SYSTEMS AND METHODS FOR DETECTING BIOMARKERS OF INTEREST

• US 20140302486A1
• Filed: 12/23/2013
• Published: 10/09/2014
• Est. Priority Date: 09/02/2011
• Status: Abandoned Application

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), University of Washington
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation), University of Washington
• Inventors: Seelig, Georg, Chen, Xi, Zhang, David Yu
• Application Number:
  US14/139,464
• Publication Number:
  US 20140302486A1
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2537/1373 Displacement by a nucleic acid
• CPC Class Codes: C12Q 2537/155 Cyclic reactions
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/625 being a nucleic acid test strip device, e.g. dipsticks, strips, tapes, CD plates
• CPC Class Codes: C12Q 2600/178 miRNA, siRNA or ncRNA

Abstract

A detection probe for detecting single base mutations or alterations in a double stranded target nucleic acid molecule is provided. In some aspects, the detection probe may include a double-stranded probe nucleic acid molecule having a first end and a second end; at least one probe initiation toehold at the first end; at least one substance capable of emitting a detectable signal at the second end; and optionally, at least one dissociation toehold at the second end. The detection probe is designed to hybridize with a double stranded target nucleic acid in a reaction which proceeds at approximate thermodynamic equilibrium (ΔG≈0) when no single base mutations or alterations are present in the double stranded target nucleic acid molecule. The probe may be used in detection systems and methods to identifying the presence or absence of one or more single base mutations or alterations in a double-stranded nucleic acid target molecule.

Claims

1. A detection probe for detecting single base mutations or alterations in a double stranded target nucleic acid molecule comprising:

a double-stranded probe nucleic acid molecule having a first end and a second end;

at least one probe initiation toehold at the first end;

at least one substance capable of emitting a detectable signal at the second end; and

optionally, at least one dissociation toehold at the second end

wherein the detection probe is designed to hybridize with a double stranded target nucleic acid in a reaction which proceeds at approximate thermodynamic equilibrium (Δ

G≈

0) when no single base mutations or alterations are present in the double stranded target nucleic acid molecule.

2. The detection probe of claim 1, wherein the detection probe hybridizes with the double stranded target nucleic acid in a reaction which proceeds at Δ

G<

0 when one or more single base mutations or alterations are present in the double stranded target nucleic acid molecule.

3. The detection probe of claim 1, wherein the at least one substance capable of emitting a detectable signal comprises a fluorophore in contact with a quencher.

4. The detection probe of claim 1, wherein the at least one probe initiation toehold, the at least one dissociation toehold, or both, is between approximately 2 nucleotides (nt) and approximately 10 nt long.

5. The detection probe of claim 1, wherein each strand of the double-stranded probe nucleic acid molecule is between approximately 12 nt and approximately 1000 nt long.

6. The detection probe of claim 1, wherein the double-stranded nucleic acid molecule comprises a DNA molecule, an RNA molecule, a peptide nucleic acid (PNA) molecule, or a locked nucleic acid (LNA) molecule.

7. A single base mutation or alteration detection system comprising:

a detection probe which comprises (i) a double stranded probe nucleic acid molecule having a first end and a second end;

(ii) at least one probe initiation toehold at the first end; and

(iii) at least one substance capable of emitting a detectable signal attached to its second end;

a double stranded target nucleic acid molecule comprising (i) a first end and a second end; and

(ii) at least one target initiation toehold at its first end that is complimentary to the probe initiation toehold; and

at least one dissociation toehold at the second end of the detection probe, the second end of the target, or both;

wherein the detection probe is designed to hybridize with the double stranded target nucleic acid molecule in a reaction which proceeds at approximate thermodynamic equilibrium (Δ

G≈

0) when no single base mutations or alterations are present in the double stranded nucleic acid molecule, and

wherein the detection probe hybridizes with the double stranded target nucleic acid in a reaction which proceeds at Δ

G<

0 when one or more single base mutations or alterations are present in the double stranded target nucleic acid molecule.

8. The detection system of claim 7, wherein the at least one substance capable of emitting a detectable signal comprises a fluorophore in contact with a quencher.

9. The detection system of claim 7, wherein the at least one probe initiation toehold, the at least one dissociation toehold, or both, is between approximately 2 nucleotides (nt) and approximately 10 nt long.

10. The detection system of claim 7, wherein each strand of the double-stranded probe nucleic acid molecule is between approximately 12 nt and approximately 1000 nt long.

11. The detection system of claim 7, wherein the double-stranded probe nucleic acid molecule comprises a DNA molecule, an RNA molecule, a peptide nucleic acid (PNA) molecule, or a locked nucleic acid (LNA) molecule.

12. The detection system of claim 7, wherein the double stranded target nucleic acid molecule comprises biological DNA or RNA.

13. A method of identifying the presence or absence of one or more single base mutations or alterations in a double-stranded nucleic acid target molecule comprising:

reacting a detection probe with a double-stranded nucleic acid target molecule to produce a reaction product which produces a detectable signal;

measuring a level of the detectable signal of the reaction product;

determining a target hybridization yield via the level of detectable signal;

wherein a target hybridization yield of greater than approximately 10% indicates the absence of one or more single base mutations or alterations in the double-stranded nucleic acid target molecule, and

wherein a target hybridization yield of less than approximately 10% indicates the presence of one or more single base mutations or alterations in the double-stranded nucleic acid target molecule.

14. The method of claim 13, wherein the detection probe comprises (i) a double stranded probe nucleic acid molecule having a first end and a second end;

(ii) at least one probe initiation toehold at the first end;

(iii) at least one substance capable of emitting a detectable signal attached to its second end; and

optionally (iv) at least one dissociation toehold at the second end.

15. The detection system of claim 14, wherein the at least one substance capable of emitting a detectable signal comprises a fluorophore in contact with a quencher.

16. The detection system of claim 14, wherein the double-stranded probe nucleic acid molecule comprises a DNA molecule, an RNA molecule, a peptide nucleic acid (PNA) molecule, or a locked nucleic acid (LNA) molecule.

17. The method of claim 13, wherein the double stranded target nucleic acid molecule comprises (i) a first end and a second end;

(ii) at least one target initiation toehold at its first end that is complimentary to the probe initiation toehold; and

optionally (iii) at least one dissociation toehold at the second end of the detection probe, the second end of the target, or both.

18. The detection system of claim 13, wherein the double stranded target nucleic acid molecule comprises biological DNA or RNA.

19. The method of claim 13, wherein the detection probe is designed to hybridize with the double stranded target nucleic acid molecule in a reaction which proceeds at approximate thermodynamic equilibrium (Δ

G≈

0) when no single base mutations or alterations are present in the double stranded nucleic acid molecule.

20. The method of claim 13, wherein the detection probe hybridizes with the double stranded target nucleic acid in a reaction which proceeds at Δ

G<

0 when one or more single base mutations or alterations are present in the double stranded target nucleic acid molecule.