Sensitive and Selective Amplified Fluorescence DNA Detection Based on Exonuclease III-Aided Target Recycling

Abstract

A limitation of many traditional approaches to the detection of specific oligonucleotide sequences, such as molecular beacons, is that each target strand hybridizes with (and thus activates) only a single copy of the relevant probe sequence. This 1:1 hybridization ratio limits the gain of most approaches and thus their sensitivity. Here we demonstrate a nuclease-amplified DNA detection scheme in which exonuclease III is used to "recycle" target molecules, thus leading to greatly improved sensitivity relative to, for example, traditional molecular beacons without any significant restriction in the choice of target sequences. The exonuclease-amplified assay can detect target DNA at concentrations as low as 10 pM when performed at 37 degrees C, which represents a significant improvement over the equivalent molecular beacon alone. Moreover, at 4 degrees C we can obtain a detection limit as low as 20 aM, albeit at the cost of a 24 h incubation period. Finally, our assay can be easily interrogated with the naked eye and is thus amenable to deployment in the developing world, where fluorometric detection is more problematic.

Ultrasensitive fluorescence-based methods for nucleic acid detection: towards amplification-free genetic analysis

“…exonuclease III, 105 to cleave fluorogenic oligonucleotide probes have been proposed as ultrasensitive methods for DNA detection. The turnover of Molecular Beacons in the cleavage reactions means that multiple fluorescence signals can be generated by a single unlabelled target molecule.…”

A Method for SNP Genotyping Using MoS2 and a DNA Intercalating Dye

“…Therefore, SNPs can be used to powerful tools in the study of human sports. Up to now, a variety of methods have been used for SNPs determination including nucleotide incorporation [2] , sequence cleavage [3] , molecular beacon [4,5] , direct sequencing [6] , and fluorescence energy transfer [7] . However, some methods are limited by requirements for complicated procedures, time-consuming or expensive reagents.…”

Polystyrene Microspheres Coupled with Hybridization Chain Reaction for Dual-Amplified Chemiluminescence Detection of Specific DNA Sequences

“…In the HCR process, two complementary stable species of hairpins coexist in solution until the introduction of initiator strands triggers a cascade of hybridization events to yield nicked double helices analogous to alternating copolymers [34,35]. Competing with other amplification methods, such as polymerase chain reaction (PCR) [36,37], enzyme amplification [38][39][40] and rolling circle amplification (RCA) [41][42][43], HCR shows its superiority that it allows for selective and specific extension at room temperature without enzymes. Recently, our group has reported a label-free CL platform where the reporter is amplified via HCR, offering significant amplification for the detection of target DNA or microRNA [44,45].…”