Field-switch sequencing

• US 8,592,148 B2
• Filed: 10/31/2008
• Issued: 11/26/2013
• Est. Priority Date: 04/30/2004
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Williams, John G. K., Anderson, Jon P.
• Primary Examiner(s): BERTAGNA, ANGELA MARIE
• Application Number:
  US12/263,397
• Publication Number:
  US 20110177496A1
• Time in Patent Office: 1,852 Days
• Field of Search: None
• US Class Current: 435/6.1
• 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 method for detecting a catalytic event having improved binding kinetics, said method comprising:

(a) immobilizing a target nucleic acid, a primer nucleic acid, and a polymerase onto a surface;

(b) contacting said surface with a plurality of nanoparticles, wherein each nanoparticle comprises a plurality of nucleotide phosphates (NPs), which are substrates for the polymerase, wherein each of said plurality of nucleotide phosphates (NPs) is attached to the nanoparticle via a linker, wherein the nanoparticle size, the linker and the NP density on the nanoparticle surface are selected such that the K_m for the binding of nanoparticle-immobilized NPs to the polymerase is lowered compared to the K_m for a free nucleotide in solution binding to the polymerase; and

(c) detecting polymerase-mediated incorporation of at least one of said plurality of nucleotide phosphates into a single molecule of the primer nucleic acid to detect said catalytic event, wherein the nanoparticle is released from the nucleotide phosphate upon incorporation, and wherein the catalytic event has improved binding kinetics.

2. The method of claim 1, wherein detecting said catalytic event is by detecting one or more cleavage molecules.

3. The method of claim 1, wherein detecting said catalytic event occurs while the nucleotide phosphate is resident within the polymerase active site.

4. The method of claim 1, wherein the detection is carried out by a mechanism that provides low background and enables the detection of single molecules.

5. The method of claim 1, wherein said detection occurs while the nucleotide phosphate is resident with the polymerase, and wherein said polymerase is a member selected from the group consisting of a DNA-dependent DNA polymerase, a DNA-dependent RNA polymerase and a reverse transcriptase.

6. The method of claim 1, wherein detecting is via 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.

7. The method of claim 1, wherein the detection is 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.

8. The method of claim 1, wherein detecting is performed using a detection device, and wherein the detection device is a four color single molecule imaging microscope.

9. The method of claim 1, wherein said nanoparticle is selected from the group consisting of a nucleic acid nanoparticle, a nucleic acid analog particle, 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 light-scattering nanoparticle, an electroactive nanoparticle, a coated nanoparticle, a protein nanoparticle, a fluorescent protein, and combinations thereof.

10. The method of claim 1, wherein said nanoparticle is selected from the group consisting of a 20 nm polystyrene sphere, a semiconductor nanocrystal, a quantum dot, a resonance light scattering (RLS) metal particle, a dye-doped silica shell nanoparticle, and a silicon crystal nanoparticle.

11. The method of claim 1, wherein said nanoparticle further comprises a dye.

12. The method of claim 11, wherein said dye is attached to the surface of said nanoparticle.

13. The method of claim 11, wherein said dye is embedded in said nanoparticle.

14. The method of claim 11, wherein said dye and said nucleotide phosphates are attached to said nanoparticle through said linker.

15. The method of claim 10, wherein said nanoparticle surface is coated with a reactive functional group.

16. The method of claim 15, wherein said reactive functional group is a haloacetyl group, an amine, a thiol, a phosphate, a carboxylate, or a combination thereof.

17. The method of claim 1, wherein said nucleotide phosphates are selected from the group consisting of a nucleotide diphosphate, a nucleotide triphopshate (NTP), a nucleotide tetraphosphate, a nucleotide pentaphosphate and a nucleotide hexaphosphate.

18. The method of claim 17, wherein said nucleotide phosphates are nucleotide triphosphates (NTPs).

19. The method of claim 10, wherein said linker comprises polyethylene glycol (PEG).

20. The method of claim 11, wherein said dye is a fluorescent dye.

21. The method of claim 9, wherein said semiconductor nanoparticle is a quantum dot.