Process for cognate nucleotide detection in a nucleic acid sequencing workflow
Method and composition for identifying cognate nucleotides in a Sequencing By Binding™ procedure, wherein one or more labeled nucleotides are detected in ternary complexes but never incorporated. Labeled nucleotides can be incorporable nucleotides that contact preformed blocked primed template nucleic acids. Alternatively, labeled nucleotides are labeled non-incorporable nucleotides. Labeled nucleotides, including labeled non-incorporable nucleotides, can be detected in ternary complexes in the same reaction mixture that incorporates a reversible terminator nucleotide to create a blocked primed template nucleic acid. Detection of ternary complexes can take place in the presence of a catalytic metal ion.
1. A method comprising:
(a) providing a plurality of primed template nucleic acids immobilized to a solid support;
(b) delivering to the solid support a reaction mixture that comprises unlabeled reversible terminator nucleotides, labeled nucleotides, polymerases, and catalytic metal ions, thereby forming a plurality of stabilized ternary complexes that each comprise one of the polymerases, one of the labeled nucleotides and one of the primed template nucleic acids that is immobilized to the solid support; and
(c) detecting signals produced by the labeled nucleotides in the stabilized ternary complexes to determine the next template bases for the template nucleic acids in the stabilized ternary complexes, wherein the reversible terminator nucleotides lack labels that produce the signals.
2. The method of claim 1 , wherein each primed template nucleic acid comprises a template nucleic acid primed with a primer and wherein the primers of the primed template nucleic acids in the stabilized ternary complexes comprise 3′ termini comprising reversible terminator moieties.
3. The method of claim 2 , wherein the reversible terminator moieties of the primed template nucleic acids are the same type of reversible terminator moieties that are present in the reversible terminator nucleotides.
4. The method of claim 2 , further comprising (d) removing the stabilized ternary complexes from the primed template nucleic acids and removing the reversible terminator moieties from the primers of the primed template nucleic acids.
5. The method of claim 4 , further comprising (e) repeating steps (a) through (d), thereby sequencing the primed template nucleic acid.
6. The method of claim 4 , further comprising (e) delivering to the solid support a second reaction mixture that comprises polymerases and reversible terminator nucleotides in the absence of labeled nucleotides, wherein a reversible terminator nucleotide is incorporated into each of the primed template nucleic acids in the absence of labeled nucleotides.
7. The method of claim 6 , wherein the reversible terminator nucleotides in the second reaction mixture comprise the same type of reversible terminator moiety that is present in the reaction mixture of step (b).
8. The method of claim 1 , wherein step (b) further comprises incorporating the unlabeled reversible terminator nucleotides of the reaction mixture into primed template nucleic acids that are immobilized to the solid support.
9. The method of claim 1 , wherein the label comprises a fluorescent moiety.
10. The method of claim 1 , wherein the labeled nucleotides in the ternary complexes comprises base-specific labels that produces the signals.
11. The method of claim 1 , further comprising washing the solid support to remove non-complexed labeled nucleotides and polymerases from contact with the solid support.
12. The method of claim 11 , wherein the signals produced by the labels in the stabilized ternary complexes are detected after the removing of the non-complexed labeled nucleotides and polymerases from contact with the solid support.
13. The method of claim 1 , wherein the reaction mixture comprises a plurality of nucleotides that collectively complements four types of nucleotides in the primed template nucleic acids.
14. The method of claim 13 , wherein the plurality of nucleotides comprises fewer than four different labels.
15. The method of claim 1 , wherein the labeled nucleotides are non-incorporable nucleotides.
16. The method of claim 1 , wherein the unlabeled reversible terminator nucleotides, labeled nucleotides, polymerases, and catalytic metal ions are added to the solid support separately to form the reaction mixture in the presence of the primed template nucleic acids.
17. The method of claim 1 , wherein the unlabeled reversible terminator nucleotides, labeled nucleotides, polymerases, and catalytic metal ions are mixed prior to being added to the solid support.
18. The method of claim 1 , wherein step (b) is carried out in the absence of non-catalytic metal ions that inhibit phosphodiester bond formation by the polymerases.
19. The method of claim 1 , wherein step (b) is carried out in the absence of Ca 2+ , Zn 2+ , Co 2+ , Ni 2+ , Sr 2+ , Eu 3+ and Tb 3+ ions that inhibit phosphodiester bond formation by the polymerases.
20. The method of claim 1 , wherein the solid support comprises an array of features, wherein the primed template nucleic acids are immobilized at features of the array.
21. The method of claim 20 , wherein each of the features comprises an ensemble of several primed template nucleic acids, the ensemble at each feature comprising the same sequence or complementary sequence thereof.
22. The method of claim 1 , wherein the solid support comprises a plurality of beads, wherein the primed template nucleic acids are immobilized to the beads.
23. The method of claim 1 , wherein the labeled nucleotides of the reaction mixture comprise 3′ hydroxyl moieties that are extendable.