NUCLEIC ACID SEQUENCING METHOD AND SYSTEM EMPLOYING ENHANCED DETECTION OF NUCLEOTIDE-SPECIFIC TERNARY COMPLEX FORMATION
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 .- 64 . (canceled)
65 . A method of determining whether a test nucleotide is the next correct nucleotide comprising a base complementary to the next base in a template strand immediately downstream of a primer in a primed template nucleic acid, said method comprising the steps of:
(a) contacting the primed template nucleic acid with a first reaction mixture that comprises a DNA polymerase, thereby forming a complex comprising the primed template nucleic acid and the polymerase; and
(b) performing consecutive first and second examination steps with reaction mixtures that comprise different concentrations of at least one test nucleotide, wherein examination comprises identifying the base of the nucleotide that is complementary to the next base of the primed template nucleic acid without chemical incorporation of the labeled nucleotide molecule.
66 . The method of claim 1 , wherein a closed-complex is formed under examination conditions.
67 . The method of claim 1 , wherein the closed-complex is a stabilized ternary complex.
68 . The method of claim 3 , further comprising step (c) of chemically incorporating the test nucleotide into the primer of the primed template nucleic acid by formation of a phosphodiester bond.
69 . The method of claim 4 , further comprising repeating steps (a)-(c).
70 . The method of claim 5 , further comprising repeating steps (a)-(c) at least 50 times.
71 . The method of claim 1 , wherein the polymerase is present at the same concentration in the reaction mixtures of the first and second examination steps.
72 . The method of claim 1 , wherein the test nucleotide is absent in the reaction mixture of the second consecutive examination step.
73 . The method of claim 1 , wherein the test nucleotide is present in the reaction mixture of the second consecutive examination step at 30% or less the concentration of the test nucleotide in the reaction mixture of the first examination step.
74 . The method of claim 1 , comprising performing 4 examination steps prior to an incorporation step.
75 . The method of claim 1 , wherein the reaction mixtures of the examination steps each comprise 4 test nucleotides.
76 . The method of claim 1 , wherein the test nucleotide does not comprise an exogenous fluorescent label.
77 . The method of claim 1 , wherein the test nucleotide is a nucleotide analog, that comprises an exogenous label that is detected in step (b).
78 . The method of claim 1 , wherein the DNA polymerase is free of added fluorescent label.
79 . The method of claim 1 , wherein nucleotides present in reaction mixtures that are not sequestered in a closed-complex are removed in wash steps.
80 . The method of claim 1 , wherein step (e) comprises determining that the test nucleotide is the next correct nucleotide if the measured binding resulting from step (b) exceeds the measured binding resulting from step (a).
81 . The method of claim 1 , wherein the first reaction mixture comprises a nucleotide that is different from the test nucleotides.
82 . The method of claim 1 , wherein the complex formed in step (a) is a binary complex.