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.
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; and
(b) detecting the ternary complex while it is in the fluid comprising Li + .
3 . The method of claim 2 , further comprising (c) identifying the next correct base for the primed template nucleic acid molecule from the result of step (b).
4 . The method of claim 3 , further comprising (d) extending the primer of the primed template nucleic acid.
5 . The method of claim 4 , wherein the primer is extended by addition of a nucleotide comprising a reversible terminator moiety.
6 . The method of claim 2 , wherein the primer of the primed template nucleic acid molecule comprises a reversible terminator moiety.
7 . The method of claim 6 , further comprising (d) modifying or removing the reversible terminator moiety to deblock the primer.
8 . The method of claim 7 , further comprising (e) extending the deblocked primer by addition of a nucleotide comprising a reversible terminator moiety.
9 . The method of claim 2 , wherein ternary complex is immobilized on a solid support.
10 . The method of claim 2 , wherein the nucleotide cognate comprises an exogenous label that is detected in step (b).
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 2 , wherein the polymerase comprises an exogenous label that is detected in step (c).
14 . 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.
15 . 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.
16 . The method of claim 2 , wherein the fluid further comprises betaine.
17 . The method of claim 2 , wherein the fluid further comprises polyethylenimine.