Phospholink nucleotides for sequencing applications

• US 8,921,532 B2
• Filed: 10/07/2013
• Issued: 12/30/2014
• 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:
  US14/047,644
• Publication Number:
  US 20140065607A1
• Time in Patent Office: 449 Days
• Field of Search: 435/6.1, 435/91.1, 536/4.1
• US Class Current: 536/4.1
• 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 composition, comprising a labeled phospholink nucleotide, wherein said labeled phospholink nucleotide comprises a structure that has formula:

2. The composition of claim 1 wherein L has a diameter ranging from about 10 nm to about 500 nm.

3. The composition of claim 1 wherein the beads are spherical in shape.

4. 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.

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

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

7. The composition of claim 1, wherein said bead is encapsulated in a polymeric Material.

8. The composition of claim 1 wherein the bead comprises multiple scattering or magnetic moities.

9. The composition of claim 1 wherein multiple nucleotides are attached to the bead.

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

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;

contacting said first complex with one or more labeled phospholinked nucleotides with the composition of claim 1; and

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.

11. The method of claim 10, 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.

12. The method of claim 10 wherein L has a diameter ranging from about 10 nm to about 500 nm.

13. The method of claim 10 wherein the beads are spherical in shape.

14. 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.

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

16. The method of claim 10, wherein said bead is a latex bead.

17. The method of claim 10, wherein said bead is encapsulated in a polymeric material.

18. The method of claim 10 wherein the head comprises multiple scattering or magnetic moieties.

19. The method of claim 10 wherein multiple phospholinked nucleotides are attached to the bead.