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Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes

Nature Biotechnology volume 17, pages 292–296 (1999)Cite this article

Abstract

Flap endonucleases (FENs) isolated from archaea are shown to recognize and cleave a structure formed when two overlapping oligonucleotides hybridize to a target DNA strand. The downstream oligonucleotide probe is cleaved, and the precise site of cleavage is dependent on the amount of overlap with the upstream oligonucleotide. We have demonstrated that use of thermostable archaeal FENs allows the reaction to be performed at temperatures that promote probe turnover without the need for temperature cycling. The resulting amplification of the cleavage signal enables the detection of specific DNA targets at sub-attomole levels within complex mixtures. Moreover, we provide evidence that this cleavage is sufficiently specific to enable discrimination of single-base differences and can differentiate homozygotes from heterozygotes in single-copy genes in genomic DNA.

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Figure 1: Schematic representation of the configurations assumed by oligonucleotides with an overlap complementary to a DNA target.
Figure 2: Cleavage structures resulting from overlap of 0, 1, 3, 5, and 8 nucleotides.
Figure 3: Signal amplification by repeated invasive cleavage.
Figure 4: Detection of hCMV by invasive cleavage with AfuFEN1 nuclease, using a microplate-based readout.
Figure 5: Mutation discrimination of single-copy genes in genomic DNA extracts.

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Acknowledgements

We thank Frank Robb for providing the sequence and the DNA containing the gene for the Pfu FEN1 nuclease, and we thank Laura Heisler, Lance Fors, Dennis Blakeslee, Lloyd Smith, and James Dahlberg for discussions and for critical reading of the manuscript. This work was supported by Cooperative Agreement 70NANB5H1030 from the Department of Commerce Advanced Technology Program to L. Fors, and by grant DE-FG02-94ER81891 from the Department of Energy to M. Brow.

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  1. Third Wave Technologies, 502 South Rosa Rd., Madison, 53719-1256, WI

    Victor Lyamichev, Andrea L. Mast, Jeff G. Hall, James R. Prudent, Michael W. Kaiser, Tsetska Takova, Robert W. Kwiatkowski, Tamara J. Sander, Monika de Arruda, David A. Arco, Bruce P. Neri & Mary Ann D. Brow

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  1. Victor Lyamichev
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Correspondence to Mary Ann D. Brow.

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Lyamichev, V., Mast, A., Hall, J. et al. Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes. Nat Biotechnol 17, 292–296 (1999). https://doi.org/10.1038/7044

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