Methods and systems for monitoring reactions

• US 8,465,922 B2
• Filed: 08/24/2011
• Issued: 06/18/2013
• Est. Priority Date: 08/26/2010
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Eid, John, Turner, Stephen
• Primary Examiner(s): FORMAN, BETTY J
• Application Number:
  US13/217,112
• Publication Number:
  US 20120052490A1
• Time in Patent Office: 664 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: B01J 2219/00693 Means for quality control
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: C40B 60/10 for identifying library members
• CPC Class Codes: C40B 60/12 for screening libraries
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence

Abstract

Methods and systems for monitoring reactions by observing signals deriving from those reactions, using signal processing that allows differentiation between signals that are otherwise optically overlapping by conventional detection methods. Centroid determination is used to identify signal sources that are presenting confounding overlapping signals due to their physical proximity, and/or to identify discrete signals from different reaction centers.

Claims

1. A method of monitoring single molecule reactions at a first and a second single molecule reaction complex, comprising:

providing a first single molecule reaction complex and a second single molecule reaction complex, each at least partially immobilized on a substrate,

exposing the first and second single molecule reaction complexes to reaction conditions;

wherein said first single molecule reaction complex produces a first optically detectable signal indicative of the presence of a single molecule reaction at the first single molecule reaction complex and the second single molecule reaction complex produces a second optically detectable signal indicative of the presence of a single molecule reaction at the second single molecule reaction complex, wherein the first and second optically detectable signals are not completely optically resolvable from each other;

positionally detecting the first optically detectable signal and the second optically detectable signal;

identifying a first positional centroid for the first optically detectable signal and a second positional centroid for the second optically detectable signal; and

characterizing the first optically detectable signal as emanating from the first single molecule reaction complex based upon the first positional centroid and characterizing the second optically detectable signal as emanating from the second single molecule reaction complex based upon the second positional centroid, and;

detecting a plurality of optically detectable signals from the first and second single molecule reaction complexes and assigning each of said plurality of signals as emanating from either the first or second single molecule reaction, complex based upon a comparison of a positional centroid of each of the plurality of optically detectable signals with a positional centroid determined from the first and second single molecule reaction complexes.

2. The method of claim 1, wherein the identification of the first positional centroid is determined at the sub-pixel level.

3. The method of claim 1, wherein the first single molecule reaction complex comprises a nucleic acid polymerase/template/primer complex.

4. The method of claim 3, wherein the first optically detectable signal indicative of the presence of the first single molecule reaction complex comprises a fluorescent signal indicative of polymerase mediated extension of a primer in the complex to produce an extended primer.

5. The method of claim 4, wherein the first optically detectable signal comprises a fluorescent label on a labeled nucleotide or nucleotide analog that has been incorporated into or is being incorporated into the extended primer.

6. The method of claim 5, wherein the labeled nucleotide is labeled upon a portion of the nucleotide that is retained upon the nucleotide following incorporation.

7. The method of claim 5, wherein the labeled nucleotide is labeled upon a portion of the nucleotide that is not retained upon the nucleotide following incorporation.

8. The method of claim 1, wherein the first and second optically detectable signals comprise substantially the same spectrum.

9. The method of claim 1, wherein the first and second optically detectable signals comprise different spectra.

10. The method of claim 1, wherein the first single molecule reaction complex comprises a nucleic acid polymerase/template/primer complex, and the first optically detectable signal comprises a fluorescent signal emanating from a nucleotide that has been or is being incorporated into a primer extension reaction by the polymerase, the optically detectable signal being indicative of a type of nucleotide that is incorporated or being incorporated.

11. The method of claim 1, wherein the first and second optically detectable signals are temporally distinct.