Methods and compositions for stabilizing nucleic acid-nucleotide-polymerase complexes
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.
1. A method of detecting a primed template nucleic acid, comprising the steps of:
(a) providing a mixture comprising a ternary complex, the ternary complex comprising a primed template nucleic acid, a polymerase, and a nucleotide cognate for the next base of the primed template nucleic acid,
wherein the mixture further comprises excess nucleotide of the same type present in the ternary complex;
(b) replacing the excess nucleotide with a fluid comprising Li + ; and
(c) detecting the ternary complex while it is in contact with the fluid comprising Li + .
2. The method of claim 1 , further comprising (d) identifying the next correct base for the primed template nucleic acid molecule from the result of step (c).
3. The method of claim 2 , further comprising (e) extending the primer of primed template nucleic acid.
4. The method of claim 3 , wherein the primer is extended by addition of a nucleotide comprising a reversible terminator moiety.
5. The method of claim 1 , wherein the primer of the primed template comprises a reversible terminator moiety.
6. The method of claim 5 , further comprising modifying or removing the reversible terminator moiety to deblock the primer.
7. The method of claim 6 , further comprising extending the deblocked primer.
8. The method of claim 7 , wherein the deblocked primer is extended by addition of a nucleotide comprising a reversible terminator moiety.
9. The method of claim 1 , wherein the ternary complex is immobilized on a solid support.
10. The method of claim 1 , wherein the nucleotide cognate comprises an exogenous label that is detected in step (c).
11. The method of claim 10 , wherein the mixture comprises nucleotide cognates for at least two different base types suspected of being present in the template nucleic acid.
12. The method of claim 11 , wherein the nucleotide cognates for the different base types comprise different exogenous labels, respectively.
13. The method of claim 1 , wherein the polymerase comprises an exogenous label that is detected in step (c).
14. The method of claim 1 , wherein the mixture further comprises excess polymerase of the same type present in the ternary complex.
15. The method of claim 14 , wherein step (c) comprises replacing the excess polymerase and the excess nucleotide with the fluid comprising Li + .
16. The method of claim 14 , wherein the excess polymerase is soluble in the fluid.
17. The method of claim 1 , wherein the excess nucleotide is soluble in the fluid.
18. The method of claim 1 , wherein step (b) comprises delivering to the ternary complex a fluid that comprises Li + and lacks nucleotides of the type present in the ternary complex, thereby replacing the excess nucleotide with the fluid.
19. The method of claim 1 , wherein the Li + is present at a concentration between 1 mM and 100 mM during step (c).
20. The method of claim 1 , wherein the fluid further comprises betaine.
21. The method of claim 1 , wherein the fluid further comprises polyethylenimine.
22. The method of claim 1 , wherein the concentration of ternary complex is greater than the concentration of free nucleotide in the fluid comprising Li + during the detecting in step (c), the free nucleotide being the type of nucleotide that is present in the ternary complex.
23. The method of claim 1 , wherein the concentration of ternary complex is greater than the concentration of free polymerase in the fluid comprising Li + during the detecting in step (c), the free polymerase being the type of polymerase that is present in the ternary complex.