Fret-labeled compounds and uses therefor

• US 10,570,445 B2
• Filed: 07/30/2018
• Issued: 02/25/2020
• 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:
  US16/049,541
• Publication Number:
  US 20190032117A1
• Time in Patent Office: 575 Days
• Field of Search: 435 61
• 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 kit for performing a sequencing-by-synthesis reaction, comprising:

first and second nucleotide analogs, wherein the first nucleotide analog comprises a first fluorescent label that produces a first signal in response to excitation illumination, and the second nucleotide analog comprises a second fluorescent label that produces a second signal in response to excitation illumination, wherein the first and second signals have distinct signal intensities, and wherein the first and second signals comprise peaks at the same wavelengths wherein during incorporation of the first or second nucleotide analogs the first or second fluorescent labels are removed, and

a polymerase enzyme for performing template-dependent synthesis employing the labeled nucleotide analogs.

2. The kit 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 kit 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 kit of claim 1, 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 kit of claim 1, wherein the kit further comprises at least a third nucleotide analog, the third nucleotide analog 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 kit of claim 1, wherein the kit further comprises at least a fourth nucleotide analog, the fourth reactant comprising a fourth fluorescent label producing a fourth signal in response to excitation illumination, the fourth signal differing from each of the first, second and third signals by at least one of its emission intensity and its emission spectrum.

7. The kit of claim 1, wherein the kit further comprises at least four different nucleotide analogs, namely those that are analogous to A, T, G, and C, where each bears a detectably different labeling group to permit its individual identification in the presence of the others.

8. The kit of claim 1, wherein the kit further comprises two additional nucleotide analogs, wherein at least one of the first, second, and additional nucleotide analogs comprises a FRET label and one other comprises a non-FRET label.

9. The kit of claim 1, wherein the first and second nucleotide analogs comprise FRET labels having the same constituent FRET label moieties in different orientations.

10. The kit of claim 1, wherein the kit further comprises nucleotide analogs having multiple labeling moieties that do not undergo FRET.

11. The kit of claim 1, wherein the polymerase is a phi29 polymerases.

12. The kit of claim 1, wherein the kit further comprises a sequencing primer.

13. The kit of claim 1, wherein the kit further comprises one or more solutions for performing a sequencing reaction.

14. The kit of claim 1, wherein the kit further comprises dye standards for detector calibration.