Modular nucleotide compositions and uses therefor

• US 10,161,002 B2
• Filed: 12/13/2017
• Issued: 12/25/2018
• Est. Priority Date: 11/19/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Korlach, Jonas, Wegener, Jeffrey
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US15/840,991
• Publication Number:
  US 20180163267A1
• Time in Patent Office: 377 Days
• Field of Search: 435 61, 435 911
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Abstract

Nucleic acid compositions, methods of making and using such compositions that comprise modular functional groups that can be configured to provide desired functionality to different nucleotide types through a swappable and preferably non-covalent linkage component. Such compositions are useful in a variety of applications including nucleic acid analyses.

Claims

1. A method of sequencing comprising:

initiating a nucleic acid polymerization reaction on a template nucleic acid in a sequencing reaction mixture, wherein the sequencing reaction mixture comprises a nucleoside polyphosphate coupled to a detectable label through a phosphate group by a non-covalent linkage component;

monitoring the nucleic acid polymerization reaction for signals from the detectable label to obtain sequence data, wherein incorporation of the nucleobase of the nucleoside polyphosphate is detected as a pulse in the sequence data.

2. The method of claim 1, wherein the detectable label is removed during incorporation of the nucleobase of the nucleoside polyphosphate.

3. The method of claim 1, wherein the detectable label is coupled to the nucleoside polyphosphate through a phosphate group other than the alpha phosphate group.

4. The method of claim 3, wherein the detectable label is coupled to the nucleoside polyphosphate through the terminal phosphate group.

5. The method of claim 1, wherein the detectable label is selected from the group consisting of:

an optically detectable label, an electrically detectable label, an enzymatically detectable label, an electrochemically detectable label, a magnetic label, and a mass label.

6. The method of claim 1, wherein the detectable label is selected from the group consisting of:

a fluorescent label, a FRET label, and combinations thereof.

7. The method of claim 1, wherein the detectable label is a fluorescent label.

8. The method of claim 1, wherein the non-covalent linkage component comprises an affinity binding pair.

9. The method of claim 8, wherein the affinity binding pair comprises a biotin group.

10. The method of claim 9, wherein the affinity binding pair comprises one of avidin, streptavidin, neutravidin, or a biotin-binding subunit thereof.

11. The method of claim 1, wherein the non-covalent linkage component comprises one of an antibody or a fraction thereof, a polynucleotide, and a nucleic acid binding protein.

12. The method of claim 1, wherein the non-covalent linkage component comprises a polyvalent non-covalent linkage.

13. The method of claim 1, wherein the sequencing reaction mixture comprises multiple different nucleoside polyphosphates coupled to corresponding different detectable labels by non-covalent linkage components through a phosphate group of their respective polyphosphates.

14. The method of claim 1, wherein the method is a single-molecule, real-time sequencing method.

15. The method of claim 1, wherein the non-covalent linkage component comprises a photo-induced damage mitigating agent.

16. The method of claim 15, wherein the photo-induced damage mitigating agent comprises a triplet state quencher, an antioxidant, or combinations thereof.

17. The method of claim 13, wherein the different detectable labels are spectrally distinguishable fluorescent labels.