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Nucleic Acids Research Advance Access published online on April 15, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn182
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© 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

The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases

Hua Wei1, Caitlin Therrien2, Aine Blanchard1, Shengxi Guan1 and Zhenyu Zhu1,*

1New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938 and 2Duke University, PO Box 98790, Durham, NC 22708, USA

*To whom correspondence should be addressed. Tel: +1 978 380 7237; Fax: +1 978 380 7419; Email: zhuz{at}neb.com

Received February 29, 2008. Revised March 25, 2008. Accepted March 28, 2008.

Restriction endonucleases are the basic tools of molecular biology. Many restriction endonucleases show relaxed sequence recognition, called star activity, as an inherent property under various digestion conditions including the optimal ones. To quantify this property we propose the concept of the Fidelity Index (FI), which is defined as the ratio of the maximum enzyme amount showing no star activity to the minimum amount needed for complete digestion at the cognate recognition site for any particular restriction endonuclease. Fidelity indices for a large number of restriction endonucleases are reported here. The effects of reaction vessel, reaction volume, incubation mode, substrate differences, reaction time, reaction temperature and additional glycerol, DMSO, ethanol and Mn2+ on the FI are also investigated. The FI provides a practical guideline for the use of restriction endonucleases and defines a fundamental property by which restriction endonucleases can be characterized.


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