Restriction Endonuclease-Based Assays for DNA Detection and Isothermal Exponential Signal Amplification

Application of restriction endonuclease (REase) enzymes for specific detection of nucleic acids provides for high assay specificity, convenience and low cost. A direct restriction assay format is based on the specific enzymatic cleavage of a target-probe hybrid that is accompanied with the release of a molecular marker into the solution, enabling target quantification. This format has the detection limit in nanomolar range. The assay sensitivity is improved drastically to the attomolar level by implementation of exponential signal amplification that is based on a cascade of self-perpetuating restriction endonuclease reactions. The cascade is started by action of an amplification "trigger". The trigger is immobilized through a target-specific probe. Upon the target probe hybridization followed with specific cleavage, the trigger is released into the reaction solution. The solution is then added to the assay amplification stage, and the free trigger induces cleavage of amplification probes, thus starting the self-perpetuating cascade of REase-catalyzed events. Continuous cleavage of new amplification probes leads to the exponential release of new triggers and rapid exponential signal amplification. The proposed formats exemplify a valid isothermal alternative to qPCR with similar sensitivity achieved at a fraction of the associated costs, time and labor. Advantages and challenges of the approach are discussed.