ENHANCED PROBE BINDING

• US 20140212874A1
• Filed: 01/16/2014
• Published: 07/31/2014
• Est. Priority Date: 01/18/2013
• Status: Active Grant

• Current Assignee: NABsys, Inc.
• Original Assignee: NABsys, Inc.
• Inventors: Oliver, John S.
• Granted Patent: US 10,294,516 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore

Abstract

Methods for enhancing the binding of oligonucleotide probes to DNA and RNA are disclosed. The methods make use of thermodynamic and kinetic effects to reduce probe mismatches and failure of complementary probes to bind to DNA and RNA templates. Mapping and sequencing of the probed DNA and RNA samples are contemplated herein.

Claims

1. A method for preparing a biomolecule analyte, the method comprising:

a. providing a single-stranded DNA or RNA template;

b. hybridizing a plurality of identical, sequence-specific oligonucleotide probes to the template;

c. conducting a base extension reaction from a 3′

end of a hybridized probe;

d. terminating the base-extension reaction; and

e. allowing additional unhybridized probes from the plurality of probes to hybridize to the template to prepare the biomolecule analyte.

2. The method of claim 1, wherein the base extension reaction is allowed to produce a double-stranded region on the single-stranded template of a length approximating the resolution of a detection apparatus.

3. The method of claim 2, wherein the base extension reaction and the termination are carried out simultaneously.

4. The method of claim 1, further comprising:

following termination of the base extension reaction, maintaining the hybridized template under a set of conditions including temperature and for a time sufficient to melt probe mismatches.

5. The method of claim 4, wherein the time and temperature are sufficient to melt substantially all probe mismatches.

6. The method of claim 1, wherein at least a portion of the probes comprises tagged probes.

7. The method of claim 6, wherein the tags on the probes include at least one of double stranded DNA, gold beads, quantum dots, or fluorophores.

8. The method of claim 1, wherein at least a portion of the template or probes is coated with a protein.

9. The method of claim 8, wherein the protein includes at least one of RecA, T4 gene 32 protein, f1 geneV protein, human replication protein A, Pf3 single-stranded binding protein, adenovirus DNA binding protein, or E. coli single-stranded binding protein.

31. A method for analyzing a biomolecule analyte comprising the steps of:

a. preparing the biomolecule analyte using the method of claim 1;

b. providing an apparatus having a first fluid chamber, a second fluid chamber, a membrane positioned between the first and second chambers and a nanopore extending through the membrane such that the first and second chambers are in fluid communication via the nanopore;

c. introducing the biomolecule analyte into the first chamber;

d. translocating the biomolecule analyte from the first chamber through the nanopore and into the second chamber;

e. monitoring changes in an electrical property across the nanopore as the biomolecule analyte is translocated therethrough, the changes in the electrical property corresponding to locations along the biomolecule analyte containing probes; and

f. recording the changes in the electrical property as a function of time.

35. A method for analyzing a biomolecule analyte comprising the steps of:

a. preparing the biomolecule analyte using the method of claim 1;

b. disposing the biomolecule analyte in a fluidic nanochannel or microchannel;

c. applying a potential along the fluidic channel;

d. translocating the biomolecule analyte from a first end of the fluidic channel to a second end of the fluidic channel; and

e. detecting electrical properties as the biomolecule analyte moves through the fluidic channel, the electrical properties corresponding to at least one detector volume in the fluidic channel, each detector volume being defined by two or more sensing electrodes disposed along the length of the fluidic channel, wherein the detected electrical signals indicate locations of hybridized probes along the biomolecule analyte.

10. A method for preparing a biomolecule analyte, the method comprising:

a. providing a single-stranded DNA or RNA template comprising one or more secondary structures;

b. hybridizing a plurality of identical, sequence-specific oligonucleotide probes to the template;

c. conducting a base extension reaction from a 3′

end of a hybridized probe,

d. terminating the base-extension reaction;

e. denaturing the template to break at least a portion of said one or more secondary structures; and

f. repeating steps b, c, d, and e at least one additional time with a different plurality of identical, sequence-specific oligonucleotide probes to prepare the biomolecule analyte.

11. The method of claim 10, wherein the denaturing step comprises heating.

12. The method of claim 10, wherein at least a portion of the probes comprises tagged probes.

13. The method of claim 10, wherein at least a portion of the template or probes is coated with a protein.

14. The method of claim 10, wherein the base extension reaction and the termination are carried out simultaneously.

32. A method for analyzing a biomolecule analyte comprising the steps of:

a. preparing the biomolecule analyte using the method of claim 10;

b. providing an apparatus having a first fluid chamber, a second fluid chamber, a membrane positioned between the first and second chambers and a nanopore extending through the membrane such that the first and second chambers are in fluid communication via the nanopore;

c. introducing the biomolecule analyte into the first chamber;

d. translocating the biomolecule analyte from the first chamber through the nanopore and into the second chamber;

e. monitoring changes in an electrical property across the nanopore as the biomolecule analyte is translocated therethrough, the changes in the electrical property corresponding to locations along the biomolecule analyte containing probes; and

f. recording the changes in the electrical property as a function of time.

36. A method for analyzing a biomolecule analyte comprising the steps of:

a. preparing the biomolecule analyte using the method of claim 10;

b. disposing the biomolecule analyte in a fluidic nanochannel or microchannel;

c. applying a potential along the fluidic channel;

d. translocating the biomolecule analyte from a first end of the fluidic channel to a second end of the fluidic channel; and

e. detecting electrical properties as the biomolecule analyte moves through the fluidic channel, the electrical properties corresponding to at least one detector volume in the fluidic channel, each detector volume being defined by two or more sensing electrodes disposed along the length of the fluidic channel, wherein the detected electrical signals indicate locations of hybridized probes along the biomolecule analyte.

15. A method for preparing a biomolecule analyte, the method comprising:

a. providing a single-stranded DNA or RNA template;

b. providing a first plurality of identical, sequence-specific oligonucleotide probes having a first melting temperature, and a second plurality of identical, sequence-specific oligonucleotide probes having a second melting temperature, the first melting temperature being higher than the second melting temperature and the first plurality of probes having a different sequence than the second plurality of probes;

c. hybridizing probes from the first plurality of identical, sequence-specific oligonucleotide probes to the template at a temperature between the second melting temperature and the first melting temperature;

d. conducting a first base extension reaction from a 3′

end of a hybridized probe from the first plurality of probes;

e. terminating the first base-extension reaction;

f. allowing additional unhybridized probes from the first plurality of probes to hybridize to the template;

g. conducting a second base-extension reaction from a 3′

end of a hybridized probe from the first plurality of probes;

h. terminating the second base-extension reaction; and

i. hybridizing probes from the second plurality of identical, sequence-specific oligonucleotide probes to the template at a temperature equal to or below the second melting temperature to prepare the biomolecule analyte.

16. The method of claim 15, wherein at least a portion of the probes comprises tagged probes.

17. The method of claim 15, wherein at least a portion of the template or probes is coated with a protein.

18. The method of claim 15, wherein the first base extension reaction and its termination are carried out simultaneously.

19. The method of claim 15, wherein the second base extension reaction and its termination are carried out simultaneously

20. The method of claim 15, further comprising, after probes from the second plurality of identical, sequence-specific oligonucleotide probes are hybridized to the analyte, carrying out the additional steps of:

j. conducting a third base-extension reaction in the at least one single-stranded region from a 3′

end of a hybridized probe from the second plurality of probes;

k. terminating the third base-extension reaction; and

allowing additional unhybridized probes from the second plurality of probes to hybridize to the template.

21. The method of claim 20, wherein the third base extension reaction and its termination are carried out simultaneously.

22. The method of claim 20, further comprising the steps of:

m. conducting a fourth base-extension reaction in the at least one single-stranded region from a 3′

end of a hybridized probe from the second plurality of probes, and

n. terminating the fourth base-extension reaction.

23. The method of claim 22, wherein the fourth base extension reaction and its termination are carried out simultaneously.

33. A method for analyzing a biomolecule analyte comprising the steps of:

a. preparing the biomolecule analyte using the method of claim 15;

b. providing an apparatus having a first fluid chamber, a second fluid chamber, a membrane positioned between the first and second chambers and a nanopore extending through the membrane such that the first and second chambers are in fluid communication via the nanopore;

c. introducing the biomolecule analyte into the first chamber;

d. translocating the biomolecule analyte from the first chamber through the nanopore and into the second chamber;

e. monitoring changes in an electrical property across the nanopore as the biomolecule analyte is translocated therethrough, the changes in the electrical property corresponding to locations along the biomolecule analyte containing probes; and

f. recording the changes in the electrical property as a function of time.

37. A method for analyzing a biomolecule analyte comprising the steps of:

a. preparing the biomolecule analyte using the method of claim 15;

b. disposing the biomolecule analyte in a fluidic nanochannel or microchannel,

c. applying a potential along the fluidic channel;

d. translocating the biomolecule analyte from a first end of the fluidic channel to a second end of the fluidic channel; and

e. detecting electrical properties as the biomolecule analyte moves through the fluidic channel, the electrical properties corresponding to at least one detector volume in the fluidic channel, each detector volume being defined by two or more sensing electrodes disposed along the length of the fluidic channel, wherein the detected electrical signals indicate locations of hybridized probes along the biomolecule analyte.

24. A method for preparing a biomolecule analyte, the method comprising:

a. providing a single-stranded DNA or RNA template;

b. providing a first plurality of identical, sequence-specific oligonucleotide probes and a second plurality of identical oligonucleotide probes, the first plurality being different from the second plurality;

c. hybridizing the first plurality of probes and the second plurality of probes to the template;

d. conducting an enzymatic ligation reaction to ligate hybridized probes from the first plurality to adjacent probes from the second plurality; and

e. allowing additional unhybridized probes from the first plurality of probes to hybridize to the template to prepare the biomolecule analyte.

25. The method of claim 24 wherein the probes from the second plurality each include at least one degenerate or universal site.

26. The method of claim 24, wherein at least a portion of the first plurality of probes comprises tagged probes.

27. The method of claim 24, wherein at least a portion of the second plurality of probes comprises tagged probes.

28. The method of claim 24, wherein at least a portion of the template is coated with a protein.

29. The method of claim 24, wherein at least a portion of the first plurality of probes is coated with a protein.

30. The method of claim 24, wherein at least a portion of the second plurality of probes is coated with a protein.

34. A method for analyzing a biomolecule analyte comprising the steps of:

a. preparing the biomolecule analyte using the method of claim 24;

b. providing an apparatus having a first fluid chamber, a second fluid chamber, a membrane positioned between the first and second chambers and a nanopore extending through the membrane such that the first and second chambers are in fluid communication via the nanopore;

c. introducing the biomolecule analyte into the first chamber;

d. translocating the biomolecule analyte from the first chamber through the nanopore and into the second chamber;

e. monitoring changes in an electrical property across the nanopore as the biomolecule analyte is translocated therethrough, the changes in the electrical property corresponding to locations along the biomolecule analyte containing probes; and

f. recording the changes in the electrical property as a function of time.

38. A method for analyzing a biomolecule analyte comprising the steps of:

a. preparing the biomolecule analyte using the method of claim 24;

b. disposing the biomolecule analyte in a fluidic nanochannel or microchannel;

c. applying a potential along the fluidic channel;

d. translocating the biomolecule analyte from a first end of the fluidic channel to a second end of the fluidic channel; and

e. detecting electrical properties as the biomolecule analyte moves through the fluidic channel, the electrical properties corresponding to at least one detector volume in the fluidic channel, each detector volume being defined by two or more sensing electrodes disposed along the length of the fluidic channel, wherein the detected electrical signals indicate locations of hybridized probes along the biomolecule analyte.