Method for detecting reactants using fluorescent signal intensity

• US 9,551,660 B2
• Filed: 03/22/2012
• Issued: 01/24/2017
• Est. Priority Date: 03/30/2009
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Kong, Xiangxu, Shen, Gene
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/427,611
• Publication Number:
  US 20130071849A1
• Time in Patent Office: 1,769 Days
• Field of Search: 435/4.1, 435/6.1, 435/7.1, 435/7.2, 422/68.1
• US Class Current: 1/1
• CPC Class Codes: C07H 19/207 the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: G01N 2021/6441 with two or more labels
• 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 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

FRET-labeled compounds are provided for use in analytical reactions. In certain embodiments, FRET-labeled nucleotide analogs are used in place of naturally occurring nucleoside triphosphates or other analogs in analytical reactions comprising nucleic acids, for example, template-directed nucleic acid synthesis, DNA sequencing, RNA sequencing, single-base identification, hybridization, binding assays, and other analytical reactions.

Claims

1. A method of monitoring reactions, comprising:

providing a reaction mixture comprising first and second reactants, wherein the first and second reactants comprise reactants selected from nucleic acids, nucleotides, nucleotide analogs, nucleic acid segments and primers, wherein the first reactant comprises a first fluorescent label that produces a first signal in response to excitation illumination, and the second reactant comprises a second fluorescent label that produces a second signal in response to excitation illumination, wherein reaction of the first and second reactants is detectable by the first and second signals, respectively, wherein the first and second signals have distinct signal intensities, and wherein the first and second signals comprise peaks at the same wavelengths;

reacting one of the first and second reactants;

detecting one of the first and second signals;

identifying the first or second signal detected based upon its signal intensity; and

correlating the first or second signal identified to the reaction of the first or second reactant in the reaction mixture, respectively.

2. The method of claim 1, wherein at least one of the first and second signals comprises distinct emission signal intensities at a plurality of different wavelengths.

3. The method of claim 1, wherein the first fluorescent label comprises a first FRET label having a first FRET efficiency, and the second fluorescent label comprises a second FRET label having a second FRET efficiency different from the first FRET efficiency, wherein the first FRET label and the second FRET label each comprise at least two chromophores that engage in FRET.

4. The method of claim 3, wherein the first and second FRET efficiencies are from about 0 to about 100% of a maximum FRET efficiency for each of the first and second FRET labels.

5. The method of claim 1, wherein the reaction mixture comprises at least a third reactant present in the reaction mixture, the third reactant comprising a third fluorescent label producing a third signal in response to excitation illumination, the third signal differing from each of the first and second signals by at least one of its emission intensity and its emission spectrum.

6. The method of claim 5, wherein the reaction mixture comprises at least a fourth reactant present in the reaction mixture, the fourth reactant comprising a fourth fluorescent label producing a fourth signal, the fourth signal in response to excitation illumination differing from each of the first, second and third signals by at least one of its emission intensity and its emission spectrum.

7. The method of claim 1, wherein the first and second reactants comprise first and second types of nucleotides or nucleotide analogs.

8. The method of claim 7, wherein the reaction of the first or second reactant in the reaction mixture correlated to the first or second signal detected in the detecting step, comprises incorporation of a first or second type of nucleotide or nucleotide analog incorporated in a primer extension reaction carried out in the reaction volume.

9. The method of claim 1, wherein said reaction mixture comprises said first and second reactants and two additional reactants, and further wherein at least one of said first, second, and additional reactants comprises a FRET label and one other comprises a non-FRET label.

10. The method of claim 1, wherein said first and second reactants comprise FRET labels having the same constituent FRET label moieties in different orientations.

11. The method of claim 1, wherein said reaction mixture further comprises reactants having multiple labeling moieties that do not undergo FRET.