismatch was systematically investigated by fluorescence analysis and force spectroscopy analysis. The same hairpin probe was used. The uneven fluorescence analysis sensitivity was obviously influenced by the guanine-cytosine (GC) contents as well as the location of the mismatched base. However, we found that force spectroscopy analysis distinguished itself, displaying a high and even sensitivity in detecting differently mismatched targets. This could therefore be an alternative and novel way to minimize the sequence-dependent effect of the hairpin probe. The advantage offered by force spectroscopy analysis could mainly be attributed to the percentage of rupture force reduction, which could be directly and dramatically influenced by the percentage of secondary structure disruption contributed by each mismatched base pair, regardless of its location and identity. This yes-or-no detection mechanism should both contribute to a comprehensive understanding of the sensitivity source of different mutation analyses and extend the application range of hairpin probes.


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