PHOSPHOLINK NUCLEOTIDES FOR SEQUENCING APPLICATIONS

• US 20100167299A1
• Filed: 11/17/2009
• Published: 07/01/2010
• 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
• Granted Patent: US 8,367,813 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 composition, comprising a labeled phospholink nucleotide, wherein said labeled phospholink nucleotide comprises a structure that has formula:

2. The composition of claim 1, 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.

3. The composition of claim 1, wherein said linker moiety comprises poly(ethylene glycol).

4. The composition of claim 1, wherein said bead is a latex bead.

5. The composition of claim 1, wherein said bead is a fluorescent bead.

6. The composition of claim 1, wherein said bead comprises a fluorophore.

7. The composition of claim 6, wherein said bead is encapsulated in an organic or inorganic material.

8. The composition of claim 6, wherein said bead is encapsulated in a polymeric material.

9. The composition of claim 8, wherein said polymeric material is a member selected from polyethylene glycol and alginate.

20. A kit comprising at least four differently labeled nucleotide analogs each comprising said structure of said composition of claim 1, a polymerase enzyme source, and instructions for using said composition with said polymerase enzyme in template directed nucleic acid synthesis.

10. A composition comprising a microfluidic flow cell, said composition further comprising:

a. a nucleic acid polymerase immobilized on a surface of a substrate, wherein said substrate is contained within said microfluidic flow cell; and

b. at least four differentially labeled nucleotides, wherein said labeled nucleotides comprise beads comprising a fluorophore.

11. 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, 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.

12. The method of claim 11, wherein said first complex is contained in a microfluidic flow cell, and wherein said detecting step comprises detecting a temporary retention of said bead at a stationary location of said flow cell.

13. The method of claim 12, 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.

14. The method of claim 11, wherein said first complex is contained in a zero-mode waveguide.

15. The method of claim 11, wherein said bead comprises multiple fluorophores.

16. The method of claim 15, 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.

17. The method of claim 11, wherein multiple nucleotides are attached to said bead.

18. The method of claim 17, wherein subsequent to said detecting step, said method further comprises cleaving said bead from said phospholink nucleotide.

19. The method of claim 18, 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.

21. A nucleic acid sequencing system, comprising:

a body structure comprising a plurality of microfluidic channels;

a source of one or more template nucleic acid, which template nucleic acid source is capable of being fluidly coupled to at least a first one of the microfluidic channels;

a source of one or more sequencing reagent, which sequencing reagent source is capable of being fluidly coupled to the at least first microfluidic channel;

wherein the source of one or more sequencing reagent comprises a set of at least four differently labeled nucleotide analogs, wherein said labeled nucleotide analogs comprise a fluorescent bead, and a fluid flow controller that flows the template nucleic acid and the one or more sequencing reagents into contact in the at least first microfluidic channel, whereby one or more sequencing product is formed, wherein a detector detects the one or more sequencing product, thereby determining at least a subsequence of the template nucleic acid.