NUCLEOTIDE COMPOSITIONS AND USES THEREOF

• US 20100240045A1
• Filed: 02/04/2010
• Published: 09/23/2010
• 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
• Granted Patent: US 8,927,211 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 analyses. The compositions and methods embodied in the present invention are particularly useful for nucleic acid analyses 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 mixture containing at least one labeled nucleotide or nucleotide analog;

treating the mixture with an agent that selectively renders unlabeled nucleotides or nucleotide analogs substantially incapable of participating in an analytical reaction of interest in which a labeled nucleotide or nucleotide analog participates.

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 in 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, transferse, 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 mixture are unlabeled nucleotides or nucleotide analogs.

11. The method of claim 1, wherein following the treating step, less than 5% of the nucleotides or nucleotide analogs in the mixture are unlabeled nucleotides or nucleotide analogs.

12. The method of claim 1, wherein following the treating step, less than I% of the nucleotides or nucleotide analogs in the mixture are unlabeled nucleotides or nucleotide analogs.

13. The method of claim 1, wherein following the treating step, less than 0.1% of the nucleotides or nucleotide analogs in the mixture are unlabeled nucleotides or nucleotide analogs.

14. The method of claim 1, wherein following the treating step, less than 0.01% of the nucleotides or nucleotide analogs in the mixture are unlabeled nucleotides or nucleotide analogs.

15. A method of sequencing a nucleic acid, comprising:

providing a reaction mixture comprising a template nucleic acid, a primer sequence complementary to the template nucleic acid sequence, a polymerase enzyme, and a nucleotide composition comprising a labeled nucleotide or nucleotide analog capable of incorporation into a nascent nucleic acid strand and said composition being substantially free of unlabeled nucleotides or nucleotide analogs capable of incorporation into the nascent nucleic acid strand; and

detecting incorporation of the labeled nucleotide or nucleotide analog by the polymerase, into the nascent nucleic acid strand.

16. The method of claim 15, wherein the providing step, comprises first treating the nucleotide composition with an agent that selectively alters unlabeled nucleotides or nucleotide analogs so that they become substantially unincorporatable by the polymerase enzyme.

17. The method of claim 15, wherein within the reaction mixture, less than 5% of the nucleotides or nucleotide analogs are unlabeled nucleotides or nucleotide analogs.

18. The method of claim 15, wherein within the reaction mixture, less than 1% of the nucleotides or nucleotide analogs are unlabeled nucleotides or nucleotide analogs.

19. The method of claim 15, wherein within the reaction mixture, less than 0.1% of the nucleotides or nucleotide analogs are unlabeled nucleotides or nucleotide analogs.

20. The method of claim 15, wherein within the reaction mixture, less than 0.01% of the nucleotides or nucleotide analogs are unlabeled nucleotides or nucleotide analogs.

21. The method of claim 15, wherein the reaction mixture comprises at least two different labeled nucleotides or nucleotide analogs.

22. The method of claim 15, wherein the reaction mixture comprises at least four different labeled nucleotides or nucleotide analogs.

24. The method of claim 15, wherein the detecting step involves detecting an optical signal from the labeled nucleotide or nucleotide analog.

25. The method of claim 15, wherein the template nucleic acid and/or the polymerase is immobilized to a support.

26. The method of claim 15, wherein the reaction mixture comprises a single complex of a template nucleic acid, a polymerase enzyme, and a nascent strand that is complementary to the template nucleic acid.

27. The method of claim 26, wherein the detecting step involves detecting incorporation of the labeled nucleotide or nucleotide analog into the single complex.

23. The method of 15, wherein the detecting step is performed during incorporation of the labeled nucleotide or nucleotide analog by the polymerase into the nascent nucleic acid strand.

28. A method for identifying a nucleotide incorporated into a nascent strand during template-directed synthesis, said method comprising:

(a) providing a complex comprising a polymerase and a template nucleic acid such that said polymerase is capable of incorporating a nucleotide complementary to a nucleotide on the template nucleic acid, wherein the complex is immobilized to a solid support;

(b) providing a nucleotide composition comprising at least one incorporatable labeled nucleotide or nucleotide analog to the complex to effect polymerizing a nucleotide into a nascent strand that is complementary to the target nucleic acid, wherein the nucleotide composition is substantially free of incorporatable unlabeled nucleotides or nucleotides analogs; and

(c) optically detecting the incorporated nucleotide by sensing its detectable label, thereby identifying the incorporated nucleotide.

29. The method of claim 28, wherein said detecting is performed by passing radiation through the complex at a spatial location of the complex where said label is expected to occur.

30. The method of claim 28, further comprising repeating, with the complex immobilized on the support, steps (b) through (c) so that a plurality of incorporated nucleotides is identified and, by complementarity, a sequence of the template nucleic acid is determined.

31. The method of claim 28, wherein the solid support comprises an array of individually optically resolvable complexes.