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.
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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). https://doi.org/10.1038/nprot.2006.78