title: An autonomous fueled machine that replicates catalytic nucleic acid templates for the amplified optical analysis of DNA.
abstract: 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 x 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.
authors.affiliation: Institute of Chemistry and the Farkas Center for Light-Induced Processes, Hebrew University of Jerusalem, Jerusalem 91904, Israel.
authors.forename: Yossi
authors.initials: Y
authors.lastname: Weizmann
authors.forename: Zoya
authors.initials: Z
authors.lastname: Cheglakov
authors.forename: Valeri
authors.initials: V
authors.lastname: Pavlov
authors.forename: Itamar
authors.initials: I
authors.lastname: Willner
mesh_terms: D002384:Catalysis; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation* / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D012680:Sensitivity and Specificity; D013661:Tay-Sachs Disease / Q000235:genetics; D013698:Templates, Genetic
chemical_list: D005456:Fluorescent Dyes; D009841:Oligonucleotides; D004247:DNA; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific