Real-time sequencing methods and systems
First Claim
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1. A method of sequencing an mRNA transcript, the method comprising:
- a) circularizing the mRNA transcript and, after the circularizing, 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 complementarity 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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18 Claims
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1. A method of sequencing an mRNA transcript, the method comprising:
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a) circularizing the mRNA transcript and, after the circularizing, 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 complementarity the sequence of the mRNA transcript based upon the sequence of incorporation of nucleotides into the nascent polynucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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- 8. 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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15. 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, wherein the RNA template molecule is a circular 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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16. 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 to prevent incorporation of the differentially labeled nucleotides; 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 (17, 18)
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Specification