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

Patent metadata

Field	Value
Publication number	US-11242557-B2
Application number	US-201916516808-A
Country	US
Kind code	B2
Filing date	Jul 19, 2019
Priority date	Apr 26, 2018
Publication date	Feb 8, 2022
Grant date	Feb 8, 2022

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, a polymerase, and a nucleotide cognate for the next correct base for the primed template nucleic acid molecule, wherein the fluid further comprises free nucleotide at a concentration that is lower than the concentration of ternary complex in the fluid, the free nucleotide being the same type present in the ternary complex.

2. A method of detecting a primed template nucleic acid, comprising the steps of:
(a) providing a fluid comprising a ternary complex and Li + , wherein the ternary complex comprises a primed template nucleic acid molecule, a polymerase, and a nucleotide cognate for the next correct base for the primed template nucleic acid molecule;
(b) detecting the ternary complex while it is in the fluid comprising Li + ;
(c) identifying the next correct base for the primed template nucleic acid molecule from the result of step (b); and
(d) extending the primer of the primed template nucleic acid, wherein the primer is extended by addition of a nucleotide comprising a reversible terminator moiety.

3. The method of claim 2 , wherein the primer of the primed template nucleic acid molecule comprises a reversible terminator moiety.

4. The method of claim 3 , further comprising (d) modifying or removing the reversible terminator moiety to deblock the primer.

5. The method of claim 4 , further comprising (e) extending the deblocked primer by addition of a nucleotide comprising a reversible terminator moiety.

6. The method of claim 2 , wherein ternary complex is immobilized on a solid support.

7. The method of claim 2 , wherein the nucleotide cognate comprises an exogenous label that is detected in step (b).

8. The method of claim 7 , wherein the mixture comprises nucleotide cognates for at least two different base types suspected of being present in the template nucleic acid.

9. The method of claim 8 , wherein the nucleotide cognates for the different base types comprise different exogenous labels, respectively.

10. The method of claim 2 , wherein the polymerase comprises an exogenous label that is detected in step (c).

11. The method of claim 2 , wherein the fluid further comprises free nucleotide at a concentration that is lower than the concentration of ternary complex in the fluid, the free nucleotide being the same type present in the ternary complex.

12. The method of claim 2 , wherein the fluid further comprises free polymerase at a concentration that is lower than the concentration of ternary complex in the fluid, the free polymerase being the same type present in the ternary complex.

13. The method of claim 2 , wherein the fluid further comprises betaine.

14. The method of claim 2 , wherein the fluid further comprises polyethylenimine.

15. A composition, comprising a fluid, the fluid comprising a ternary complex and Li + , wherein the ternary complex comprises a primed template nucleic acid molecule, a polymerase, and a nucleotide cognate for the next correct base for the primed template nucleic acid molecule, wherein the fluid further comprises free polymerase at a concentration that is lower than the concentration of ternary complex in the fluid, the free polymerase being the same type present in the ternary complex.

Assignees

• Omniome 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 Primary

  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