Field-switch sequencing

• US 7,462,452 B2
• Filed: 04/29/2005
• Issued: 12/09/2008
• 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): Horlick; Kenneth R.
• Assistant Examiner(s): BERTAGNA, ANGELA MARIE
• Application Number:
  US11/118,031
• Publication Number:
  US 20050266456A1
• Time in Patent Office: 1,320 Days
• Field of Search: None
• US Class Current: 435/6.11
• 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 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) applying an electric field to contact said surface with a plurality of charged nucleic acid nanoparticles comprising nucleotide phosphate (NP);

(c) removing said nucleotide phosphate (NP) from said charged nucleic acid nanoparticle, wherein said nucleotide phosphate is a substrate for said polymerase;

(d) reversing said electric field to transport unbound charged nucleic acid nanoparticles away from the surface; and

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

2. The method according to claim 1, wherein each of said plurality of charged nucleic acid nanoparticles comprises a plurality of NPs.

3. The method according to claim 1, wherein said surface comprises a metal, a metal silicide or metal oxide.

4. The method according to claim 3, 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.

5. The method according to claim 3, wherein said surface comprises an indium-tin oxide (ITO) electrode.

6. The method according to claim 5, wherein said ITO electrode is coated by a permeation layer.

7. The method according to claim 6, wherein said permeation layer is between about 5 nm to about 30 nm thick.

8. The method according to claim 1, wherein said plurality of charged nucleic acid nanoparticles is 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 method according to claim 1, wherein said plurality of charged nucleic acid nanoparticles further comprises at least one dye.

10. The method according to claim 9, wherein said plurality of charged nucleic acid nanoparticles comprises multiple fluorescent dyes.

11. The method according to claim 1, wherein detecting comprises detecting a unique signal from the nucleotide 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.

12. The method according to claim 11, wherein the incorporation of a nucleotide phosphate is detected by a total internal reflection fluorescence microscope.

13. The method according to claim 12, wherein said total internal reflection fluorescence microscope uses evanescent wave excitation or surface plasmon excitation.

14. The method according to claim 1, wherein the detecting comprises detecting one or more cleavage molecules.

15. The method according to claim 1, wherein the detecting comprises detecting said nucleotide phosphate while associated with the polymerase.

16. The method according to claim 1, wherein said detecting is carried out by a mechanism that enables the detection of single particles.

17. The method according to claim 1, 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.

18. The method according to claim 1, wherein polymerase catalyzed nucleotide incorporation is synchronized with the application of said electric field.