Method and system employing distinguishable polymerases for detecting ternary complexes and identifying cognate nucleotides

Abstract

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.