Serial formation of ternary complex species

Abstract

A method for identifying a nucleotide in a primed template nucleic acid, including the steps of (a) providing a vessel having a primed template nucleic acid, polymerase and a nucleotide cognate of a first base type; (b) examining the vessel for a stabilized ternary complex including the polymerase and the nucleotide cognate of the first base type bound at a base position of the primed template nucleic acid; (c) delivering a nucleotide cognate of a second base type to the vessel, whereby the vessel retains the primed template nucleic acid and the polymerase from step (b); (d) examining the vessel for a stabilized ternary complex including the polymerase and the nucleotide cognate of the second base type bound at the base position of the primed template nucleic acid; and (e) identifying the type of nucleotide at the base position of the primed template nucleic acid.

1 .- 16 . (canceled). 
     
     
         17 . A sequencing method comprising cycles of:
 (i) an examination step; and   (ii) a primer extension step;   wherein the examination step comprises detecting a ternary complex;   wherein the ternary complex comprises a polymerase, a primed template nucleic acid, and a nucleotide cognate;   wherein the primed template nucleic acid comprises a template nucleic acid and a primer;   wherein the nucleotide cognate comprises an exogenous label;   wherein the nucleotide cognate is removed under conditions that dissociate the nucleotide cognate from the ternary complex without causing removal of the polymerase;   wherein the nucleotide is dissociated from a ternary complex using chemical conditions that disrupt non-covalent forces that bind the components of the ternary complex; and   wherein the examination step is carried out separately from the primer extension step.   
     
     
         18 . The sequencing method of  claim 17 , wherein the chemical condition for dissociating the nucleotide cognate is the presence of at least 10% organic solvent. 
     
     
         19 . The sequencing method of  claim 18 , wherein the organic solvent comprises an alcohol. 
     
     
         20 . The sequencing method of  claim 19 , wherein the alcohol is ethanol. 
     
     
         21 . The sequencing method of  claim 19 , wherein the alcohol is isopropanol. 
     
     
         22 . The sequencing method of  claim 19 , wherein the alcohol is a diol. 
     
     
         23 . The sequencing method of  claim 17 , wherein the exogenous label comprises a fluorophore moiety. 
     
     
         24 . The sequencing method of  claim 17 , wherein the cognate nucleotide comprises streptavidin and biotin. 
     
     
         25 . The sequencing method of  claim 17 , wherein the nucleotide cognate is a non-incorporable nucleotide. 
     
     
         26 . The sequencing method of  claim 17 , wherein different nucleotide types are serially delivered to a vessel containing one or more primed template nucleic acids under conditions that are amenable to formation of the ternary complex. 
     
     
         27 . The sequencing method of  claim 17 , wherein multiple nucleotide types are delivered in one step, wherein each type of nucleotide comprises a different label, and wherein the examination step comprises distinguishing between the different labels of the multiple nucleotide types. 
     
     
         28 . The sequencing method of  claim 17 , wherein the ternary complex of the examination step is stabilized by the presence of a blocking group that precludes enzymatic incorporation of an incoming nucleotide into the primer. 
     
     
         29 . The sequencing method of  claim 28 , wherein the blocking group is a reversible terminator moiety on the 3′ nucleotide of the primer. 
     
     
         30 . The sequencing method of  claim 17 , wherein the ternary complex of the examination step is stabilized by the absence of a catalytic metal ion. 
     
     
         31 . The sequencing method of  claim 17 , wherein the ternary complex of the examination step is stabilized by a non-catalytic ion. 
     
     
         32 . The sequencing method of  claim 31 , wherein the non-catalytic metal ion is strontium. 
     
     
         33 . The sequencing method of  claim 17 , wherein the primed template nucleic acid is immobilized on a solid support. 
     
     
         34 . The sequencing method of  claim 17 , wherein polymerase is immobilized on a solid support. 
     
     
         35 . The sequencing method of  claim 17 , wherein the primer extension step comprises adding a reversibly terminated nucleotide to produce a reversibly terminated primer. 
     
     
         36 . The sequencing method of  claim 35 , wherein the reversibly terminated nucleotide does not comprise a label. 
     
     
         37 . The sequencing method of  claim 36 , further comprising deblocking to convert the reversibly terminated primer into an extendable primer, wherein repeated cycles of extension, examination and deblocking reveal the sequence of the template nucleic acid. 
     
     
         38 . The sequencing method of  claim 17 , comprising at least 5 repeated cycles. 
     
     
         39 . The sequencing method of  claim 38 , comprising at least 100 repeated cycles. 
     
     
         40 . The sequencing method of  claim 17 , wherein the template nucleic acid is a common template sequence of an ensemble. 
     
     
         41 . The sequencing method of  claim 40 , wherein the method comprises sequencing an array of ensembles. 
     
     
         42 . The sequencing method of  claim 41 , wherein the array of ensembles are clusters amplified on a surface by rolling circle amplification.