Simultaneous background reduction and complex stabilization in binding assay workflows
Method and apparatus to facilitate separation of solution-phase components surrounding an immobilized multicomponent complex while stabilizing association of the components within the complex. The technique can be used for reducing background signal arising from the presence of non-complexed components harboring detectable labels, thereby enhancing signal-to-background ratios and allowing enhanced detection of the multicomponent complex.
1. A method of identifying a cognate nucleotide, said method comprising the steps of:
(a) contacting an immobilized primed template nucleic acid molecule with a first polymerase and a first test nucleotide to form, without incorporation, an immobilized ternary complex,
wherein the immobilized ternary complex comprises the immobilized primed template nucleic acid molecule and the first polymerase in reversible association with the first test nucleotide;
(b) contacting the immobilized ternary complex with a stabilizing fluid,
wherein the first test nucleotide is substantially insoluble in the stabilizing fluid;
(c) detecting the immobilized ternary complex in contact with the stabilizing fluid; and
(d) identifying the cognate nucleotide from the result of step (c).
2. The method of claim 1 , wherein the first test nucleotide comprises a detectable label and wherein step (c) comprises detecting the immobilized ternary complex in contact with the stabilizing fluid by detecting the detectable label.
3. The method of claim 2 , wherein the detectable label comprises a fluorescent moiety.
4. The method of claim 2 , wherein the first test nucleotide comprises the detectable label, and the first polymerase does not comprise any chemical moiety in energy transfer relationship with the detectable label of the first test nucleotide.
5. The method of claim 1 , wherein step (a) further comprises contacting the immobilized primed template nucleic acid molecule with a second test nucleotide different from the first test nucleotide.
6. The method of claim 1 , further comprising, after step (c), a step of destabilizing the immobilized ternary complex by replacing the stabilizing fluid with a wash solution that removes the first polymerase and the first test nucleotide from contact with the immobilized primed template nucleic acid molecule.
7. The method of claim 6 , further comprising, after the destabilizing step, a step of incorporating a cognate nucleotide into the primer strand of the immobilized primed template nucleic acid molecule.
8. The method of claim 7 , wherein the cognate nucleotide incorporated into the primer strand of the immobilized primed template nucleic acid molecule is a reversible terminator nucleotide comprising a reversible terminator moiety.
9. The method of claim 1 , wherein the immobilized primed template nucleic acid molecule of step (a) is a blocked primed template nucleic acid molecule comprising a reversible terminator moiety.
10. The method of claim 6 , wherein the immobilized primed template nucleic acid molecule of step (a) is a blocked primed template nucleic acid molecule comprising a reversible terminator moiety, and wherein after the destabilizing step there are the further steps of:
cleaving the reversible terminator moiety from the blocked primed template nucleic acid molecule to result in a de-blocked primed template nucleic acid molecule; and
incorporating a cognate nucleotide into the de-blocked primed template nucleic acid molecule.
11. The method of claim 10 , wherein the cognate nucleotide incorporated into the de-blocked primed template nucleic acid molecule is a reversible terminator nucleotide.
12. The method of claim 11 , further comprising repeating all of the steps to determine the identities of consecutive nucleotides in the immobilized primed template nucleic acid molecule.
13. The method of claim 1 , wherein the immobilized primed template nucleic acid molecule is contained within a flow cell.
14. The method of claim 1 , wherein the immobilized primed template nucleic acid molecule is immobilized to the surface of a bead.
15. The method of claim 1 , wherein the stabilizing fluid is a non-aqueous fluid.
16. The method of claim 15 , wherein the non-aqueous fluid is selected from the group consisting of a gas and an oil.
17. The method of claim 1 , wherein step (b) occurs for a duration of at least 10 seconds and at most 10 minutes prior to step (c).
18. The method of claim 1 , wherein the first polymerase comprises a detectable label and wherein step (c) comprises detecting the immobilized ternary complex in contact with the stabilizing fluid by detecting the detectable label of the first polymerase.
19. The method of claim 18 , wherein the detectable label comprises an exogenous fluorescent label attached to the first polymerase.
20. The method of claim 1 ,
wherein the immobilized primed template nucleic acid molecule is contained within a flow cell,
wherein step (a) comprises flowing into the flow cell a first reagent solution that comprises the first polymerase and the first test nucleotide, and
wherein step (b) comprises flowing the stabilizing fluid into the flow cell to replace any of the first reagent solution contained therein.
21. The method of claim 20 , wherein the first reagent solution further comprises a second test nucleotide different from the first test nucleotide.