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

Ghindilis AL, Smith MW, Simon HM, Seoudi IA, Yazvenko NS, Murray IA, Fu X, Smith K, Jen-Jacobson L, Xu SY - Sci Rep (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Cascade Biosystems, Inc., E7279 State Road 170, Colfax, WI 54730, USA.

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.

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Testing of the REase-oligonucleotide conjugates, S17C BamHI-MCA-BG-Bio and K249C EcoRI-MCA-BG-Bio.(a) The REase conjugates were tested for the presence of oligonucleotide parts and immobilization as described by the schematic in Figure 2. The X-axis shows the target AMC-BG-40 concentration (the negative controls had no target added); and the Y-axis shows the HRP reporter signals quantified colorimetrically as OD655. Dark and light grey bars show the data obtained for S17C BamHI and K249C EcoRI, respectively. The tests were performed in triplicate for calculations of the mean values and standard deviations (error bars). (b) Titration of free REase conjugates using the HRP reporter systems. The X-axis shows the conjugate concentrations. Negative controls were prepared by addition of the restriction buffer without conjugates and used for background subtraction. The Y-axis shows the background-subtracted HRP signals (OD655) generated using the two mutant enzyme conjugates, S17C BamHI and K249C EcoRI. The inset shows the data corresponding to the REase conjugate concentrations between 0 and 80 μM.
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f3: Testing of the REase-oligonucleotide conjugates, S17C BamHI-MCA-BG-Bio and K249C EcoRI-MCA-BG-Bio.(a) The REase conjugates were tested for the presence of oligonucleotide parts and immobilization as described by the schematic in Figure 2. The X-axis shows the target AMC-BG-40 concentration (the negative controls had no target added); and the Y-axis shows the HRP reporter signals quantified colorimetrically as OD655. Dark and light grey bars show the data obtained for S17C BamHI and K249C EcoRI, respectively. The tests were performed in triplicate for calculations of the mean values and standard deviations (error bars). (b) Titration of free REase conjugates using the HRP reporter systems. The X-axis shows the conjugate concentrations. Negative controls were prepared by addition of the restriction buffer without conjugates and used for background subtraction. The Y-axis shows the background-subtracted HRP signals (OD655) generated using the two mutant enzyme conjugates, S17C BamHI and K249C EcoRI. The inset shows the data corresponding to the REase conjugate concentrations between 0 and 80 μM.

Mentions: As shown in Figure 3a, for both S17C BamHI (dark grey bars) and K249C EcoRI (light grey bars), the HRP signal generated with the target exceeded the mean negative control approximately 5-fold. Apparently, both conjugates were able to (i) bind to the agarose beads through their oligonucleotide parts, and (ii) stay enzymatically active.


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

Ghindilis AL, Smith MW, Simon HM, Seoudi IA, Yazvenko NS, Murray IA, Fu X, Smith K, Jen-Jacobson L, Xu SY - Sci Rep (2015)

Testing of the REase-oligonucleotide conjugates, S17C BamHI-MCA-BG-Bio and K249C EcoRI-MCA-BG-Bio.(a) The REase conjugates were tested for the presence of oligonucleotide parts and immobilization as described by the schematic in Figure 2. The X-axis shows the target AMC-BG-40 concentration (the negative controls had no target added); and the Y-axis shows the HRP reporter signals quantified colorimetrically as OD655. Dark and light grey bars show the data obtained for S17C BamHI and K249C EcoRI, respectively. The tests were performed in triplicate for calculations of the mean values and standard deviations (error bars). (b) Titration of free REase conjugates using the HRP reporter systems. The X-axis shows the conjugate concentrations. Negative controls were prepared by addition of the restriction buffer without conjugates and used for background subtraction. The Y-axis shows the background-subtracted HRP signals (OD655) generated using the two mutant enzyme conjugates, S17C BamHI and K249C EcoRI. The inset shows the data corresponding to the REase conjugate concentrations between 0 and 80 μM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4291554&req=5

f3: Testing of the REase-oligonucleotide conjugates, S17C BamHI-MCA-BG-Bio and K249C EcoRI-MCA-BG-Bio.(a) The REase conjugates were tested for the presence of oligonucleotide parts and immobilization as described by the schematic in Figure 2. The X-axis shows the target AMC-BG-40 concentration (the negative controls had no target added); and the Y-axis shows the HRP reporter signals quantified colorimetrically as OD655. Dark and light grey bars show the data obtained for S17C BamHI and K249C EcoRI, respectively. The tests were performed in triplicate for calculations of the mean values and standard deviations (error bars). (b) Titration of free REase conjugates using the HRP reporter systems. The X-axis shows the conjugate concentrations. Negative controls were prepared by addition of the restriction buffer without conjugates and used for background subtraction. The Y-axis shows the background-subtracted HRP signals (OD655) generated using the two mutant enzyme conjugates, S17C BamHI and K249C EcoRI. The inset shows the data corresponding to the REase conjugate concentrations between 0 and 80 μM.
Mentions: As shown in Figure 3a, for both S17C BamHI (dark grey bars) and K249C EcoRI (light grey bars), the HRP signal generated with the target exceeded the mean negative control approximately 5-fold. Apparently, both conjugates were able to (i) bind to the agarose beads through their oligonucleotide parts, and (ii) stay enzymatically active.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Cascade Biosystems, Inc., E7279 State Road 170, Colfax, WI 54730, USA.

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.

Show MeSH