Field switch sequencing

• US 9,045,798 B1
• Filed: 03/15/2006
• Issued: 06/02/2015
• 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:
  US11/377,136
• Time in Patent Office: 3,366 Days
• US Class Current: 1/1
• 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 increasing the signal to noise ratio (S/N) for single particle detection while 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) providing a sequencing reaction mixture including single particles, which are dendrimers, each said single particle comprising both i) a plurality of dyes and ii) a plurality of incorporatable substrate nucleotide phosphates for the polymerase, wherein a plurality of said single particles sequentially reacts with an immobilized complex, wherein in each said sequential reaction one of the plurality of nucleotide phosphates reacts with the polymerase, is cleaved from the dendrimer, and is incorporated into the primer nucleic acid, thereby releasing the dendrimer from the complex; and

(c) detecting said single particle while incorporating each nucleotide phosphate, thereby sequencing the target nucleic acid.

2. The method of claim 1, further comprising increasing the detection speed for said single particle comprising a plurality of dyes compared to a single particle comprising a single dye.

3. The method of claim 1, wherein each of said plurality of dyes is a fluorescent dye.

4. The method of claim 3, wherein said fluorescent dyes are selected from the group consisting of a cyanine dye, a cyanine dye dimer and a combination thereof.

5. The method of claim 1, wherein detecting comprises detecting a unique signal from said single particle comprising a plurality of dyes, 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.

6. The method of claim 1, wherein said detecting is carried out by a mechanism that provides low background and enables the detection of single particles.

7. The method of 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.

8. The method of claim 1, wherein each of said plurality of incorporatable substrate nucleotide phosphates is a member selected from the group consisting of a nucleotide triphosphate, a nucleotide tetraphosphate, a nucleotide pentaphosphate, and a nucleotide hexaphosphate.

9. The method of claim 1, wherein the detecting occurs during association of the incorporatable substrate nucleotide phosphate within the active site of the polymerase.

10. The method of claim 9, wherein the polymerase is selected from the group consisting of a DNA polymerase, an RNA polymerase, and a reverse transcriptase.

11. The method of claim 1, wherein each of the plurality of incorporatable substrate nucleotide phosphates is attached to at least one of the plurality of dyes by a linker.