Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

An autonomous fueled machine that replicates catalytic nucleic acid templates for the amplified optical analysis of DNA


Here we describe a protocol for the amplified detection of a target DNA using a DNA/FokI-based replicating cutting machine. The protocol is based on the design of a sensing hairpin oligonucleotide that is opened upon hybridization with the analyte DNA. The endonuclease FokI binds to the double-stranded complex and cleaves it to a “cutter” unit. The “cutter” unit reacts with a fuel oligonucleotide to generate and amplify the signal. The fuel molecule is an oligonucleotide in a hairpin configuration with a fluorophore/quencher pair attached to the 5′ and 3′ ends. Formation of the duplex between the cutter and the fuel leads to the scission of the duplex by FokI, leading to a second, replicated “cutter”, a fluorescent waste product, and to the regeneration of the original “cutter” unit. The autonomous replication of the “cutter” unit, as a result of the primary recognition of the analyte DNA, leads to the amplified fluorescent detection of the analyte DNA with a sensitivity limit of 1 × 10−14 M. The operation of the machine and the sensing process are monitored by the fluorescence generated by the waste product. Here we apply the protocol, which takes about 2 h to complete, to analyze a Tay-Sachs genetic disorder mutant DNA.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Scheme of the amplified detection of DNA using the FokI/DNA complex as a biocatalytic template for the scission of the fuel substrate (3), yielding the fluorescent waste product, (4) (F = FAM; Q = TAMRA).
Figure 2
Figure 3: Time-dependent changes in fluorescence intensities (λem = 520 nm) upon analyzing different concentrations of the mutant DNA (1): (a) 1 × 10−6 M; (b) 1 × 10−11 M; (c) analysis of the normal gene (1a) (5′- GGGCCATAGGATATACGGTTCAGG-3′, 1 × 10−6 M); (d) analysis of a foreign DNA (5′-CTCCCGTCTAATGCGCTTCCCTGTTTT-3′, 1 × 10−6 M).
Figure 4: Nondenaturating PAGE analysis of the mutant DNA (1) by the hairpin-sensing nucleic acid (2) by the FokI-induced mechanical scission of the fuel (3).

Similar content being viewed by others


  1. Weizmann, Y., Cheglakov, Z., Pavlov, V. & Willner, I. Autonomous fueled mechanical replication of nucleic acid templates for the amplified optical detection of DNA. Angew. Chem. Int. Edn. Engl. 45, 2238–2242 (2006).

    Article  CAS  Google Scholar 

  2. Szybalski, W. Universal restriction endonucleases: designing novel cleavage specificities by combining adaptor oligodeoxynucleotide and enzyme moieties. Gene 40, 169–173 (1985).

    Article  CAS  Google Scholar 

  3. Lyamichev, V. et al. Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes. Nat. Biotechnol. 17, 292–296 (1999).

    Article  CAS  Google Scholar 

  4. Notomi, T. et al. Loop-mediated isothermal amplification. Nucleic Acids Res. 28, E63 (2000).

    Article  CAS  Google Scholar 

  5. Lizardi, P.M. et al. Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat. Genet. 19, 225–232 (1998).

    Article  CAS  Google Scholar 

  6. Solinas, A. et al. Duplex Scorpion primers in SNP analysis and FRET applications. Nucleic Acids Res. 29, E96 (2001).

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations


Corresponding author

Correspondence to Itamar Willner.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Weizmann, Y., Cheglakov, Z., Pavlov, V. et al. An autonomous fueled machine that replicates catalytic nucleic acid templates for the amplified optical analysis of DNA. Nat Protoc 1, 554–558 (2006).

Download citation

  • Published:

  • Issue Date:

  • DOI:


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing