Phospholink nucleotides for sequencing applications

• US 8,580,539 B2
• Filed: 12/31/2012
• Issued: 11/12/2013
• Est. Priority Date: 11/17/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Korlach, Jonas
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/731,493
• Publication Number:
  US 20130122490A1
• Time in Patent Office: 316 Days
• Field of Search: 435/6.1, 435/91.1
• US Class Current: 435/91.1
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence

Abstract

The present invention provides labeled phospholink nucleotides that can be used in place of naturally occurring nucleotide triphosphates or other analogs in template directed nucleic acid synthesis reactions and other nucleic acid reactions and various analyses based thereon, including DNA sequencing, single base identification, hybridization assays, and others.

Claims

1. A method of determining an identity of a nucleotide in a template nucleic acid sequence, said method comprising:

a. providing said template nucleic acid sequence complexed with a polymerase enzyme capable of template dependent synthesis of a complementary nascent sequence as a first complex;

b. contacting said first complex with a labeled phospholink nucleotide, wherein said labeled phospholink nucleotide comprises a bead comprising at least one fluorophore, wherein said bead is encapsulated in an organic, inorganic, or polymeric material, and wherein said labeled phospholink nucleotide is complementary to a known nucleotide; and

c. detecting whether said labeled phospholink nucleotide is incorporated into said nascent sequence, wherein incorporation of said labeled phospholink nucleotide is indicative that said complementary nucleotide is in a position in the template nucleic acid that is being processed by the polymerase enzyme.

2. The method of claim 1, wherein said first complex is contained in a microfluidic flow cell.

3. The method of claim 2, wherein said microfluidic flow cell comprises a surface, and wherein said polymerase of said first complex is immobilized on said surface of said microfluidic flow cell.

4. The method of claim 1, wherein said first complex is contained in a zero-mode waveguide.

5. The method of claim 1, wherein said bead comprises multiple fluorophores.

6. The method of claim 5, wherein said detecting step comprises detecting a signal from said bead comprising multiple fluorophores, and wherein said signal from said bead has an improved signal relative to a bead comprising a single fluorophore.

7. The method of claim 1, wherein multiple nucleotides are attached to said bead.

8. The method of claim 7, wherein subsequent to said detecting step, said method further comprises cleaving said bead from said phospholink nucleotide incorporated into said nascent sequence.

9. The method of claim 8, wherein said providing, contacting, detecting and cleaving steps are repeated a number of times to identify a desired number of nucleotides in said template nucleic acid sequence.

10. The method of claim 1, wherein said labeled phospholink nucleotide comprises a structure that has a formula:

11. The method of claim 10, wherein said linker moiety comprises poly(ethylene glycol).

12. The method of claim 10, wherein said linker moiety is a member selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl.

13. The method of claim 1, wherein said bead is a latex bead.

14. The method of claim 1, wherein said bead is a fluorescent bead.

15. The method of claim 1, wherein said polymeric material is a member selected from polyethylene glycol and alginate.