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Nucleic Acids Research Advance Access originally published online on September 29, 2006
Nucleic Acids Research 2006 34(19):e128; doi:10.1093/nar/gkl679
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Nucleic Acids Research, 2006, Vol. 34, No. 19 e128
© 2006 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

A novel endonuclease IV post-PCR genotyping system

Igor V. Kutyavin*, Dave Milesi, Yevgeniy Belousov, Mikhail Podyminogin**, Alexei Vorobiev, Vladimir Gorn, Eugeny A. Lukhtanov, Nicolaas M. J. Vermeulen*** and Walt Mahoney

Nanogen, 21720 23rd Drive SE Suite 150, Bothell, WA 98021, USA

***To whom correspondence should be addressed. Tel: +1 425 482 5153; Fax: +1 425 482 5550; Email: nvermeulen{at}nanogen.com

Received May 22, 2006. Revised August 29, 2006. Accepted September 5, 2006.

Here we describe a novel endonuclease IV (Endo IV) based assay utilizing a substrate that mimics the abasic lesions that normally occur in double-stranded DNA. The three component substrate is characterized by single-stranded DNA target, an oligonucleotide probe, separated from a helper oligonucleotide by a one base gap. The oligonucleotide probe contains a non-fluorescent quencher at the 5' end and fluorophore attached to the 3' end through a special rigid linker. Fluorescence of the oligonucleotide probe is efficiently quenched by the interaction of terminal dye and quencher when not hybridized. Upon hybridization of the oligonucleotide probe and helper probe to their complementary target, the phosphodiester linkage between the rigid linker and the 3' end of the probe is efficiently cleaved, generating a fluorescent signal. In this study, the use of the Endo IV assay as a post-PCR amplification detection system is demonstrated. High sensitivity and specificity are illustrated using single nucleotide polymorphism detection.

Present addresses: *Geneureka LLC, 1631 220th Street SE Suite 202, Bothell, WA 98021, **USA Integrated DNA Technologies, 1710 Commercial Park, Coralville, IA 52241, USA


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