Real-time analytical methods and systems

• US 9,063,156 B2
• Filed: 06/11/2010
• Issued: 06/23/2015
• Est. Priority Date: 06/12/2009
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Korlach, Jonas, Turner, Stephen, Flusberg, Benjamin, Chaisson, Mark, Schadt, Eric, Wegener, Jeffrey
• Primary Examiner(s): BERTAGNA, ANGELA MARIE
• Application Number:
  US12/814,075
• Publication Number:
  US 20100323912A1
• Time in Patent Office: 1,838 Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: G01N 2333/705 Assays involving receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: G01N 2458/10 Oligonucleotides as tagging agents for labelling antibodies
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6875 Nucleoproteins

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, and for determining various characteristics of the 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 stimulate, enhance, or inhibit such reactions.

Claims

1. A method of determining a consensus binding site of a binding partner on a random array of optically resolvable polymer molecules, said determining comprising:

(i) providing optically resolvable reaction sites arrayed on a substrate and synthesizing a plurality of different polymer molecules wherein a single polymer molecule is synthesized at each of the optically resolvable reaction sites, wherein the synthesizing comprises exposing the reaction sites to a reaction mixture comprising a plurality of different types of monomers, wherein each type is linked to a detectably distinct label, under conditions that promote enzyme-mediated incorporation of the monomers into the single polymer molecule, and further wherein the synthesizing is performed in the presence of the binding partner, thereby generating a random array of optically resolvable polymer molecules, and further wherein the different polymer molecules are polynucleotides or polypeptides and the binding partner is selected from the group consisting of a protein, a carbohydrate, or a nucleic acid;

(ii) monitoring each of the reaction sites during said synthesizing to generate a set of time sequences of incorporations of the monomers at each of the reaction sites, thereby determining the sequences of the optically resolvable polymer molecules and mapping their locations on the substrate, wherein the monitoring comprises detecting the detectably distinct label;

(iii) monitoring each of the reaction sites during said synthesizing to detect binding of the binding partner to a subset of the optically resolvable polymer molecules at the optically resolvable reaction sites on the substrate; and

(iv) using the sequences of the optically resolvable polymer molecules determined in (ii), identifying a common monomer sequence in the sequences of the subset of the optically resolvable polymer molecules to which the binding partner bound in (iii), wherein the common monomer sequence is the consensus binding site for the binding partner, thereby identifying the consensus binding site of the binding partner.

2. The method of claim 1, wherein the single polymer molecules are polypeptides.

3. The method of claim 1, wherein the single polymer molecules are nucleic acids and the binding partner is selected from the group consisting of a transcription factor, a histone, an antibody, an RNA, an enzyme, and a nucleic acid binding protein.

4. The method of claim 1, wherein the binding partner comprises a detectable label.

5. The method of claim 4, wherein the detectable label on the binding partner comprises a fluorophore.

6. The method of claim 1, wherein the binding partner undergoes a conformational change upon binding that changes its intrinsic fluorescence.

7. The method of claim 1, wherein the reaction mixture further comprises an agent that alters binding between the common monomer sequence and the binding partner relative to binding in the absence of the agent.

8. The method of claim 1, further comprising monitoring the reaction site under a first set of reaction conditions to detect a first binding result for the binding partner, changing the first set of reaction conditions to a second set of reaction conditions, and monitoring the reaction site under the second set of conditions to detect a second binding result for the binding partner.

9. The method of claim 1, wherein the different polymer molecules are nucleic acid products of template-directed synthesis reactions.

10. The method of claim 9, wherein polymerase enzymes catalyze the template-directed synthesis reactions.

11. The method of claim 1, further comprising immobilizing a plurality of molecular complexes at the reaction sites, wherein the molecular complexes each comprise an enzyme and a nucleic acid template, wherein a plurality of different nucleic acid templates are present in the plurality of molecular complexes, and further wherein the sequence of the nucleic acid template in each of the molecular complexes is unknown during the immobilizing, wherein the plurality of different polymer molecules is synthesized by the enzyme in the molecular complex.

12. The method of claim 11, wherein the enzyme is a polymerase enzyme, the monomers comprise nucleotides, and the polymer molecule is a polynucleotide complementary to the nucleic acid template.

13. The method of claim 11, wherein the enzyme is a ribosome, the monomers comprise amino acids, and the polymer is a polypeptide encoded by the nucleic acid template.

14. The method of claim 1, wherein the optically resolvable reaction sites are within optical confinements.

15. The method of claim 14, wherein the optical confinements are zero mode waveguides.