Restriction Cascade Exponential Amplification (RCEA) assay with an attomolar detection limit: a novel, highly specific, isothermal alternative to qPCR

• Published in: Scientific reports Vol. 5; no. 1; p. 7737
• Main Authors: Ghindilis, Andrey L., Smith, Maria W., Simon, Holly M., Seoudi, Ihab A., Yazvenko, Nina S., Murray, Iain A., Fu, Xiaoqing, Smith, Kenneth, Jen-Jacobson, Linda, Xu, Shuang-yong
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 13.01.2015; Nature Publishing Group
• Subjects: 45/71; 631/1647/1888; 631/61/32; 692/420/254; 9/10; Base Sequence; Biological Assay; Calibration; Catalysis; Colorimetry; Deoxyribonucleic acid; DNA; DNA - metabolism; DNA Restriction Enzymes - metabolism; Enzymes, Immobilized - metabolism; Genetic Engineering; Horseradish peroxidase; Humanities and Social Sciences; Hybridization; Hybrids; Limit of Detection; multidisciplinary; Mutant Proteins - metabolism; Mutation - genetics; Nucleic acids; Nucleotide sequence; Oligonucleotides; Oligonucleotides - metabolism; Peroxidase; Real-Time Polymerase Chain Reaction - methods; Science; Sensitivity and Specificity; Temperature
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep07737

An alternative to qPCR was developed for nucleic acid assays, involving signal rather than target amplification. The new technology, Restriction Cascade Exponential Amplification (RCEA), relies on specific cleavage of probe-target hybrids by restriction endonucleases (REase). Two mutant REases for amplification (Ramp), S17C BamHI and K249C EcoRI, were conjugated to oligonucleotides and immobilized on a solid surface. The signal generation was based on: (i) hybridization of a target DNA to a Ramp-oligonucleotide probe conjugate, followed by (ii) specific cleavage of the probe-target hybrid using a non-immobilized recognition REase. The amount of Ramp released into solution upon cleavage was proportionate to the DNA target amount. Signal amplification was achieved through catalysis, by the free Ramp, of a restriction cascade containing additional oligonucleotide-conjugated Ramp and horseradish peroxidase (HRP). Colorimetric quantification of free HRP indicated that the RCEA achieved a detection limit of 10 aM (10 −17  M) target concentration, or approximately 200 molecules, comparable to the sensitivity of qPCR-based assays. The RCEA assay had high specificity, it was insensitive to non-specific binding and detected target sequences in the presence of foreign DNA. RCEA is an inexpensive isothermal assay that allows coupling of the restriction cascade signal amplification with any DNA target of interest.