Method and system for sequencing nucleic acids

Abstract

Provided are compositions, methods and systems for determining the sequence of a template nucleic acid using a polymerase-based, sequencing-by-binding procedure. An examination step involves monitoring the interaction between a polymerase and template nucleic acid in the presence of one or more nucleotides. Identity of the next correct nucleotide in the sequence is determined without incorporation of any nucleotide into the structure of the primer by formation of a phosphodiester bond. An optional incorporation step can be used after the examination step to extend the primer by one or more nucleotides, thereby incrementing the template nucleotides that can be examined in a subsequent examination step. The sequencing-by-binding procedure does not require the use of labeled nucleotides or polymerases, but optionally can employ these reagents.

1 . A method of determining the identity of the next correct nucleotide for a primed template nucleic acid, the method comprising:
 (a) contacting the primed template nucleic acid with a series of different reaction mixtures, thereby forming stabilized ternary complexes that comprise the primed template nucleic acid molecule, a polymerase and a next correct nucleotide; and   (b) detecting the stabilized ternary complexes, thereby determining the identity of the next correct nucleotide for the primed template nucleic acid;   wherein each of the different reaction mixtures comprises one or more nucleotides; and   wherein no nucleotide is incorporated at the 3′ terminal end of the primer at the primed template nucleic acid during or between steps (a) and (b).   
     
     
         2 . The method of  claim 1 , wherein the series of reaction mixtures comprises at least two different reaction mixtures. 
     
     
         3 . The method of  claim 2 , wherein the at least two different reaction mixtures comprises four different reaction mixtures. 
     
     
         4 . The method of  claim 1 , further comprising repeating steps (a) and (b) for a series of next base positions to identify a sequence of the template nucleic acid. 
     
     
         5 . The method of  claim 1 , wherein the determining in step (b) comprises determining whether a nucleotide that is common to two of the different reaction mixtures is the next correct nucleotide for the primed template nucleic acid. 
     
     
         6 . The method of  claim 1 , wherein the ternary complex is stabilized by the presence of a reversible terminator moiety on the 3′ terminal nucleotide of the primer of the primed template nucleic acid. 
     
     
         7 . The method of  claim 6 , further comprising, after step (b), removing the reversible terminator moiety on the 3′ terminal nucleotide of the primer. 
     
     
         8 . The method of  claim 7 , further comprising adding a reversible terminator nucleotide to the primer after the removing of the reversible terminator moiety on the 3′ terminal nucleotide of the primer. 
     
     
         9 . The method of  claim 1 , wherein one or more of the different reaction mixtures comprises an unlabeled nucleotide. 
     
     
         10 . The method of  claim 1 , wherein each of the different reaction mixtures comprises one or more nucleotides having an exogenous label. 
     
     
         11 . The method of  claim 10 , wherein the exogenous label comprises a fluorophore. 
     
     
         12 . The method of  claim 1 , wherein the next correct nucleotide comprises an exogenous label that is detected in step (b). 
     
     
         13 . The method of  claim 1 , wherein the polymerase comprises an exogenous label that is detected in step (b). 
     
     
         14 . The method of  claim 1 , further comprising incorporating a nucleotide at the 3′ terminal end of the primer after step (b). 
     
     
         15 . The method of  claim 14 , wherein the nucleotide that is incorporated lacks an exogenous labels. 
     
     
         16 . The method of  claim 15 , wherein the nucleotide that is incorporated further comprises a reversible terminator moiety. 
     
     
         17 . The method of  claim 1 , wherein the primed template nucleic acid is contacted with the polymerase before the primed template nucleic acid is contacted with one of the different reaction mixtures in step (a). 
     
     
         18 . The method of  claim 1 , wherein the primed template nucleic acid is attached to a solid support. 
     
     
         19 . The method of  claim 18 , wherein the primed template nucleic acid is a member of a clonal population of template nucleic acids. 
     
     
         20 .- 21 . (canceled) 
     
     
         22 . The method of  claim 19 , wherein the clonal population of template nucleic acids comprising rolling-circle amplified DNA. 
     
     
         23 . The method of  claim 1 , wherein the ternary complex is stabilized by the presence of a non-catalytic metal ion. 
     
     
         24 . The method of  claim 1 , wherein a first reaction mixture of the series of reaction mixtures further comprises the polymerase. 
     
     
         25 . The method of  claim 1 , further comprising contacting the stabilized ternary complex with a catalytic metal ion to covalently add the nucleotide sequestered in the ternary complex to the 3′ terminal end of the primer after step (b) of detecting. 
     
     
         26 . The method of  claim 1 , wherein a first ternary complex formed from a first reaction mixture of the series of different reaction mixtures is detected prior to contacting the primed template nucleic acid with a second reaction mixture of the series of different reaction mixtures. 
     
     
         27 . The method of  claim 1 , wherein step (a) of contacting comprises stabilizing the ternary complexes while destabilizing binary complexes. 
     
     
         28 . The method of  claim 1 , wherein the primed template nucleic acid is immobilized and step (a) further comprises contacting the immobilized primed template nucleic acid with a non-immobilized primed template nucleic acid molecule. 
     
     
         29 . A method of sequencing a primed template nucleic acid, comprising:
 (a) contacting a primed template nucleic acid with a first mixture of one or more nucleotides, thereby forming stabilized ternary complexes between the primed template nucleic acid, a polymerase and a nucleotide from the first mixture of nucleotides that is complementary to the next base of the primed template nucleic acid;   (b) detecting the stabilized ternary complexes while precluding incorporation the nucleotide into the primer;   (c) repeating steps (a) and (b) at least once using the primed template nucleic acid and a second mixture of one or more nucleotides, wherein the second mixture of nucleotides is different from the first mixture of nucleotides;   (d) incorporating into the primer, after step (c), a nucleotide that is complementary to the next base of the primed template nucleic acid; and   (e) repeating steps (a) through (d) to identify a sequence of the primed template nucleic acid.