title: Restriction Cascade Exponential Amplification (RCEA) assay with an attomolar detection limit: a novel, highly specific, isothermal alternative to qPCR.
abstract: 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.
authors.affiliation: Cascade Biosystems, Inc., E7279 State Road 170, Colfax, WI 54730, USA.
authors.forename: Andrey L
authors.initials: AL
authors.lastname: Ghindilis
authors.affiliation: 1] Cascade Biosystems, Inc., E7279 State Road 170, Colfax, WI 54730, USA [2] Center for Coastal Margin Observation and Prediction, and Institute of Environmental Health, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA.
authors.forename: Maria W
authors.initials: MW
authors.lastname: Smith
authors.affiliation: Center for Coastal Margin Observation and Prediction, and Institute of Environmental Health, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA.
authors.forename: Holly M
authors.initials: HM
authors.lastname: Simon
authors.affiliation: Hamad Medical Corporation, P.O. Box 3050 Doha, Qatar.
authors.forename: Ihab A
authors.initials: IA
authors.lastname: Seoudi
authors.affiliation: Cascade Biosystems, Inc., E7279 State Road 170, Colfax, WI 54730, USA.
authors.forename: Nina S
authors.initials: NS
authors.lastname: Yazvenko
authors.affiliation: New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Iain A
authors.initials: IA
authors.lastname: Murray
authors.affiliation: New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Fu
authors.affiliation: Cascade Biosystems, Inc., E7279 State Road 170, Colfax, WI 54730, USA.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Smith
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, 320 Clapp Hall, 4249 Fifth Avenue, Pittsburgh, PA 15260, USA.
authors.forename: Linda
authors.initials: L
authors.lastname: Jen-Jacobson
authors.affiliation: New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Shuang-Yong
authors.initials: SY
authors.lastname: Xu
mesh_terms: D001483:Base Sequence; D001681:Biological Assay; D002138:Calibration; D004247:DNA / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000378:metabolism; D005818:Genetic Engineering; D057230:Limit of Detection; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009841:Oligonucleotides / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013696:Temperature
chemical_list: D004800:Enzymes, Immobilized; D050505:Mutant Proteins; D009841:Oligonucleotides; D004247:DNA; D004262:DNA Restriction Enzymes