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

Maria Smith,Kenneth Smith,Alan Olstein,Andrey Ghindilis,Andrew Oleinikov

doi:10.3390/s20143873

Abstract

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.

Highlights

Nucleic acid assays of different formats provide a core for modern-day biotechnology and diagnostics
Addition of a second biorecognition event based on Class II restriction endonucleases (REases) has numerous advantages, first and foremost due to the nearly absolute specificity of these enzymes for particular double-stranded DNA recognition sites
Both approaches are discussed in detail below, and they provide for the development of simple, low-cost, isothermal DNA hybridization assay platforms with exponential signal amplification that can achieve sensitivity similar to PCR applications

Summary

Introduction

Nucleic acid assays of different formats provide a core for modern-day biotechnology and diagnostics. The trigger molecule is initially attached to the surface through an assay probe, where upon cleavage it is released into the reaction solution. Both approaches are discussed in detail below, and they provide for the development of simple, low-cost, isothermal DNA hybridization assay platforms with exponential signal amplification that can achieve sensitivity similar to PCR applications. A single-stranded (ss) target DNA (i.e., dsDNA denatured to ssDNA, or cDNA) is added to the reaction solution and hybridizes to the probe forming dsDNA helix (Figure 1B). The probe–target hybrid carries a specific restriction site, the corresponding specific REase added to the reaction solution cleaves the helix. A part of the probe labeled with the molecular marker is released from the solid carrier into the reaction solution (Figure 1C).

General

Tandem

REase Based Assays

Conclusions