Arrays of optical confinements and uses thereof

• US 7,906,284 B2
• Filed: 10/26/2007
• Issued: 03/15/2011
• Est. Priority Date: 09/17/2004
• 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
• Primary Examiner(s): Martinell; James
• Application Number:
  US11/925,607
• Publication Number:
  US 20080156974A1
• Time in Patent Office: 1,236 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 2021/6463 Optics
• CPC Class Codes: G01N 2021/7786 Fluorescence
• CPC Class Codes: G01N 21/00 Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light G01N3/00 - G01N19/00 take precedence
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/648 using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
• CPC Class Codes: G01N 21/77 by observing the effect on a chemical indicator
• CPC Class Codes: G01N 2201/06113 Coherent sources; lasers

Abstract

The present invention relates to optical confinements, methods of preparing and methods of using them for analyzing molecules and/or monitoring chemical reactions. The apparatus and methods embodied in the present invention are particularly useful for high-throughput and low-cost single-molecular analysis.

Claims

1. A method of determining a nucleic acid sequence of a sequence segment of interest, comprising:

providing an individual concatemer molecule comprising a plurality of copies of the sequence segment of interest;

determining nucleotide sequences of the plurality of copies to generate redundant sequence information for said sequence segment of interest, wherein the determining comprises detecting a plurality of luminescent signals, each of which identifies a base in the sequence segment of interest, and further wherein the luminescent signals are indicative of interactions between the individual concatemer molecule and labeled reaction components emitting the luminescent signals; and

analyzing the redundant sequence information to determine a nucleic acid sequence of the sequence segment of interest.

2. The method of claim 1, wherein the individual concatemer molecule is a linear template.

3. The method of claim 1, further comprising amplifying a circular template bearing the sequence segment of interest to generate the individual concatemer molecule.

4. The method of claim 1, wherein the plurality of copies of the sequence segment of interest are tandem copies.

5. The method of claim 1, wherein the individual concatemer molecule is immobilized upon a substrate.

6. The method of claim 5, wherein the individual concatemer molecule is covalently immobilized upon the substrate.

7. The method of claim 5, wherein the individual concatemer molecule is noncovalently immobilized upon the substrate.

8. The method of claim 5, wherein the individual concatemer molecule is immobilized upon the substrate via interaction with at least one of the group consisting of a streptavidin/biotin linkage, an avidin/biotin linkage, a carbamate linkage, an ester linkage, an amide, a thiolester, an (N)-functionalized thiourea, a functionalized maleimide, an amino, a disulfide, an amide, a hydrazone linkage, an antibody, a silyl moiety, and an enzyme bound to the substrate.

9. The method of claim 8, wherein the enzyme is a polymerase enzyme.

10. The method of claim 5, wherein a plurality of different individual concatemer molecules are immobilized upon the substrate in a manner that permits optical resolution of the plurality of different individual concatemer molecules from one another.

11. The method of claim 5, wherein the individual concatemer molecule is immobilized in an ordered array.

12. The method of claim 11, wherein the ordered array is an ordered array of optical confinements.

13. The method of claim 12, wherein the optical confinements are zero mode waveguides.

14. The method of claim 1, wherein the luminescent signals are fluorescent signals.

15. The method of claim 1, wherein the detecting is performed using an optical system comprising an excitation source and a photon detector.

16. The method of claim 15, wherein the photon detector is selected from the group consisting of a camera, a fluorescence microscope, a photodiode, an optical reader, a charge-coupled device, and electron-multiplying charge-coupled device, and an intensified charge-coupled device.

17. The method of claim 1, wherein the labeled reaction components are labeled nucleic acids.

18. The method of claim 17, wherein the labeled nucleic acids are labeled nucleotides or nucleotide analogs.

19. The method of claim 1, wherein the determining nucleotide sequences of the plurality of copies comprises a template-directed polymerization reaction.

20. A method for generating redundant sequence information from a single molecule, comprising:

providing a linear template strand comprising multiple repeated copies of a nucleic acid sequence; and

determining a nucleotide sequence of the multiple repeated copies in the linear template strand, thereby generating redundant sequence information from a single molecule, wherein the determining comprises detecting a plurality of luminescent signals, each of which identifies a base in the sequence segment'"'"'of interest, and further wherein the luminescent signals are indicative of interactions between the individual concatemer molecule and labeled reaction components emitting the luminescent signals.

21. The method of claim 20, wherein the nucleic acid sequence is derived from a circular nucleic acid sequence, and wherein the method further comprises synthesizing the linear template strand from the circular nucleic acid sequence such that the linear template strand comprises multiple repeated copies of the circular nucleic acid sequence.

22. The method of claim 21, wherein said synthesizing is performed using a single-molecule amplification strategy.

23. The method of claim 20, wherein the determining comprises immobilization of the linear template strand upon a substrate.

24. The method of claim 23, wherein a plurality of different linear template strands are immobilized upon the substrate in a manner that permits optical resolution of the plurality of different linear template strands from one another.

25. The method of claim 23, wherein the linear template strand is immobilized in an ordered array.

26. The method of claim 20, wherein the detecting is performed using an optical system comprising an excitation source and a photon detector.

27. The method of claim 20, wherein the determining comprises a template-directed polymerization reaction.

28. A method of performing analyses on a plurality of concatemeric nucleic acid templates, comprising:

providing a plurality of concatemeric nucleic acid templates;

providing a substrate;

immobilizing each of the concatemeric nucleic acid templates at a different position on the substrate;

subjecting the substrate to conditions suitable for formation of products of the analyses; and

detecting the formation of the products with an optical system, wherein the detecting comprises detecting a plurality of luminescent signals emitted from each of said products, and wherein each of the luminescent signals identifies a base in one of the concatemeric nucleic acid templates, and further wherein the luminescent signals are indicative of interactions between the concatemeric nucleic acid templates and labeled reaction components emitting the luminescent signals, thereby performing analyses on said plurality of concatemeric nucleic acid templates.

29. The method of claim 28, wherein the detecting further comprises illuminating each of said products.

30. The method of claim 28, wherein the luminescent signals emitted from each of said products are optically resolvable from each other.

31. The method of claim 28, wherein the optical system generates and transmits an incident wavelength to the sequencing reactions, and collects and analyzes the luminescent signals emitted from the products.

32. The method of claim 28, wherein the optical system comprises an excitation source and a photon detector.

33. The method of claim 28, wherein the substrate is an ordered array.

34. The method of claim 33, wherein the ordered array comprises a plurality of rows of confinements on a surface of the substrate.

35. The method of claim 34, wherein each of the confinements contains no more than a single of the concatemeric nucleic acid templates.