Nucleic acid sequencing method and system employing enhanced detection of nucleotide-specific ternary complex formation

Abstract

Provided are methods and systems for detecting formation of nucleotide-specific ternary complexes comprising a DNA polymerase, a nucleic acid, and a nucleotide complementary to the templated base of the primed template nucleic acid. The methods and systems facilitate determination of the next correct nucleotide without requiring chemical incorporation of the nucleotide into the primer. This advantageously improves signal-to-noise ratios and increases the quality of results obtainable in a sequencing-by-binding protocol, and enables extended read lengths. These results can even be achieved in procedures employing unlabeled, native nucleotides.

1 - 52 . (canceled) 
     
     
         53 . A method of incorporating a nucleotide with a base complementary to the next base in a template strand immediately downstream of a primer in a primed template nucleic acid molecule, the method comprising the steps of:
 (a) contacting the primed template nucleic acid molecule with a first reaction mixture that comprises a DNA polymerase, thereby forming a binary complex comprising the primed template nucleic acid molecule and the DNA polymerase;   (b) contacting the binary complex from step (a) with a second reaction mixture that comprises the DNA polymerase and a first test nucleotide, without incorporating the first test nucleotide into the primer, thereby forming a second complex comprising the primed template nucleic acid molecule and the DNA polymerase;   (c) measuring binding of the primed template nucleic acid molecule to the DNA polymerase at one or more points during each of steps (a) and (b), without chemical incorporation of the first test nucleotide into the primer, to establish whether the second complex that formed in step (b) is a ternary complex comprising the first test nucleotide;   (d) selecting one of the following two options,   (i) if it is established in step (c) that the second complex is not the ternary complex, then contacting the second complex from step (b) with a third reaction mixture that comprises the DNA polymerase in combination with the first test nucleotide and a second test nucleotide, the second test nucleotide being different from the first test nucleotide; and   (ii) if it is established in step (c) that the second complex is the ternary complex, then performing an incorporation reaction to incorporate the first test nucleotide into the primer without first contacting the primed template nucleic acid molecule from step (b) with the third reaction mixture,   
       wherein either
 the first reaction mixture is free of the first test nucleotide, or 
 the first and second reaction mixtures each comprise the first test nucleotide, the first test nucleotide of the first reaction mixture being present at a concentration lower than the concentration of the first test nucleotide of the second reaction mixture. 
 
     
     
         54 . The method of  claim 53 , wherein the primed template nucleic acid molecule is immobilized to a solid support. 
     
     
         55 . The method of  claim 54 , wherein the fourth reaction mixture comprises a polymerase different from the polymerase of the first and second reaction mixtures. 
     
     
         56 . The method of  claim 54 , wherein the fourth reaction mixture comprises a polymerase different from the polymerase of the first and second reaction mixtures, and wherein the nucleotide incorporated into the primer in step (d)(ii) comprises a reversible terminator moiety. 
     
     
         57 . The method of  claim 53 , wherein the primed template nucleic acid molecule is immobilized to a solid support, and wherein contacting steps (a) and (b) each comprise flowing the reaction mixtures over the primed template nucleic acid molecule immobilized to the solid support. 
     
     
         58 . The method of  claim 53 , wherein the primed template nucleic acid molecule is immobilized to a solid support, and wherein contacting steps (a) and (b) each comprise moving the solid support from a vessel containing the first reaction mixture to a vessel containing the second reaction mixture. 
     
     
         59 . The method of  claim 53 , wherein the first and second reaction mixtures each comprise the same concentration of the polymerase. 
     
     
         60 . The method of  claim 53 , wherein the fourth reaction mixture comprises a polymerase different from the polymerase of the first and second reaction mixtures. 
     
     
         61 . The method of  claim 53 , wherein step (c) comprises measuring by either of interferometry or surface plasmon resonance sensing. 
     
     
         62 . The method of  claim 53 , wherein the first test nucleotide is a native nucleotide. 
     
     
         63 . The method of  claim 53 , wherein the first test nucleotide comprises a reversible terminator moiety. 
     
     
         64 . The method of  claim 53 , wherein the nucleotide incorporated in step (d)(ii) is selected from the group consisting of a native nucleotide and a nucleotide comprising a reversible terminator. 
     
     
         65 . The method of  claim 53 , wherein the test nucleotide is a native nucleotide that does not comprise any added fluorescent label, and wherein step (c) does not comprise measuring differences in fluorescence or absorbance signals from a conformationally sensitive dye that changes optical properties as the result of the DNA polymerase binding to the test nucleotide. 
     
     
         66 . The method of  claim 53 , wherein the first reaction mixture is free of nucleotides. 
     
     
         67 . The method of  claim 53 , further comprising repeating steps (a)-(d). 
     
     
         68 . The method of  claim 67 , wherein steps (a)-(d) are repeated at least 50 times. 
     
     
         69 . The method of  claim 53 , wherein the DNA polymerase of the first reaction mixture is free of added fluorescent label that changes properties after the DNA polymerase binds to a nucleotide. 
     
     
         70 . The method of  claim 66 , wherein step (c) comprises continuously measuring binding of the primed template nucleic acid to the DNA polymerase during each of steps (a) and (b). 
     
     
         71 . The method of  claim 66 , wherein step (c) comprises measuring optical signals indicating binding of the primed template nucleic acid to the DNA polymerase and the calculating the difference between the measured optical signals to determine whether the measured binding resulting from step (b) exceeds the measured binding resulting from step (a). 
     
     
         72 . The method of  claim 66 , wherein step (c) comprises measuring optical signals indicating binding of the primed template nucleic acid to the DNA polymerase and then calculating the time dependent rate of change of the measured optical signals to determine whether the measured binding resulting from step (b) exceeds the measured binding resulting from step (a).