METHOD AND SYSTEM EMPLOYING DISTINGUISHABLE POLYMERASES FOR DETECTING TERNARY COMPLEXES AND IDENTIFYING COGNATE NUCLEOTIDES
Method of identifying a cognate nucleotide (i.e., the “next correct nucleotide”) for a primed template nucleic acid molecule. In some embodiments, an ordered or random array of primed target nucleic acids characterized by different cognate nucleotides can be evaluated using a single imaging step to identify different cognate nucleotides for a collection of different primed template nucleic acid molecules. An optional incorporation step can follow the identifying step. A polymerase different from the ones used in the binding and examination steps can be used to incorporate a nucleotide, such as a reversible terminator nucleotide, preliminary to identification of the next cognate nucleotide.
1 - 35 . (canceled)
36 . A method of sequencing by binding (SBB) comprising:
(i) an examination step that comprises detecting the steady state interaction of a ternary complex stabilized by a trivalent lanthanide cation,
wherein the ternary complex comprises polymerase, a primed template nucleic acid, and a next correct nucleotide,
wherein the examination step does not comprise chemical incorporation of the next correct nucleotide molecule into a primer of the primed template nucleic acid molecule, and
wherein the trivalent lanthanide cation is non-catalytic; and
(ii) a subsequent incorporation step that comprises replacing a nucleotide from the examination step and incorporating another nucleotide into the 3′-end of the primer of the primed template nucleic acid molecule.
37 . The method of claim 36 , wherein the next correct nucleotide of the ternary complex detected in the examination step is a labeled nucleotide.
38 . The method of claim 37 , wherein the labeled nucleotide comprises a fluorescent label.
39 . The method of claim 36 , wherein the nucleotide incorporated in the incorporation step is a reversible terminator nucleotide.
40 . The method of claim 39 , wherein the reversible terminator nucleotide is an unlabeled reversible terminator nucleotide that does not include a detectable fluorophore.
41 . The method of claim 40 , wherein the next correct nucleotide of the ternary complex detected in the examination step is a labeled nucleotide.
42 . The method of claim 36 , wherein only the next correct nucleotide of the ternary complex comprises a detectable label.
43 . The method of claim 36 , wherein only the polymerase of the ternary complex comprises a detectable label.
44 . The method of claim 36 , wherein chemical incorporation in the examination step is prevented by substitution of a catalytic metal ion with a non-catalytic metal ion.
45 . The method of claim 44 , wherein the non-catalytic metal ion is the trivalent lanthanide ion.
46 . The method of claim 44 , wherein the catalytic metal ion is magnesium.
47 . The method of claim 36 , wherein chelation of a catalytic metal ion prevents the chemical incorporation of the nucleotide into the primer of the primed template nucleic acid.
48 . The method of claim 47 , wherein the catalytic metal ion is magnesium.
49 . The method of claim 36 , wherein the trivalent lanthanide cation inhibits polymerization.
50 . The method of claim 36 , wherein the trivalent lanthanide cation is Europium or Terbium.
51 . The method of claim 36 , further comprising repeating the examination step prior to the incorporation step.
52 . The method of claim 36 , further comprising repeating the examination and incorporation steps for a number of cycles.