Sequencing method employing ternary complex destabilization to identify cognate nucleotides
First Claim
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1. A method of identifying a nucleotide comprising a base complementary to the next base of a template strand immediately downstream of a primer in a primed template nucleic acid molecule, said method comprising the steps of:
- (a) providing a blocked primed template nucleic acid molecule comprising a reversible terminator moiety that precludes the 3′
-terminus of the blocked primed template nucleic acid molecule from participating in phosphodiester bond formation;
(b) contacting the blocked primed template nucleic acid molecule with a first reaction mixture that comprises a polymerase, and a plurality of different nucleotide molecules,whereby a stabilized ternary complex forms, said stabilized ternary complex comprising one of the plurality of different nucleotide molecules;
(c) contacting the stabilized ternary complex with a second reaction mixture that comprises at least one of the different nucleotide molecules and that does not comprise a first nucleotide molecule of the plurality of different nucleotide molecules;
(d) monitoring interaction of the polymerase and the blocked primed template nucleic acid molecule in contact with the second reaction mixture to detect any of the stabilized ternary complex remaining after step (c); and
(e) identifying the nucleotide that comprises the base complementary to the next base of the template strand using results from step (d).
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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. These results can even be achieved in procedures employing unlabeled, native nucleotides.
60 Citations
42 Claims
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1. A method of identifying a nucleotide comprising a base complementary to the next base of a template strand immediately downstream of a primer in a primed template nucleic acid molecule, said method comprising the steps of:
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(a) providing a blocked primed template nucleic acid molecule comprising a reversible terminator moiety that precludes the 3′
-terminus of the blocked primed template nucleic acid molecule from participating in phosphodiester bond formation;(b) contacting the blocked primed template nucleic acid molecule with a first reaction mixture that comprises a polymerase, and a plurality of different nucleotide molecules, whereby a stabilized ternary complex forms, said stabilized ternary complex comprising one of the plurality of different nucleotide molecules; (c) contacting the stabilized ternary complex with a second reaction mixture that comprises at least one of the different nucleotide molecules and that does not comprise a first nucleotide molecule of the plurality of different nucleotide molecules; (d) monitoring interaction of the polymerase and the blocked primed template nucleic acid molecule in contact with the second reaction mixture to detect any of the stabilized ternary complex remaining after step (c); and (e) identifying the nucleotide that comprises the base complementary to the next base of the template strand using results from step (d). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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22. A method of identifying a nucleotide comprising a base complementary to the next base of a template strand immediately downstream of a primer in a primed template nucleic acid molecule, said method comprising:
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(a) providing the primed template nucleic acid molecule; (b) contacting the primed template nucleic acid molecule with a first reaction mixture that comprises a polymerase and a plurality of different nucleotide molecules, whereby a stabilized ternary complex forms, said stabilized ternary complex comprising one of the plurality of different nucleotide molecules; (c) contacting the primed template nucleic acid molecule, after step (b), with a second reaction mixture that comprises at least one of the different nucleotide molecules and that does not comprise a first nucleotide molecule of the plurality of different nucleotide molecules; (d) monitoring interaction of the polymerase and the primed template nucleic acid molecule in the second reaction mixture, without incorporating any nucleotide into the primer, to detect any of the stabilized ternary complex remaining after step (c); and (e) identifying the nucleotide that comprises the base complementary to the next base of the template strand using results from step (d). - View Dependent Claims (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)
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23. A method of identifying a nucleotide comprising a base complementary to the next base of a template strand immediately downstream of a primer in a primed template nucleic acid molecule, said method comprising:
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(a) providing the primed template nucleic acid molecule; (b) contacting the primed template nucleic acid molecule with a first reaction mixture that comprises a polymerase, but does not comprise any nucleotide, whereby a binary complex forms; (c) contacting the binary complex with a second reaction mixture that comprises a plurality of different nucleotide molecules, whereby a stabilized ternary complex forms if one of the plurality of different nucleotide molecules comprises the base complementary to the next base of the template strand; (d) detecting, without incorporating any nucleotide into the primer, any of the stabilized ternary complex that may have formed; (e) contacting the primed template nucleic acid molecule, after step (d), with a third reaction mixture that comprises at least one of the different nucleotide molecules and that does not comprise a first nucleotide molecule of the plurality of different nucleotide molecules; (f) detecting, without incorporating any nucleotide into the primer, any of the stabilized ternary complex remaining after step (e); and (g) identifying the nucleotide that comprises the base complementary to the next base of the template strand using results from both of detecting steps (d) and (f). - View Dependent Claims (35, 36, 37, 38, 39, 40, 41, 42)
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Specification