Ultra High-Throughput Opti-Nanopore DNA Readout Platform

• US 20090029477A1
• Filed: 08/12/2005
• Published: 01/29/2009
• Est. Priority Date: 08/13/2004
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Mathe, Jerome, Eid, John S., Meller, Amit
• Granted Patent: US 7,972,858 B2
• Time in Patent Office: Days
• US Class Current: 436/94
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2535/131 Allele specific probes
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 33/48721 Investigating individual macromolecules, e.g. by translocation through nanopores Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68
• CPC Class Codes: Y10T 436/14 Heterocyclic carbon compound [i.e. , O, S, N, Se, Te, as only ring hetero atom]
• CPC Class Codes: Y10T 436/142222 Hetero-O [e.g., ascorbic acid, etc.]
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Described herein are methods for analyzing polymer molecules. These methods are employed for the high throughput readout of DNA and RNA molecules with single molecule sensitivity. The method of the present invention comprises (1) the electrically controlled unzipping of DNA (or RNA) double strands, and (2) the readout of the molecule'"'"'s identity (or code) using one or more molecule signal detection.

Claims

1. A method for analyzing one or more target polynucleotide molecules comprising:

(a) designing one or more Design Monomers using said target polynucleotide as a template, wherein an individual Design Monomer further corresponds to either a “

0”

or a “

1”

, wherein two individual Design Monomers correspond to a predetermined nucleotide base, and wherein said individual Design Monomer has one or more signal molecules;

(b) forming one or more Design Polymers by ligating a plurality of said Design Monomers;

(c) repeating steps (a) and (b) until one or more predetermined Design Polymers are constructed;

(d) forming a Design Polymer—

signal complex by admixing, under conditions suitable for hybridization, said Design Polymer with a signal molecule; and

(e) subjecting said complex of (d) to analysis.

2. The method of claim 1, wherein said target polynucleotide is selected from the group consisting of DNA, RNA and PNA.

3. The method of claim 2, wherein said target polynucleotide is DNA.

4. The method of claim 3, wherein said DNA is either cDNA or gDNA.

5. The method of claim 2, wherein said target polynucleotide is selected from the group consisting of single-stranded, double-stranded, or a combination of both.

6. The method of claim 1, wherein said target polynucleotide can be eukaryotic or prokaryotic.

7. The method of claim 1, wherein said target polynucleotide is either plant or animal.

8. The method of claim 1, wherein said target polynucleotide can have a size ranging from about 10 nucleotide bases to about 100,000 nucleotide bases.

9. The method of claim 1, wherein said Design Monomer comprises one or more nucleotide bases.

10. The method of claim 9, wherein said Design Monomer has a range in nucleotide bases ranging from about 1 nucleotide bases to about 100 nucleotide bases.

11. The method of claim 9, wherein said Design Monomer has a range in nucleotide bases ranging from about 5 nucleotide bases to about 10 nucleotide bases.

12. The method of claim 9, wherein said Design Monomer can comprise DNA, RNA or PNA.

13. The method of claim 1, wherein each individual unit of said target polynucleotide is represented by a binary code.

14. The method of claim 13, wherein said individual unit is selected from the group consisting of adenine, cytosine, uricil, guanine, thymine and modifications thereof.

15. The method of claim 13, wherein said binary code comprises a “

0” and

a “

1”

, wherein a particular arrangement of said “

0” and

“

1”

corresponds to said individual unit.

16. The method of claim 1, wherein said Design Polymer comprises one or more Design Monomers arrayed in a particular sequence corresponding to said target polynucleotide.

17. The method of claim 1, wherein said signal molecule is hybridized to said Design Monomer.

18. The method of claim 17, wherein percent hybridization between said signal molecule and said Design Monomer is about 90% or greater.

19. The method of claim 17, wherein percent hybridization between said signal molecule and said Design Monomer is between about 80% to about 90%.

20. The method of claim 17, wherein percent hybridization between said signal molecule and said Design Monomer is between about 70% to about 80%.

21. The method of claim 17, wherein percent hybridization between said signal molecule and said Design Monomer is between about 60% to about 70%.

22. The method of claim 1, wherein said signal molecule is a molecular beacon.

23. The method of claim 22, wherein said molecular beacon comprises one or more fluorophores and one or more quenchers.

24. The method of claim 23, wherein a nucleotide sequence on the 5′

end of said beacon is complementary to a nucleotide sequence on the 3′

end of said beacon.

25. The method of claim 1, wherein there are two molecular beacons, wherein each beacon corresponds to a particular Design Monomer.

26. The method of claim 25, wherein a first molecular beacon corresponds to a first Design Monomer, and wherein a second molecular beacon corresponds to a second Design Monomer.

27. The method of claim 1, wherein steps (a) and (b) form a cycle for producing said Design Polymer.

28. The method of claim 27, wherein said Design Polymer comprises six Design Monomers after one cycle, whereby said Design Polymer is extended by at least six Design Monomers after completion of each cycle.

29. The method of claim 28, wherein said Design Monomers are covalently inked to one another in said Design Polymer.

30. The method of claim 1, wherein said Design Polymer in step (b) is a double-stranded polynucleotide molecule.

31. The method of claim 1, wherein said Design Polymer in step (d) is partially or completely single-stranded.

32. The method of claim 1, wherein step (e) further comprises introducing said Design Polymer-signal complex to a nanopore system.

33. The method of claim 32, wherein said nanopore system comprises a plurality of nanopores and one or more detection systems.

34. The method of claim 33, wherein said nanopore comprise one or more channels and a lipid bilayer.

35. The method of claim 34, wherein said nanopore is selected from the group consisting of α

-hemolysin, receptor for bacteriophage lambda, gramicidin, valinomycin, Escherichia coli, Shigella, Tsx, F-pilus, mitochondrial porin.

36. The method of claim 35, wherein said nanopore is α

-hemolysin.

37. The method of claim 36, wherein said α

-hemolysin comprises a vestibule and a channel.

38. The method of claim 37, wherein said vestibule is about 2.5 nm in diameter.

39. The method of claim 37, wherein said channel has about a 1.5 nm diameter.

40. The method of claim 34, wherein said channel has a pore size ranging from about 0.5 nm to about 2.0 nm.

41. The method of claim 32 further comprising the unzipping of said Design Polymer-signal complex, whereby a single-stranded portion of said Design Polymer enters into and through a channel of said nanopore system.

42. The method of claim 41, wherein said unzipping is a result of an electric field established across said nanopore system.

43. The method of claim 41, wherein movement of said single-stranded Design Polymer into and through said channel is facilitated by an electric field across said nanopore system.

44. The method of claim 41, wherein the unzipping process leads to the liberation of one or more signal molecules.

45. The method of claim 44, wherein said released signal molecule comprises a molecule which emits detectable energy and a quencher molecule, wherein said released signal molecule self-hybridizes thereby quenching said emitting molecule.

46. The method of claim 45, wherein said emitting molecule is a fluorophore.

47. The method of claim 46, wherein said fluorophore is selected from the group consisting of TMR, Cy5, CPM, and alike.

48. The method of claim 46, wherein said quenching molecule is selected from the group consisting of Dabcyl, Dabsyl, methyl red, Elle Quencher, and alike.

49. The method of claim 1, wherein said analysis of step (e) comprises the detection of energy from said signal molecule.

50. The method of claim 49, wherein said detection comprises fluorescence detection.

51. The method of claim 33, wherein said detection system is an optical detection system.

52. The method of claim 51, wherein said optical detection system is a fluorescence detector.

53. A method for analyzing one or more target polynucleotide molecules comprising:

(a) designing one or more Design Monomers using said target polynucleotide as a template, wherein an individual Design Monomer further corresponds to either a “

0”

or a “

1”

, wherein two individual Design Monomers correspond to a predetermined nucleotide base, and wherein said individual Design Monomer has one or more signal molecules;

(b) forming one or more Design Polymers by ligating a plurality of said Design Monomers;

(c) repeating steps (a) and (b) until one or more predetermined Design Polymers are constructed;

(d) forming a Design Polymer—

signal complex by admixing, under conditions suitable for hybridization, said Design Polymer with a signal molecule;

(e) introducing said complex of (d) to a nanopore system, wherein said complex is unzipped, and wherein a single-stranded portion of said Design Polymer enters into and through said nanopore system; and

(f) analyzing said single-stranded Design Polymer of (e).