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 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.
Weizmann
Yossi
Y
Institute of Chemistry and the Farkas Center for Light-Induced Processes, Hebrew University of Jerusalem, Jerusalem 91904, Israel.
Cheglakov
Zoya
Z
Pavlov
Valeri
V
Willner
Itamar
I
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
D005456:Fluorescent Dyes; D009841:Oligonucleotides; D004247:DNA; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific