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 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.