REAL-TIME SEQUENCING METHODS AND SYSTEMS
First Claim
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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.
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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.
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Citations
39 Claims
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1. A method of sequencing an mRNA transcript, the method comprising:
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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. - View Dependent Claims (2, 4, 5, 6, 7, 9, 10)
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3. (canceled)
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8. (canceled)
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11. A method of sequencing an RNA molecule, comprising:
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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. - View Dependent Claims (12, 13, 14, 15, 17, 18, 19, 20)
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16. (canceled)
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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.
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24-25. -25. (canceled)
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31. (canceled)
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33. A system for RNA sequencing, comprising:
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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.
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34. A method of determining a nucleotide position in an RNA template, the method comprising:
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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. - View Dependent Claims (35, 38)
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36-37. -37. (canceled)
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39-46. -46. (canceled)
Specification