Enzyme-assisted target recycling (EATR) for nucleic acid detection

Fast, reliable and sensitive methods for nucleic acid detection are of growing practical interest with respect to molecular diagnostics of cancer, infectious and genetic diseases. Currently, PCR-based and other target amplification strategies are most extensively used in practice. At the same time, such assays have limitations that can be overcome by alternative approaches. There is a recent explosion in the design of methods that amplify the signal produced by a nucleic acid target, without changing its copy number. This review aims at systematization and critical analysis of the enzyme-assisted target recycling (EATR) signal amplification technique. The approach uses nucleases to recognize and cleave the probe-target complex. Cleavage reactions produce a detectable signal. The advantages of such techniques are potentially low sensitivity to contamination and lack of the requirement of a thermal cycler. Nucleases used for EATR include sequence-dependent restriction or nicking endonucleases or sequence independent exonuclease III, lambda exonuclease, RNase H, RNase HII, AP endonuclease, duplex-specific nuclease, DNase I, or T7 exonuclease. EATR-based assays are potentially useful for point-of-care diagnostics, single nucleotide polymorphisms genotyping and microRNA analysis. Specificity, limit of detection and the potential impact of EATR strategies on molecular diagnostics are discussed.