COMPOSITIONS AND METHODS FOR USE IN ANALYTICAL REACTIONS

• US 20090208961A1
• Filed: 02/12/2009
• Published: 08/20/2009
• Est. Priority Date: 02/12/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Bibillo, Arek, Bjornson, Keith, Sebo, Lubomir
• Granted Patent: US 8,252,911 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2527/113 Time
• CPC Class Codes: C12Q 2527/127 the enzyme inhibitor or activator used

Abstract

Compositions, methods, substrates and systems for use in analysis of single molecule reactions and particularly single molecule nucleic acid sequence analysis. Compositions that include non-reactive, distinguishable or undetectable competitive substrates for the reaction system of interest are provided, as well as their use in systems and substrates for such applications, such compounds typically preferably polyphosphate chains or analogous structures.

Claims

1. A composition, comprising:

a complex comprising a nucleic acid polymerase, a template sequence and a primer sequence complementary to at least a portion of the template sequence;

at least a first type of incorporatable labeled nucleotide analog; and

at least a first type of unincorporatable competitive polymerase reagent, said unincorporatable competitive polymerase reagent being either unlabeled or differentially labeled from the incorporatable labeled nucleotide analogs.

2. The composition of claim 1, wherein the unincorporatable competitive polymerase reagent comprises a polyphosphate compound.

3. The composition of claim 1, wherein the competitive polymerase reagent comprises an unincorporatable nucleotide analog.

4. The composition of claim 1, further comprising a plurality of types of incorporatable labeled nucleotides analogs.

5. The composition of claim 4, further comprising a plurality of types of unincorporatable nucleotide analogs.

6. The composition of claim 3, wherein the unincorporatable nucleotide analogs comprise a link between an alpha and beta phosphate groups that is unhydrolyzable by the polymerase enzyme.

7. The composition of claim 6, wherein the link between the alpha and beta phosphate groups is selected from amino, thio, or alkyl.

8. The composition of claim 1, wherein the unincorporatable competitive polymerase reagent is present at a concentration ratio to the labeled incorporatable nucleotide analogs of from about 0.1:

1 to about 20;

1.

9. The composition of claim 3, wherein the unincorporatable nucleotide analogs are unlabeled.

10. The composition of claim 1, wherein the complex is immobilized upon a solid support.

11. The composition of claim 10, wherein the solid support comprises a transparent substrate.

12. The composition of claim 10, wherein the complex is immobilized upon a solid support such that the complex is individually optically resolvable.

13. The composition of claim 10, wherein the complex is immobilized within an optically confined structure.

14. The composition of claim 1, wherein the unincorporatable competitive polymerase reagent further comprises a triplet state quencher moiety coupled to the competitive polymerase reagent.

15. The composition of claim 2, wherein the unincorporatable competitive polymerase reagent comprises a group coupled to the polyphosphate group, selected from a cycloalkyl group, an aryl group, and a carbohydrate.

16. A method of determining nucleotide sequence information from a target nucleic acid sequence, comprising:

providing the target nucleic acid sequence in a complex with a primer sequence complementary to at least a portion of the target nucleic acid sequence, and a nucleic acid polymerase enzyme capable of extending the primer sequence in a target sequence dependent manner;

contacting the complex with a mixture of labeled incorporatable nucleotide analogs and at least a first unincorporatable competitive polymerase reagent that is either unlabeled or differentially labeled from the incorporatable nucleotide analogs; and

detecting target dependent incorporation of an incorporatable nucleotide analog to identify a nucleotide in the target nucleic acid sequence.

17. The method of claim 16, wherein the unincorporatable competitive polymerase reagent comprises a polyphosphate chain.

18. The method of claim 17, wherein the polyphosphate chain comprises from 2 to 7 phosphate groups

19. The method of claim 16, wherein the unincorporatable competitive polymerase reagent comprises an unincorporatable nucleotide analog.

20. The method of claim 16, wherein the mixture of labeled incorporatable nucleotide analogs comprises a plurality of different types of labeled incorporatable nucleotide analogs and the at least one unincorporatable competitive polymerase reagent comprises a plurality of different types of unincorporatable nucleotide analogs.

21. The method of claim 16. wherein a ratio of labeled incorporatable nucleotide analogs to unincorporatable competitive polymerase reagent in the mixture is from about 0.1:

1 to about 20;

1.

22. The method of claim 16, comprising providing the complex in an individually optically resolvable configuration, and optically detecting incorporation of a labeled nucleotide analog in a primer extension reaction by the polymerase enzyme in the complex.

23. The method of claim 16, wherein the unincorporatable competitive polymerase reagent comprises an aryl or cycloalkyl group linked to a polyphosphate group.

24. A method of determining a sequence of a template nucleic acid, comprising:

providing the target nucleic acid sequence in a complex with a primer sequence complementary to at least a portion of the target nucleic acid sequence, and a nucleic acid polymerase enzyme capable of extending the primer sequence in a target sequence dependent manner;

contacting the complex with a mixture of labeled incorporatable nucleotide analogs and at least a first unincorporatable competitive polymerase reagent that is differentially labeled from the incorporatable nucleotide analogs;

detecting iterative sampling of the first unincorporatable nucleotide by the complex; and

identifying a base in the template nucleic acid based upon an identity of the first unincorporatable nucleotide analog.

25. The method of claim 24, wherein the mixture comprises a plurality of types of incorporatable nucleotide analogs bearing a first detectable label, and a plurality of types of unincorporatable nucleotide analogs, each type of unincorporatable nucleotide analog bearing a label that is distinguishable from each other type of labeled unincorporatable nucleotide analog and the first detectable label on the incorporatable nucleotide analogs.