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 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.