RESTRICTASE CASCADE EXPONENTIAL AMPLIFICATION (RCEA) DNA DETECTION - - PowerPoint PPT Presentation
1 Cascades Ventures . Any bids or questions about this offering should be directed to: Mark Magas mmagas@cascadesventures.com (847) 656-3560 RESTRICTASE CASCADE EXPONENTIAL AMPLIFICATION (RCEA) DNA DETECTION A
.
A PORTFOLIO OF PATENTS EXCLUSIVELY OFFERED FOR SALE OR LICENSE BY CASCADES VENTURES.
Non-confidential Marketing Package Not for public distribution without explicit permission from Cascades Ventures
Any bids or questions about this offering should be directed to: Mark Magas mmagas@cascadesventures.com (847) 656-3560
This marketing package constitutes an offer for sale of patents on behalf of the owner by Cascades Ventures, Inc. (Cascades), an IP licensing and brokerage firm. The information is provided solely for the purpose of assisting the independent evaluation of the portfolio by prospective buyers. The information provided herein or exchanged pursuant to the sales process is not intended to be notice or accusation of infringement of any of the patents or portfolios offered for sale. Nothing in this document shall constitute or be interpreted as legal analysis regarding the scope of the patents or other intellectual property
regarding this sales opportunity, potential purchasers must rely on their own examination and evaluation of the patents and portfolios including the merits and risks involved. No representation or warranties regarding the patents or portfolios are provided or implied. This marketing package and any other documents or information provided by Cascades related to the patents or portfolios are intended for use by the receiving party solely for its use in engaging in the sales process and in determining whether to purchase the patents or portfolios. Any distribution of these materials or information outside of the receiving party's organization without Cascades permission is strictly prohibited. Cascades reserves the right to modify or discontinue the sales process at any time including accepting offers prior to the completion of the due diligence period.
This Intellectual Property (IP) portfolio discloses “Restrictase Cascade Exponential Amplification” (RCEA) DNA detection technology.
This disruptive technology will revolutionize the existing, laboratory-based polymerase chain reaction (PCR) market, allowing for in-field testing and even greater reliability.
This portfolio has international coverage and includes 50 total assets across five patent families:
Fourteen issued U.S. patents and five pending U.S. applications;
Twenty-five issued and six pending foreign counterparts;
All patents and applications are assigned to Cascade Biosystems.
The portfolio is technically and financially beneficial for offensive and defensive strategies for any company in the DNA testing market.
➢
Polymerase chain reaction (PCR) technology dominates the current DNA testing market.
➢
PCR itself is a patented technology that was successfully licensed, earning billions in royalties.
➢
The DNA testing market is expected to achieve significant growth, expanding to over $10 Billion by 2022.
Source: Grand View Research 2016
2 4 6 8 10 12 2014 2022
➢
Collected DNA test samples often contain only trace amounts of a target DNA segment.
➢
In order for the DNA segment to be detectable, the segment generally must be amplified.
➢
PCR is currently the dominant DNA amplification technology.
➢
After amplification, detection can be performed using known techniques such as electrophoresis, hybridization, or sequencing.
➢
Polymerase chain reaction (PCR) is a laboratory-based technique that generates millions of copies of a target DNA sequence.
➢
Primers are used in combination with DNA polymerase to exponentially amplify (copy) the target DNA segment.
➢
Primers: Short DNA fragments containing complementary sequences to the target DNA segment.
➢
DNA polymerase: enzymes which “read” the existing DNA segment to produce duplicate segments.
➢
PCR generally requires thermal cycling— approximately 20-30 repeated cycles of heating and cooling—which allows the DNA segment replication process to occur.
Step 1: Heating separates the target DNA strands. Step 2: Cooling allows primers to anneal to the separated strands.
DNA REPLICATION DURING A PCR THERMAL CYCLE
Step 3: DNA polymerase elongates the new DNA chains after the primer. Then REPEAT until a sufficient number of copies are generated.
➢ Must be performed in a controlled laboratory environment. ➢ Expensive due to high costs of expensive equipment and highly trained
personnel.
➢ Subject to amplification bias due to the exponential nature of the reaction
and altered priming events.
➢ Generally, can only be used with low-throughput assays.
➢
Many other DNA amplification techniques have been attempted, such as:
➢ Transcription mediated amplification; ➢ Strand displacement amplification; ➢ Rolling circle amplification; ➢ Loop-mediated isothermal amplification; ➢ Florescent in situ hybridization; ➢ Etc.
➢
However, all of the alternative techniques fall short.
➢ The main problem with these techniques is a possible sequence-dependent bias, when
certain sequences have much higher amplification efficiency than the other.
➢ These techniques also complicate the design and validation of the multiple primers needed.
➢
Rather than amplifying the target DNA segment like PCR, the “Restrictase Cascade Exponential Amplification” (RCEA) technology uses a series of enzymatic reactions to detect the presence of the initial non-amplified target DNA.
➢
If the target DNA is present, it will trigger a cascading reaction releasing different enzymes which can then be detected.
➢
Thus, the enzymes are amplified rather than the target DNA segment;
➢
This avoids many of the problems of PCR.
https://youtu.be/aCSx_7YjAEU
Clicking the above image or the link will open the video in your web browser. A text explanation can also be found in the Appendix section.
Watch the following video for a detailed explanation of how RCEA DNA detection works:
Attribute PCR RCEA
Assay cost Highly expensive Nearly 1/3 less Assay sensitivity Very high Very high Assay specificity Moderate Extremely high Use with high throughput assay Yes, but complex and expensive Yes Use with portable equipment No Yes Low sample volume requirement Poor Excellent Used for SNP assay Limited by primers Limited by endonucleases Used for cancer methylation No Yes
Bacterial infection or pathogen detection (i.e., MRSA); Cancer detection; Agricultural or veterinary diagnostics; Food safety testing; Environmental testing (air and water); Genetic or epigenetic testing; Single-nucleotide polymorphism (SNP) analysis; Forensics.
Assay development:
Completed enzyme activity assays in various formats.
Optimized protocols for testing of horseradish peroxidase (HRP) and restrictase activity.
Proof-of-concept experiments:
Developed two protocols: direct RC assay with no amplification, and RCEA with exponential amplification.
Preliminary experiments demonstrated the exponential scheme resulted in an assay sensitivity with a 10-17 attomolar detection level, already equivalent to PCR levels. Optimization would lead to even better sensitivity.
Ghindilis AL, Smith MW, Simon HM, et al., Restriction Cascade Exponential Amplification (RCEA) assay with an attomolar detection limit: a novel, highly specific, isothermal alternative to qPCR, SCI. REP. 2015;5:7737. doi:10.1038/srep07737 (Jan. 13, 2015). (View Publication)
Confirmation of signal amplification principle
Application:
Preliminary development of a Methicillin-resistant Staphylococcus aureus (MRSA) assay.
The testing successfully detected MRSA within limits similar to PCR.
The proposed assay had high specificity, was insensitive to non-specific binding, and detected target sequences in the presence of foreign DNA.
Smith MW, Ghindilis AL, Seoudi IA, Smith K, Billharz R, et al., A New Restriction Endonuclease-Based Method for Highly-Specific Detection of DNA Targets from Methicillin-Resistant Staphylococcus aureus, PLOS ONE 9(5): e97826. doi:10.1371/journal.pone.0097826 (May 15, 2014). (View Publication)
Typical calibration curve of the restriction enzyme assay
This portfolio includes five patent families with 56 total assets:
Primary patent family: Detecting Nucleic Acid (22 assets);
Key patents: U.S. 8,278,048; U.S. 8,632,974; U.S. 8,865,407; and U.S. 9,677,120.
Four other patent families were created adapting the base technology to specific practice areas:
Methods and Materials for Detecting Contaminated Food Products (8 assets);
Methods and Materials for Detecting Viral or Microbial Infections (8 assets);
Methods and Materials for Detecting Genetic or Epigenetic Elements (8 assets);
Methods and Materials for Assessing RNA Expressions (4 assets).
Earliest priority date of August 5, 2008.
Patent term lasts until 2031.
A complete list of patents in this portfolio can be found in the Appendix section.
U.S. Patent 9,677,120 – Detecting Nucleic Acid Abstract: This document provides methods and materials for detecting target nucleic
target nucleic acid, methods and materials for detecting the amount of target nucleic acid present within a sample, kits for detecting the presence or absence of target nucleic acid, kits for detecting the amount of target nucleic acid present within a sample, and methods for making such kits are provided.
U.S. Patent 9,677,120 – Detecting Nucleic Acid
Claim 1. A kit for assessing a sample for target nucleic acid, said kit comprising:
(a) a probe nucleic acid comprising a nucleotide sequence complementary to a sequence of said target nucleic acid, wherein said probe nucleic acid comprises a restriction endonuclease, wherein, when said sample is contacted with said probe nucleic acid, at least a portion of said target nucleic acid hybridizes to at least a portion of said probe nucleic acid to form a double- stranded portion of nucleic acid comprising a restriction endonuclease cut site if said target nucleic acid is present in said sample, and wherein a recognition restriction endonuclease cleaves said double-stranded portion of nucleic acid at said restriction endonuclease cut site when said recognition restriction endonuclease is contacted with said probe nucleic acid, thereby separating a portion of said probe nucleic acid comprising said restriction endonuclease from at least another portion of said probe nucleic acid, and (b) a signal expansion nucleic acid comprising an amplifying restriction endonuclease, a label, and a double-stranded portion of nucleic acid comprising a restriction endonuclease cut site of said restriction endonuclease, wherein, when said portion of said probe nucleic acid comprising said restriction endonuclease is contacted with said signal expansion nucleic acid, said restriction endonuclease cleaves said double-stranded portion of nucleic acid at said restriction endonuclease cut site of said restriction endonuclease, thereby separating a first portion of said signal expansion nucleic acid from at least another portion of said signal expansion nucleic acid, wherein said first portion comprises said label, and wherein the presence of said first portion indicates that said sample contains said target nucleic acid, and wherein the absence of said first portion indicates that said sample does not contain said target nucleic acid.
I-SENS, INC.
ASCENSIA DIABETES CARE HOLDINGS AG (PREVIOUSLY KNOWN AS BAYER DIABETES CARE)
APEX BIOTECHNOLOGY CORPORATION
AGAMATRIX INC
TAIDOC TECHNOLOGY CORPORATION
UNIVERSAL BIOSENSORS
77 ELEKTRONIKA KFT
BEIJING YICHENG ELECTRONIC CO., LTD.
SANNUO BIOSENSOR INC.
SHANGHAI MICROSENSE TM INC.
ACON LABORATORIES, INC.
ALLMEDICUS
NATIONAL DIAGNOSTIC PRODUCTS
HMD BIOMEDICAL
YUWELL-JIANGSU YUYUE MEDICAL EQUIPMENT & SUPPLY CO., LTD.
Comparable technology: PCR
Like PCR, RCEA represents a revolutionary advance in the DNA detection field rather than just a mere enhancement of an existing technology.
The PCR patents sold for $300 million in 1991 to Hoffmann-La Roche, eventually earning the company over $2 Billion in royalties.
See: Joe Fore Jr., Ilse R. Wiechers & Robert Cook-Deegan, The effects of business practices, licensing, and intellectual property on development and dissemination of the polymerase chain reaction: case study, J BIOMED DISCOV COLLAB. 2006;1:7. 10.1186/1747-5333-1-7. (View Publication)
There has already been substantial investment in research and development:
Millions have been spent on assay development, proof of concept experiments, and potential applications.
56 patents have been or currently are being prosecuted by a leading patent law firm, Fish & Richardson.
The owner reserves the right to accept a bid at anytime.
We will keep all parties who have notified us of their interest in submitting a bid, or who have submitted a bid, reasonably informed throughout the process.
All information on interested parties – including bidder names, bid amounts, terms, and the winning bidder – will remain confidential.
Any bids or questions about this offering should be directed to:
Mark Magas Cascades Ventures Director mmagas@cascadesventures.com (847) 656-3560
Detecting Nucleic Acid:
Methods and Materials for Detecting Contaminated Food Products:
Methods and Materials for Detecting Viral or Microbial Infections:
Methods and Materials for Detecting Genetic or Epigenetic Elements:
Methods and Materials for Assessing RNA Expressions:
US 8,278,048; US 8,632,974; US 8,865,407; US 9,677,120; Pending US application 15/587,944; AU 2009282246; CA 2734244; Pending CA application 2965207; CN 102186997; CN 104673888; EP 2318549 (national entries: DE, FR, GB); EP 2586876 (national entries: DE, FR, GB); EP 2808403 (national entries: DE, FR, GB); Pending IN application 1666/CHENP/2011; HK 1157823; HK 1204659;
Detecting Nucleic Acid:
Methods and Materials for Detecting Contaminated Food Products:
Methods and Materials for Detecting Viral or Microbial Infections:
Methods and Materials for Detecting Genetic or Epigenetic Elements:
Methods and Materials for Assessing RNA Expressions:
US 8,623,616; US 9,150,932; US 9,399,802; Pending US application 15/218,415; Pending CA application 2790006; EP 2536843 (national entries: DE, FR, GB);
Detecting Nucleic Acid:
Methods and Materials for Detecting Contaminated Food Products:
Methods and Materials for Detecting Viral or Microbial Infections:
Methods and Materials for Detecting Genetic or Epigenetic Elements:
Methods and Materials for Assessing RNA Expressions:
US 8,597,886; US 9,150,935; US 9,428,814; Pending US application 15/225,365; Pending CA application 2790123; EP 2536846 (national entries: DE, FR, GB);
Detecting Nucleic Acid:
Methods and Materials for Detecting Contaminated Food Products:
Methods and Materials for Detecting Viral or Microbial Infections:
Methods and Materials for Detecting Genetic or Epigenetic Elements:
Methods and Materials for Assessing RNA Expressions:
US 8,551,701; US 9,150,921; US 9,689,037; Pending US application 15/604,112; Pending CA application 2790008; EP 2536848 (national entries: DE, FR, GB);
Detecting Nucleic Acid:
Methods and Materials for Detecting Contaminated Food Products:
Methods and Materials for Detecting Viral or Microbial Infections:
Methods and Materials for Detecting Genetic or Epigenetic Elements:
Methods and Materials for Assessing RNA Expressions:
US 8,470,533; Pending US application 14/627,742; Pending CA application 2789874; EP 2536847 (pending designations);
Step 4: The cleaving by the RREC enzyme releases the RAMP_1 enzyme, which will trigger the amplification process. Step 1: The target DNA segment is added to a solution containing probe
DNA sequences attached to a first amplification restriction enzyme (RAMP_1).
Step 2: The target DNA segment hybridizes with a probe oligonucleotide, creating a target-probe hybrid. Step 3: A recognition restriction enzyme (RREC) is added, which cleaves the target-probe hybrid.
Step 6: The released RAMP_1 cleaves the RAMP_2 conjugates.
Step 5: The released RAMP_1 is added to a solution containing: Step 7: The cleaving by the RAMP_1 releases free RAMP_2 enzymes into the solution.
conjugates;
restriction enzyme conjugates (RAMP_2); and
horseradish peroxidase (HRP), that will ultimately be detected.
Step 8: The released RAMP_2 cleaves both the additional RAMP_1 conjugates and the HRP conjugates. Step 10: The released HRP can then be detected using any known technique, such as a colorimetric test. Step 9: The released RAMP_1 and RAMP_2 enzymes continue to release each other in a cascading exponential
RAMP_2, more HRP is released.