Abstract: A sensor having a first electrode and a second electrode operably connected by a conduction channel, wherein a polymerase, primed template nucleic acid and nucleotide form a stabilized ternary complex that is immobilized in or on the conduction channel, whereby association and dissociation of the ternary complex is detected due to changes in electrical properties of the sensor. Identification of the nucleotide type that participates in the complex indicates the identity of the next template base in the template. Repeated cycles of extending the primer and detecting stabilized ternary complexes can allow determination of the sequence of nucleotides for the template.

Abstract: Method of identifying a cognate nucleotide (i.e., the “next correct nucleotide”) for a primed template nucleic acid molecule. In some embodiments, an ordered or random array of primed target nucleic acids characterized by different cognate nucleotides can be evaluated using a single imaging step to identify different cognate nucleotides for a collection of different primed template nucleic acid molecules. An optional incorporation step can follow the identifying step. A polymerase different from the ones used in the binding and examination steps can be used to incorporate a nucleotide, such as a reversible terminator nucleotide, preliminary to identification of the next cognate nucleotide.

Abstract: Methods, compositions, kits and apparatuses that include a fluid, the fluid containing a ternary complex and Li+, wherein the ternary complex includes a primed template nucleic acid, a polymerase, and a nucleotide cognate for the next correct base for the primed template nucleic acid molecule. As an alternative or addition to Li+, the fluid can contain betaine or a metal ion that inhibits polymerase catalysis such as Ca2+. In addition to Li+, the fluid can contain polyethylenimine (PEI) with or without betaine.

Abstract: Provided are nucleic acids encoding engineered polymerases comprising at least one modification in a motif A and/or at least one modification in a motif B of the polymerase and engineered polymerases encoded by the nucleic acids. Also provided are engineered DNA polymerases comprising a variant of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, the variant being at least 80% identical to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 and comprising an amino acid substitution at one or more positions selected from the group consisting of L408, Y409, P410, R484, A/L485, and I486. Methods, vectors, kits, and compositions comprising the nucleic acids and compositions, methods and kits comprising the engineered polymerases are also provided.

Abstract: A method for identifying a nucleic acid template that include (a) providing a plurality of primer-template hybrids, wherein a first hybrid of the plurality includes a first template hybridized to a first primer, and wherein a second hybrid of the plurality includes a second template hybridized to a second primer, the second primer having a ternary complex inhibitor moiety at the 3? end; (b) delivering polymerases and nucleotides to the plurality, whereby the first hybrid binds a polymerase and nucleotide to form a stabilized ternary complex and whereby the second hybrid does not bind a polymerase and nucleotide to form a stabilized ternary complex; and (c) detecting the stabilized ternary complex to identify the first template.

Abstract: Method and apparatus to facilitate separation of solution-phase components surrounding an immobilized multicomponent complex while stabilizing association of the components within the complex. The technique can be used for reducing background signal arising from the presence of non-complexed components harboring detectable labels, thereby enhancing signal-to-background ratios and allowing enhanced detection of the multicomponent complex.

Abstract: Method of identifying a cognate nucleotide (i.e., the “next correct nucleotide”) for a primed template nucleic acid molecule. In some embodiments, an ordered or random array of primed target nucleic acids characterized by different cognate nucleotides can be evaluated using a single imaging step to identify different cognate nucleotides for a collection of different primed template nucleic acid molecules. An optional incorporation step can follow the identifying step. A polymerase different from the ones used in the binding and examination steps can be used to incorporate a nucleotide, such as a reversible terminator nucleotide, preliminary to identification of the next cognate nucleotide.

Abstract: Methods, compositions, kits and apparatuses that include a fluid, the fluid containing a ternary complex and Li+, wherein the ternary complex includes a primed template nucleic acid, a polymerase, and a nucleotide cognate for the next correct base for the primed template nucleic acid molecule. As an alternative or addition to Li+, the fluid can contain betaine or a metal ion that inhibits polymerase catalysis such as Ca2+. In addition to Li+, the fluid can contain polyethylenimine (PEI) with or without betaine.

Abstract: Provided are nucleic acids encoding engineered polymerases comprising at least one modification in a motif A and/or at least one modification in a motif B of the polymerase and engineered polymerases encoded by the nucleic acids. Also provided are engineered DNA polymerases comprising a variant of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, the variant being at least 80% identical to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 and comprising an amino acid substitution at one or more positions selected from the group consisting of L408, Y409, P410, R484, A/L485, and I486. Methods, vectors, kits, and compositions comprising the nucleic acids and compositions, methods and kits comprising the engineered polymerases are also provided.

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.

Abstract: Method and apparatus to facilitate separation of solution-phase components surrounding an immobilized multicomponent complex while stabilizing association of the components within the complex. The technique can be used for reducing background signal arising from the presence of non-complexed components harboring detectable labels, thereby enhancing signal-to-background ratios and allowing enhanced detection of the multicomponent complex.

Abstract: Provided herein and methods and apparatuses for sequencing nucleic acids. For example, provided is an analytical detection apparatus, including (a) a stage configured to support a flow cell; (b) a detector configured to observe a detection channel of the flow cell when the flow cell is supported by the stage; (c) a plurality of fluid delivery channels, wherein each of the fluid delivery channels fluidically connects a reservoir to the detection channel of the flow cell; and (d) a first heater configured to heat the plurality of fluid delivery channels.

Abstract: A method for identifying a nucleic acid template that includes (a) providing a plurality of primer-template hybrids, wherein a first hybrid of the plurality includes a first template hybridized to a first primer, and wherein a second hybrid of the plurality includes a second template hybridized to a second primer, the second primer having a ternary complex inhibitor moiety at the 3? end; (b) delivering polymerases and nucleotides to the plurality, whereby the first hybrid binds a polymerase and nucleotide to form a stabilized ternary complex and whereby the second hybrid does not bind a polymerase and nucleotide to form a stabilized ternary complex; and (c) detecting the stabilized ternary complex to identify the first template.

Abstract: A method for separating a target allele from a mixture of nucleic acids by (a) providing a mixture of nucleic acids in fluidic contact with a stabilized ternary complex that is attached to a solid support, wherein the stabilized ternary complex includes a polymerase, primed nucleic acid template, and next correct nucleotide, wherein the template has a target allele, wherein the next correct nucleotide is a cognate nucleotide for the target allele, and wherein the stabilized ternary complex is attached to the solid support via a linkage between the polymerase and the solid support or via a linkage between the next correct nucleotide and the solid support; and (b) separating the solid support from the mixture of nucleic acids, thereby separating the target allele from the mixture of nucleic acids.

Abstract: Disclosed herein, inter alia, are methods for modifying a nucleotide, for example including reacting a nucleotide having a 3?-O-oxime moiety such as with a reagent having an —ONH2 moiety to produce a nucleotide having a 3?-O—NH2 moiety such as wherein the reagent having the —ONH2 moiety further comprises alkyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl, or (heteroalicyclyl)alkyl.

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.

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.

Abstract: A method for identifying target alleles, that includes steps of (a) forming a plurality of stabilized ternary complexes at a plurality of features on an array, wherein the stabilized ternary complexes each has a polymerase, a template nucleic acid having a target allele of a locus, a primer hybridized to the locus, and a next correct nucleotide having a cognate in the locus, wherein either (i) the primer is an allele-specific primer having a 3? nucleotide that is a cognate nucleotide for the target allele, or (ii) the primer is a locus-specific primer and the next correct nucleotide hybridizes to the target allele; and (b) detecting stabilized ternary complexes at the features, thereby identifying the target alleles.

Abstract: Method and composition for identifying cognate nucleotides in a Sequencing By Binding™ procedure, wherein one or more labeled nucleotides are detected in ternary complexes but never incorporated. Labeled nucleotides can be incorporable nucleotides that contact preformed blocked primed template nucleic acids. Alternatively, labeled nucleotides are labeled non-incorporable nucleotides. Labeled nucleotides, including labeled non-incorporable nucleotides, can be detected in ternary complexes in the same reaction mixture that incorporates a reversible terminator nucleotide to create a blocked primed template nucleic acid. Detection of ternary complexes can take place in the presence of a catalytic metal ion.

Abstract: Provided herein and methods and apparatuses for sequencing nucleic acids. For example, provided is an analytical detection apparatus, including (a) a stage configured to support a flow cell; (b) a detector configured to observe a detection channel of the flow cell when the flow cell is supported by the stage; (c) a plurality of fluid delivery channels, wherein each of the fluid delivery channels fluidically connects a reservoir to the detection channel of the flow cell; and (d) a first heater configured to heat the plurality of fluid delivery channels.