REAL-TIME SEQUENCING METHODS AND SYSTEMS

• US 20100311061A1
• Filed: 04/26/2010
• Published: 12/09/2010
• Est. Priority Date: 04/27/2009
• 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, SCHADT, ERIC
• Granted Patent: US 8,501,405 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/702 for retroviruses
• CPC Class Codes: C12Q 2547/101 by confinement to a single tube/container
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

The present invention is generally directed to compositions, methods, and systems for performing single-molecule, real-time analysis of a variety of different biological reactions. The ability to analyze such reactions provides an opportunity to study those reactions as well as to potentially identify factors and/or approaches for impacting such reactions, e.g., to either enhance or inhibit such reactions. In certain preferred embodiments, RNA templates are used in single-molecule real-time sequencing reactions.

Claims

1. A method of sequencing an mRNA transcript, the method comprising:

a) providing a complex comprising the mRNA transcript and a sequencing engine, wherein the complex is immobilized in an optical confinement;

b) introducing a reaction mixture to the optical confinement, wherein the reaction mixture comprises a set of labeled nucleotides;

c) performing a sequencing-by-synthesis reaction;

d) detecting a sequence of incorporation of the labeled nucleotides into a nascent polynucleotide complementary to the mRNA transcript; and

e) determining by complementarily the sequence of the mRNA transcript based upon the sequence of incorporation of nucleotides into the nascent polynucleotide.

2. The method of claim 1, wherein the sequencing engine is an RNA-dependent polymerase or a reverse transcriptase.

4. The method of claim 1, wherein the sequencing engine is bound, directly or indirectly, to a surface of the optical confinement.

5. The method of claim 1, wherein the mRNA transcript is bound, directly or indirectly, to a surface of the optical confinement.

6. The method of claim 5, wherein the mRNA transcript is hybridized to an oligonucleotide bound to a surface of the optical confinement.

7. The method of claim 6, wherein the mRNA transcript is hybridized to a sequence-specific region or a poly-T region of the oligonucleotide.

9. The method of claim 1, further comprising circularizing the mRNA transcript prior to providing the complex.

10. The method of claim 9, wherein the sequencing engine processes the mRNA transcript multiple times during the sequencing-by-synthesis reaction.

3. (canceled)

8. (canceled)

11. A method of sequencing an RNA molecule, comprising:

a) providing a complex comprising the RNA molecule and a sequencing engine;

b) introducing a reaction mixture to the complex, wherein the reaction mixture comprises a set of unincorporatable labeled nucleotides and a set of incorporatable unlabeled nucleotides;

c) performing a sequencing-by-synthesis reaction, wherein only the set of incorporatable unlabeled nucleotides are incorporated into a nascent polynucleotide complementary to the RNA molecule;

d) detecting a sequence of associations of the set of unincorporatable labeled nucleotides with the complex, wherein each of the associations is indicative of complementarity of a particular unincorporatable labeled nucleotide with a particular complementary position in the RNA molecule; and

e) determining by complementarity with the sequence of associations of the set of unincorporatable labeled nucleotides a sequence of the RNA molecule.

12. The method of claim 11, wherein the complex is immobilized in an optical confinement.

13. The method of claim 11, wherein multiple associations of the set of unincorporatable labeled nucleotides with the complex are detected for every incorporation of an incorporatable unlabeled nucleotide into the nascent polynucleotide.

14. The method of claim 11, wherein the sequencing engine is a wild-type sequencing engine.

15. The method of claim 11, wherein the sequencing engine is an RNA-dependent polymerase, or a reverse transcriptase.

17. The method of claim 11, wherein the RNA molecule is a circular RNA molecule.

18. The method of claim 17, wherein the sequencing engine processes the circular RNA molecule multiple times during the sequencing-by-synthesis reaction.

19. The method of claim 11, wherein the RNA molecule comprises a modified base.

20. The method of claim 19, wherein the modified base is a pseudouridine or N²-methylguanosine.

16. (canceled)

21-22. -22. (canceled)

23. A composition comprising an RNA-dependent polymerase bound to an RNA template in an optical confinement, and a plurality of differentially-labeled phospholinked nucleotide analogs, wherein the differentially-labeled phospholinked nucleotide analogs are unincorporatable into a nascent polynucleotide strand during template-directed synthesis by the RNA-dependent polymerase.

26. The composition of claim 23, further comprising unlabeled phospholinked nucleotide analogs that are incorporatable into a nascent polynucleotide strand during template-directed synthesis by the RNA-dependent polymerase.

27. The composition of claim 23, wherein the RNA-dependent polymerase is a reverse transcriptase.

28. The composition of claim 27, wherein the reverse transcriptase is HIV reverse transcriptase.

29. The composition of claim 23, wherein the RNA-dependent polymerase is an RNA-dependent RNA polymerase.

30. The composition of claim 29, wherein the RNA-dependent RNA polymerase is phi6 polymerase.

32. The composition of claim 23, wherein the optical confinement is a zero mode waveguide.

24-25. -25. (canceled)

31. (canceled)

33. A system for RNA sequencing, comprising:

a) a substrate comprising a reaction site within an optical confinement, wherein a single RNA-dependent polymerase is immobilized at the reaction site and is further bound to a single RNA template molecule;

b) a reaction mixture in contact with the reactions site comprising differentially labeled nucleotide analogs capable of incorporation into a nascent strand during template-directed polymerization by the single RNA-dependent polymerase on the single RNA template molecule;

c) an illumination source that provides excitation illumination;

d) an optical detector;

e) an optical train that guides the illumination from the excitation illumination source to the reaction site and guides emission signals from the reaction site to an optical detector; and

f) a recording device to record the emission signals detected by the optical detector.

34. A method of determining a nucleotide position in an RNA template, the method comprising:

a) providing an immobilized complex comprising a single RNA-dependent polymerase and a single RNA template molecule;

b) contacting the immobilized complex with a reaction mixture that comprises a set of differentially labeled nucleotides and Ca²⁺ ions, and wherein the reaction mixture is deficient in Mg²⁺ ions;

c) binding one of the differentially labeled nucleotides to the immobilized complex to form a bound labeled nucleotide;

d) detecting an emission signal from the bound labeled nucleotide;

e) based upon the emission signal, determining a nucleobase in the bound labeled nucleotide and, by complementarity, an identity of the nucleotide position in the RNA template.

35. The method of claim 34, further comprising removing the Ca²⁺ ions and adding Mg²⁺ ions to promote incorporation of the bound labeled nucleotide into a nascent strand complementary to the RNA template.

38. The method of claim 34, wherein the method is repeated to promote incorporation of multiple of the differentially labeled nucleotides into the nascent strand complementary to the RNA template.

36-37. -37. (canceled)

39-46. -46. (canceled)