Enzymatic Nucleic Acid Detection via Restriction Endonuclease Cascade

The patent replaces the mechanical/thermal cycling system (PCR-based techniques requiring thermal cyclers) with a biochemical cascade system using restriction endonucleases. The detection mechanism substitutes thermal amplification with enzymatic cleavage reactions that occur at constant temperature, eliminating the need for complex thermal cycling equipment while maintaining detection sensitivity through the cascade amplification effect of multiple restriction enzymes acting sequentially on the target nucleic acid.

The patent implements continuous enzymatic action through a cascade of restriction endonucleases that act sequentially and continuously on the target nucleic acid molecules. Unlike discrete thermal cycling steps, the enzymatic cascade proceeds continuously at constant temperature, with each enzyme cleaving substrates and generating products that serve as substrates for subsequent enzymes, maintaining uninterrupted useful action and reducing total diagnosis time while preserving sensitivity.

The patent changes the operational parameters from thermal cycling (temperature variation over time) to constant temperature enzymatic reactions. By shifting from time-temperature profile control to constant temperature biochemical catalysis, the system reduces equipment complexity and cost while increasing detection speed through the rapid, continuous enzymatic cleavage reactions that occur at optimal constant temperatures without the delays inherent in thermal cycling processes.