METHOD TO IMPROVE SINGLE MOLECULE ANALYSES

• US 20110217698A1
• Filed: 02/01/2011
• Published: 09/08/2011
• Est. Priority Date: 02/04/2010
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Peluso, Paul, Rank, David
• Granted Patent: US 8,518,643 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/25 involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: G01N 21/64 Fluorescence; Phosphorescence
• 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 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/75 Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated systems in which material is burnt in a flame or plasma G01N21/72, G01N21/73

Abstract

The quality of information from single molecule analyses is improved by employing a method in which a single molecule reaction carried out within an optical confinement is monitored, the single molecule reaction is halted, and data from the optical confinement is obtained while the reaction is not occurring. Characteristic optical behavior observed while the reaction is halted is used to improve the quality of information obtained during the single molecule reaction, for example, by correcting the reaction data, excluding the reaction data, or providing a confidence level to the reaction data.

Claims

1. An analytical method comprising:

localizing a plurality of active molecules into optical confinements on a substrate whereby a plurality of the optical confinements comprise a single active molecule;

exposing the single active molecules in the optical confinements to a reagent solution, whereby either the single active molecules or a reagent in the reagent solution or both comprise one or more fluorescent labels;

initiating a reaction of the single active molecules;

measuring fluorescence from the plurality of optical confinements over time to monitor the reaction and to obtain reaction data from each of the plurality of confinements;

halting the reaction;

measuring fluorescence from the plurality of optical confinements over time while the reaction is halted to obtain sticking data; and

combining the sticking data with reaction data to provide a more accurate measurement of the reaction than if the sticking data was not used.

2. The method of claim 1 wherein the sticking data is used to determine a set of optical confinements that are most likely to provide accurate information about the reaction.

3. The method of claim 1 wherein the active molecules comprise catalysts.

4. The method of claim 1 wherein the active molecules comprise enzymes.

5. The method of claim 4 wherein the enzymes comprise polymerases, reverse transcriptases, or ribosomes.

6. The method of claim 1 wherein pH, temperature, or light is used for halting of the reaction.

7-9. -9. (canceled)

10. A method of improving accuracy in single molecule sequencing comprising:

localizing a plurality of polymerase enzyme complexes into optical confinements whereby a plurality of the optical confinements comprise a single active polymerase enzyme complex;

exposing the polymerase enzyme complexes to a reagent solution comprising the components necessary for polymerase activity including a plurality of types of labeled nucleotides or nucleotide analogs, each type comprising a different label;

initiating a sequencing reaction;

measuring fluorescence from the plurality of optical confinements over time during the sequencing reaction to obtain sequencing data;

halting the sequencing reaction;

measuring fluorescence from the plurality of optical confinements over time while the sequencing reaction is halted to obtain sticking data; and

using both the sticking data and the sequencing data to provide sequencing information that is more accurate than if the sticking data was not used.

11. The method of claim 10 further comprising combining data from multiple optical confinements to produce combined sequencing data.

12. The method of claim 11 wherein the sticking data is used to determine a set of optical confinements that are least likely to provide accurate information about the reaction, and eliminating the sequencing data from these optical confinements in the combined sequencing data.

13. The method of claim 11 wherein the sticking data is used to determine a set of optical confinements that are most likely to provide accurate sequencing information and higher weight is given to the data from these optical confinements in producing the combined sequencing data.

14. The method of claim 10 wherein the sequencing data and sticking data comprise data from four or more optical channels, each corresponding to a label on a type of nucleotide or nucleotide analog.

15. The method of claim 14 wherein the sticking data in one channel, two channels, three channels, or in four channels is used to improve the accuracy of sequencing information in that channel or set of channels.

16. The method of claim 10 further comprising, after halting the reaction to obtain sticking data, initiating the reaction again and measuring fluorescence from the plurality of optical confinements over time during the ensuing sequencing reaction to obtain subsequent sequencing data, and using the subsequent sequencing data along with the sticking data and the sequencing data to provide sequencing information.

17. The method of claim 10 wherein each polymerase enzyme complex comprises a nucleic acid template.

18. The method of claim 17 wherein the nucleic acid template comprises DNA or RNA.

19. The method of claim 17 wherein the nucleic acid template is derived from genomic DNA, BACs, cDNA libraries, or PCR products.

20. The method of claim 10 wherein halting the sequencing reaction comprises inhibiting the polymerase enzyme.

21. The method of claim 20 wherein the polymerase enzyme is inhibited by the addition of a polymerase enzyme inhibitor.

22. The method of claim 21 wherein the polymerase enzyme inhibitor comprises a reversible inhibitor.

23. The method of claim 21 wherein the polymerase enzyme inhibitor comprises an irreversible inhibitor.

24. The method of claim 10 wherein halting the sequencing reaction comprises denaturing or degrading the polymerase enzyme.

25. The method of claim 10 wherein halting the sequencing reaction comprises changing the pH, changing the temperature, irradiating the enzyme with electromagnetic radiation, or adding a chelating agent.

26-27. -27. (canceled)

28. An analysis instrument comprising:

a substrate comprising optical confinements having a plurality of active molecules localized therein whereby a plurality of the optical confinements comprise a single active molecule;

the substrate having a substrate reservoir which allows for exposing the single active molecules in the optical confinements to a reagent solution, whereby either the single active molecules or a reagent in the reagent solution or both comprise one or more fluorescent labels;

an optical system for measuring fluorescence from the plurality of optical confinements over time to monitor the reaction and to obtain reaction data from each of the plurality of confinements and for measuring fluorescence from the plurality of optical confinements over time while the reaction is halted to obtain sticking data;

a flow-cell in fluidic contact with the substrate for delivering agents to halt the reaction without moving the substrate; and

a computer for combining the sticking data with reaction data.

29. (canceled)