Methods and compositions for stabilizing nucleic acid-nucleotide-polymerase complexes

Patent metadata

Field	Value
Publication number	US-12404549-B2
Application number	US-202217568077-A
Country	US
Kind code	B2
Filing date	Jan 4, 2022
Priority date	Apr 26, 2018
Publication date	Sep 2, 2025
Grant date	Sep 2, 2025

Abstract

Methods, compositions, kits and apparatuses that include a fluid, the fluid containing a ternary complex and Li + , wherein the ternary complex includes a primed template nucleic acid, a polymerase, and a nucleotide cognate for the next correct base for the primed template nucleic acid molecule. As an alternative or addition to Li + , the fluid can contain betaine or a metal ion that inhibits polymerase catalysis such as Ca 2+ . In addition to Li + , the fluid can contain polyethylenimine (PEI) with or without betaine.

First claim

What is claimed is:

1. A composition, comprising a fluid, the fluid comprising a ternary complex and Li + , wherein the ternary complex comprises a primed template nucleic acid molecule comprising reversible terminator moiety, a polymerase, and a nucleotide cognate for the next correct base for the primed template nucleic acid molecule.

2. A kit, comprising:
labeled nucleotides;
unlabeled reversibly terminated nucleotides; and
a stabilizing fluid comprising Li + .

3. The kit of claim 2 , wherein the stabilizing fluid further comprises betaine.

4. The kit of claim 2 , wherein the stabilizing fluid further comprises polyethylenimine (PEI).

5. The kit of claim 2 , wherein the stabilizing fluid further comprises betaine and polyethylenimine (PEI).

6. The kit of claim 2 , wherein the stabilizing fluid further comprises an inhibitory metal ion.

7. The kit of claim 6 , wherein the inhibitory metal ion is Ca 2+ .

8. The kit of claim 2 , further comprising a deblocking reagent.

9. The kit of claim 2 , further comprising a polymerase.

10. The kit of claim 9 , wherein the polymerase is unlabeled.

11. The kit of claim 9 , wherein the polymerase is an exo minus Klenow fragment.

12. The kit of claim 2 , wherein the unlabeled reversibly terminated nucleotides are in an extension reaction mixture.

13. The kit of claim 2 , wherein the labeled nucleotides are in a reaction mixture for ternary complex formation.

14. The kit of claim 2 , wherein the labeled nucleotides comprise labels that are detectible by fluorescence emission.

15. The kit of claim 2 , wherein the labeled nucleotides comprise nucleotides of each of 4 different bases.

16. The kit of claim 2 , further comprising a cartridge having reservoirs to contain the labeled nucleotides, unlabeled reversibly terminated nucleotides, and the stabilizing fluid.

17. The kit of claim 16 , wherein the cartridge further comprises fluidic components for transferring reagents from the reservoirs to a detection instrument.

18. A system comprising:
a flow cell;
reagents comprising labeled nucleotides, unlabeled reversibly terminated nucleotides, and a stabilizing fluid comprising Li + ;
a fluidic system configured to deliver the reagents to the flow cell; and
a detection apparatus configured to detect the flow cell.

19. The system of claim 18 , wherein the fluidic system comprises a cartridge having reservoirs to contain the labeled nucleotides, unlabeled reversibly terminated nucleotides, and the stabilizing fluid.

Assignees

• Pacific Biosciences California Inc

Inventors

• Middleton Morassa Mohseni
• Wallen Mark C
• Iyidogan Pinar
• Schmidt Michael James
• Rohrman Brittany A
• Liu ying lin
• Block Fabian
• Oliphant Arnold

Classifications

• C12Q1/6832

  Enhancement of hybridisation reaction · CPC title

• C12Q1/6816

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title