Nucleotide compositions and uses thereof

• US 8,927,211 B2
• Filed: 02/04/2010
• Issued: 01/06/2015
• Est. Priority Date: 02/09/2005
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Turner, Stephen, Korlach, Jonas
• Primary Examiner(s): Kim, Young J
• Application Number:
  US12/700,593
• Publication Number:
  US 20100240045A1
• Time in Patent Office: 1,797 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2527/127 the enzyme inhibitor or activator used
• CPC Class Codes: C12Q 2533/101 Primer extension

Abstract

The present invention relates to preparation of nucleotide compositions and uses thereof for conducting nucleic acid analyzes. The compositions and methods embodied in the present invention are particularly useful for nucleic acid analyzes that require high-resolution detection of labeled nucleotides or labeled nucleic acid targets.

Claims

1. A method for producing a nucleotide composition, comprising:

providing a solution containing at least one labeled nucleotide or nucleotide analog, wherein the labeled nucleotide or nucleotide analog emits a detectable optical signal;

treating the solution with an agent that selectively renders unlabeled nucleotides or nucleotide analogs substantially incapable of participating in an analytical reaction of interest in which the labeled nucleotide or nucleotide analog participates, wherein the agent displays preferential activity toward alteration or modification of the unlabeled nucleotides or nucleotide analogs as compared to the labeled nucleotide or nucleotide analog; and

subsequent to said treating, performing an additional step of adding said solution to a reaction mixture for the analytical reaction of interest, wherein the reaction mixture comprises a primer, a template nucleic acid, and a polymerase.

2. The method of claim 1, wherein the treating step renders the unlabeled nucleotides or nucleotide analogs unincorporatable by a polymerase enzyme in a template-dependent polymerization reaction, wherein an unincorporatable unlabeled nucleotide or nucleotide analog so rendered is no longer available for incorporation into a nascent strand in the template-dependent polymerization reaction.

3. The method of claim 1, wherein the agent comprises an enzyme.

4. The method of claim 3, wherein the enzyme selectively alters the unlabeled nucleotide or nucleotide analogs via a hydrolysis, transferase, phosphorylase, or polymerase reaction.

5. The method of claim 3, wherein the enzyme comprises a polymerase that selectively scavenges unlabeled nucleotides or nucleotide analogs.

6. The method of claim 3, wherein the agent comprises a polymerase enzyme and Mg²⁺.

7. The method of claim 3, wherein the enzyme comprises a phosphatase.

8. The method of claim 7, wherein the phosphatase comprises shrimp alkaline phosphatase.

9. The method of claim 7, wherein the phosphatase comprises a calf intestinal phosphatase.

10. The method of claim 1, wherein following the treating step, less than 10% of the nucleotides or nucleotide analogs in the solution are unlabeled nucleotides or nucleotide analogs that are capable of participating in an analytical reaction of interest in which a labeled nucleotide or nucleotide analog participates.

11. The method of claim 1, wherein following the treating step, less than 5% of the nucleotides or nucleotide analogs in the solution are unlabeled nucleotides or nucleotide analogs that are capable of participating in an analytical reaction of interest in which a labeled nucleotide or nucleotide analog participates.

12. The method of claim 1, wherein following the treating step, less than 1% of the nucleotides or nucleotide analogs in the solution are unlabeled nucleotides or nucleotide analogs that are capable of participating in an analytical reaction of interest in which a labeled nucleotide or nucleotide analog participates.

13. The method of claim 1, wherein following the treating step, less than 0.1% of the nucleotides or nucleotide analogs in the solution are unlabeled nucleotides or nucleotide analogs that are capable of participating in an analytical reaction of interest in which a labeled nucleotide or nucleotide analog participates.

14. The method of claim 1, wherein following the treating step, less than 0.01% of the nucleotides or nucleotide analogs in the solution are unlabeled nucleotides or nucleotide analogs that are capable of participating in an analytical reaction of interest in which a labeled nucleotide or nucleotide analog participates.

15. The method of claim 1, wherein the analytical reaction of interest is a sequencing reaction.

16. The method of claim 1, wherein the solution comprises at least two different labeled nucleotides or nucleotide analogs.

17. The method of claim 1, wherein the solution comprises at least four different labeled nucleotides or nucleotide analogs.

18. The method of claim 1, wherein the at least one labeled nucleotide or nucleotide analog comprises a nucleoside tetraphosphate, a nucleoside pentaphosphate, or a nucleoside hexaphosphate.

19. The method of claim 1, wherein at least one of the polymerase and the template nucleic acid is immobilized to a solid support.