Field-switch sequencing

• US 20050266456A1
• Filed: 04/29/2005
• Published: 12/01/2005
• Est. Priority Date: 04/30/2004
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Li & S Company Limited (Yesco Holdings Co., Ltd.)
• Inventors: Williams, John G.K., Anderson, Jon P.
• Granted Patent: US 7,462,452 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2523/307 Denaturation or renaturation by electric current/voltage
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

The present invention provides novel compositions, methods and apparatus for DNA sequencing that can be performed, e.g., in a two-electrode chamber. The present invention also provides a method for sequencing a nucleic acid comprising immobilizing a plurality of complexes comprising a target nucleic acid, a primer nucleic acid, and a polymerase onto a surface, contacting the surface with a plurality of charged particles comprising a nucleotide phosphate by applying an electric field, reversing the electric field to transport unbound charged particles away from the surface, and detecting the incorporation of a nucleotide phosphate into a single molecule of the primer nucleic acid.

Claims

1. A particle, said particle comprising:

a particle surface;

at least one nucleotide phosphate attached to said particle surface via a linker; and

a dye, wherein said particle carries a charge.

2. The particle according to claim 1, wherein the at least one nucleotide phosphate is at least 2 nucleotide phosphates.

3. The particle according to claim 1, wherein the dye is attached to the surface of said particle.

4. The particle according to claim 1, wherein said dye is embedded in said particle.

5. The particle according to claim 1, wherein said dye and said nucleotide phosphate are attached to said particle through said linker.

6. The particle according to claim 1, wherein said particle is selected from the group consisting of a nucleic acid nanoparticle, a semiconductor nanoparticle, a nanocrystal, a silicon nanocrystal, a metallic nanoparticle, a silica-shell nanoparticle, a dye-doped silica-shell nanoparticle, a nanoparticle made of an organic polymer, a dendrimer, a light-scattering nanoparticle, an electroactive nanoparticle, a coated nanoparticle, a lipid nanoparticle, a protein nanoparticle, a fluorescent protein, and combinations thereof.

7. The particle according to claim 6, wherein said particle is a nucleic acid nanoparticle.

8. The particle according to claim 7, wherein said particle is a nucleic acid nanoparticle selected from the group consisting of a hairpin structure consisting of tandem inverted repeat DNA sequences, a DNA nanoparticle, a RNA nanoparticle, a silica-DNA nanoparticle, a nucleic acid analog nanoparticle, a DNA nanoparticle comprising multiple complementary and non-complementary strands of single stranded DNA, an RNA nanoparticle comprising multiple complementary and non-complementary strands of single stranded RNA, and a combination thereof.

9. The particle according to claim 8, wherein said particle is a hairpin structure consisting of tandem inverted repeat DNA sequences.

10. The particle according to claim 1, wherein said particle surface is coated with a reactive functional group.

11. The particle according to claim 10, wherein said reactive functional group is a haloacetyl group, an amine, a thiol, a phosphate, a carboxylate, and combination thereof.

12. The particle according to claim 1, wherein said particle further comprises an oligonucleotide.

13. The particle according to claim 12, wherein said oligonucleotide is a plurality of oligonucleotides.

14. The particle according to claim 12, wherein said dye is attached to said oligonucleotide.

15. The particle according to claim 1, wherein said nucleotide phosphate is a member selected from the group consisting of a nucleotide diphosphate, a nucleotide triphopshate (NTP), a nucleotide tetraphosphate, a nucleotide pentaphosphate and a nucleotide hexaphosphate.

16. The particle according to claim 15, wherein said nucleotide phosphate is a nucleotide triphopshate (NTP).

17. The particle according to claim 16, wherein said NTP is attached to said oligonucleotides via covalent bonding.

18. The particle according to claim 1, wherein said linking moiety comprises polyethylene glycol (PEG).

19. The particle according to claim 1, wherein said dye is a fluorescent dye.

20. The particle according to claim 19, wherein said fluorescent dye is an intercalating dye.

21. The particle according to claim 20, wherein said intercalating dye is selected from the group consisting of YOYO-1, JOJO-1, LOLO-1, YOYO-3, BOBO-3, SYBR, SYTO, SYTOX, PicoGreen, OilGreen and a combination thereof.

22. The particle according to claim 19, wherein said fluorescent dye is a non-intercalating dye.

23. The particle according to claim 12, wherein said dye is attached to said oligonucleotides via covalent bonding.

24. The particle according to claim 12, wherein said dye is attached to said at least one of said plurality of oligonucleotides via non-covalent bonding.

25. The particle according to claim 12, wherein said dye is attached to said oligonucleotides via ionic bonding.

26. The particle according to claim 12, wherein said dye is attached to said oligonucleotides via hydrophobic interactions.

27. The particle according to claim 1, wherein said particle has a diameter of about 10 nanometers to about 10 microns.

28. The particle according to claim 27, wherein said particle has a diameter of about 20 nanometers to about 100 nanometers.

29. A method for sequencing a nucleic acid, said method comprising:

(a) immobilizing a plurality of complexes comprising a target nucleic acid, a primer nucleic acid, and a polymerase onto a surface;

(b) contacting said surface with a plurality of charged particles comprising a nucleotide phosphate (NP) by applying an electric field;

(c) reversing said electric field to transport unbound charged particles away from the surface; and

(d) detecting the incorporation of said nucleotide phosphate into a single molecule of the primer nucleic acid.

30. The method according to claim 29, wherein each of said plurality of particles comprises a plurality of NPs.

31. The method according to claim 29, wherein said surface comprises a metal, a metal silicide or metal oxide.

32. The method according to claim 31, wherein said metal or metal oxide is selected from the group consisting of indium, tin, indium-tin, indium-tin oxide, silver, silver oxide, platinum, palladium and gold.

33. The method according to claim 31, wherein said surface comprises an indium-tin oxide (ITO) electrode.

34. The method according to claim 33, wherein said ITO electrode is coated by a permeation layer.

35. The method according to claim 34, wherein said permeation layer is between about 5 nm to about 30 nm thick.

36. The method according to claim 29, wherein said plurality of charged particles are selected from the group consisting of nucleic acid nanoparticles, nucleic acid analog particles, semiconductor nanoparticles, nanocrystals, silicon nanocrystals, metallic nanoparticles, silica-shell nanoparticles, dye-doped silica-shell nanoparticles, nanoparticles made of organic polymers, dendrimers, light-scattering nanoparticles, electroactive nanoparticles, coated nanoparticles, lipid nanoparticles, protein nanoparticles, fluorescent proteins, particles comprising multiple fluorescent dyes and combinations thereof.

37. The method according to claim 36, wherein said plurality of charged particles are nucleic acid nanoparticles selected from the group consisting of a hairpin structure consisting of tandem inverted repeat DNA sequences, a DNA nanoparticle, a RNA nanoparticle, a silica-DNA nanoparticle, a nucleic acid analog nanoparticle, a DNA nanoparticle comprising multiple complementary and non-complementary strands of single stranded DNA, an RNA nanoparticle comprising multiple complementary and non-complementary strands of single stranded RNA, and a combination thereof.

38. The method according to claim 29, wherein said plurality of charged particles further comprise at least one dye.

39. The method according to claim 29, wherein detecting comprises detecting a unique signal from the nucleoside phosphate using a system or device selected from the group consisting of an optical reader, a high-efficiency photon detection system, a photodiode, a camera, a charge couple device, an intensified charge couple device, an on-chip multiplication gain charge coupled device, a near-field scanning microscope, a far-field confocal microscope, a microscope that detects wide-field epi-illumination, and a total internal reflection fluorescence microscope.

40. The method according to claim 39, wherein the incorporation of a nucleotide phosphate is detected by a total internal reflection fluorescence microscope.

41. The method according to claim 40, wherein said total internal reflection fluorescence microscope uses evanescent wave excitation or surface plasmon excitation.

42. The method according to claim 29, wherein the detecting comprises detecting one or more cleavage molecules.

43. The method according to claim 29, wherein the detecting comprises detecting said nucleotide phosphate while associated with the polymerase.

44. The method according to claim 29, wherein said detecting is carried out by a mechanism that provides low background and enables the detection of single particles.

45. The method according to claim 29, wherein said detecting is carried out by a mechanism selected from the group consisting of fluorescence resonance energy transfer, an electron transfer mechanism, an excited-state lifetime mechanism and a ground-state complex quenching mechanism.

46. The method according to claim 29, wherein polymerase catalyzed nucleotide incorporation is synchronized with the application of said electric field.

47. A sequencing chamber, said sequencing chamber comprising:

a first electrode on one side of said chamber;

a second electrode on another side said chamber; and

a spacer separating said first electrode and second electrode.

48. The sequencing chamber according to claim 47, wherein said first electrode and said second electrode independently are selected from a metal, a metal silicide, carbon and a metal oxide.

49. The sequencing chamber according to claim 48, wherein said metal is selected from the group consisting of indium, tin, indium-tin, silver, platinum, palladium, titanium, aluminum and gold.

50. The sequencing chamber according to claim 47, wherein said metal oxide is selected from the group consisting of indium oxide, tin oxide, indium-tin oxide (ITO), titanium oxide, silver oxide and aluminum oxide.

51. The sequencing chamber according to claim 47, wherein said spacer comprises a polymer, insulator, dielectric, semiconductor or a combination thereof.

52. The sequencing chamber according to claim 47, wherein said spacer comprises PDMS, SU-8 or PET.

53. The sequencing chamber according to claim 47, wherein said first electrode comprises a permeation layer.

54. The sequencing chamber according to claim 47, wherein said second electrode comprises a permeation layer.

55. The sequencing chamber according to claim 47, wherein said first electrode on one side of said chamber is the bottom of the chamber and said second electrode on said other side of said chamber is the top of said chamber.