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: Labeled nucleotide analogs comprising at least one avidin protein, at least one dye-labeled compound, and at least one nucleotide compound are provided. The analogs are useful in various fluorescence-based analytical methods, including the analysis of highly multiplexed optical reactions in large numbers at high densities, such as single molecule real time nucleic acid sequencing reactions. The analogs are detectable with high sensitivity at desirable wavelengths. They contain structural components that modulate the interactions of the analogs with DNA polymerase, thus decreasing photodamage and improving the kinetic and other properties of the analogs in sequencing reactions. Also provided are nucleotide and dye-labeled compounds of the subject analogs, as well as intermediates useful in the preparation of the compounds and analogs. Compositions comprising the compounds, methods of synthesis of the intermediates, compounds, and analogs, and mutant DNA polymerases are also provided.

Abstract: Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions, and/or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing or nucleic acid amplification. Such properties include enhanced performance with large nucleotide analogs, increased stability, increased readlength, and improved detection of modified bases, and can also include resistance to photodamage, enhanced metal ion coordination, reduced exonuclease activity, reduced reaction rates at one or more steps of the polymerase kinetic cycle, decreased branching fraction, altered cofactor selectivity, increased yield, increased accuracy, altered speed, increased cosolvent resistance, and the like. Also provided are nucleic acids which encode the polymerases with the aforementioned phenotypes, as well as methods of using such polymerases to make a DNA or to sequence a DNA template.

Abstract: The present disclosure provides compositions, methods and systems for sequencing a template nucleic acid using a polymerase based, nucleic acid binding reaction involving examination of the interaction between a polymerase and template nucleic acid in the presence of one or more unlabeled nucleotides. The methods rely, in part, on identifying a base of a template nucleic acid during nucleic acid synthesis by controlling the sequencing reaction conditions. Template nucleic acid bases may be identified during an examination step followed by an optional incorporation step.

Abstract: Labeled nucleotide analogs comprising at least one avidin protein, at least one dye-labeled compound, and at least one nucleotide compound are provided. The analogs are useful in various fluorescence-based analytical methods, including the analysis of highly multiplexed optical reactions in large numbers at high densities, such as single molecule real time nucleic acid sequencing reactions. The analogs are detectable with high sensitivity at desirable wavelengths. They contain structural components that modulate the interactions of the analogs with DNA polymerase, thus decreasing photodamage and improving the kinetic and other properties of the analogs in sequencing reactions. Also provided are nucleotide and dye-labeled compounds of the subject analogs, as well as intermediates useful in the preparation of the compounds and analogs. Compositions comprising the compounds, methods of synthesis of the intermediates, compounds, and analogs, and mutant DNA polymerases are also provided.

Abstract: A method of characterizing a nucleic acid that includes steps of (a) contacting a primer-template nucleic acid hybrid with a polymerase and a mixture of nucleotides to produce an extended primer hybrid and to form a stabilized ternary complex including the extended primer hybrid, the polymerase and a nucleotide cognate of the next base in the template, wherein the mixture contains nucleotide cognates of four different base types, wherein the nucleotide cognate of the first base type has a reversible terminator, and wherein nucleotide cognates of the second, third and fourth base types are extendable; (b) detecting the stabilized ternary complex to distinguish the next base from other base types in the template; and (c) determining the presence of a base multiplet in the template nucleic acid, the base multiplet including the first base type followed by the next base.

Abstract: Provided herein are engineered DNA polymerases comprising modifications improving accuracy and processivity of the polymerase including modifications in the Motif A region, optionally, along with additional modifications in the palm and/or exonuclease domains of the polymerase. Also provided are nucleic acids encoding the engineered DNA polymerases comprising modifications in motif A of the polymerase, optionally, with additional modifications. Methods, vectors, kits, and compositions comprising the nucleic acids and compositions, methods and kits comprising the engineered polymerases are also provided.

Abstract: Optical analytical devices and their methods of use are provided. The devices are useful in the analysis of highly multiplexed optical reactions in large numbers at high densities, including biochemical reactions, such as nucleic acid sequencing reactions. The devices include optical waveguides for illumination of the optical reactions. The devices further provide for the efficient coupling of optical excitation energy from the waveguides to the optical reactions. Optical signals emitted from the reactions can thus be measured with high sensitivity and discrimination using features such as spectra, amplitude, and time resolution, or combinations thereof. The devices of the invention are well suited for miniaturization and high throughput.

Abstract: Methods, compositions, and systems are provided for characterization of modified nucleic acids. In certain preferred embodiments, single molecule sequencing methods are provided for identification of modified nucleotides within nucleic acid sequences. Modifications detectable by the methods provided herein include chemically modified bases, enzymatically modified bases, abasic sites, non-natural bases, secondary structures, and agents bound to a template nucleic acid.

Abstract: Compositions, methods and systems are provided for isolating nucleic acids. A polymerase-nucleic acid complex can be formed by mixing a polymerase enzyme comprising strand displacement activity and a mixture of double stranded nucleic acids. Nucleic acid synthesis can then be initiated by the polymerase enzyme to produce a nascent strand complementary to the first strand, thereby displacing a portion of the second strand. After halting or reducing the rate of nucleic acid synthesis, a hybridizing a hook oligonucleotide can be used hybridize to the nucleic acid through a capture region on the hook oligonucleotide that is complementary to the displaced portion of the second strand. The nucleic acid can then be isolated from the mixture of nucleic acids using the hook oligonucleotide.

Abstract: The present disclosure is directed to a method for purifying a sample containing nucleic acids to obtain isolated nucleic acids of a desired size range, either above a size cut-off, below a cut-off, or within a defined band of sizes, including: a) combining a nucleic acid-containing sample with a binding buffer to provide a binding mixture; b) contacting the binding mixture with a silica nanomembrane, wherein the silica nanomembrane adsorbs nucleic acids from the binding mixture within a desired size-range; and c) separating the bound nucleic acid from the remaining sample. Kits including a silica nanomembrane, a binding buffer and one or wash buffers are also provided herein.

Abstract: Technical solutions for mapping long nucleic acid sequence reads to a target sequence are provided. A directed graph, representing all or some of a genome and comprising one or more nonlinear topological components, is obtained for an organism having a heterozygous genome. Each nonlinear topological component has an initiating node and a terminal node connected by at least a first branch and a second branch. One of these branches corresponds to the target sequence. The directed graph uses a plurality of sequence reads from a biological sample of the organism. The sequence reads are overlapped by an unrestricted overhang amount, provided there is a minimum consensus region between each two sequence reads. A query sequence, encompassing at least the initiating node or the terminal node of a first nonlinear topological component, is obtained. The directed graph is used to form a mapping of the query sequence to the directed graph.

Abstract: Labeled nucleotide analogs comprising at least one avidin protein, at least one dye-labeled compound, and at least one nucleotide compound are provided. The analogs are useful in various fluorescence-based analytical methods, including the analysis of highly multiplexed optical reactions in large numbers at high densities, such as single molecule real time nucleic acid sequencing reactions. The analogs are detectable with high sensitivity at desirable wavelengths. They contain structural components that modulate the interactions of the analogs with DNA polymerase, thus decreasing photodamage and improving the kinetic and other properties of the analogs in sequencing reactions. Also provided are nucleotide and dye-labeled compounds of the subject analogs, as well as intermediates useful in the preparation of the compounds and analogs. Compositions comprising the compounds, methods of synthesis of the intermediates, compounds, and analogs, and mutant DNA polymerases are also provided.

Abstract: Reaction mixtures are provided having at least a first nucleotide analog and a second nucleotide analog that produce signals in response to excitation illumination. The signals produced by the analogs have peaks at the same wavelengths, but have distinct signal intensities. The distinct intensities allow for identification of the analogs in nucleic acid sequencing. In some embodiments, FRET-labeled compounds are provided. In certain embodiments, FRET-labeled nucleotide analogs are used, for example, in DNA sequencing or RNA sequencing.

Abstract: Compositions comprising covalently modified and mutated biotin-binding proteins, particularly biotin-binding proteins having a negative charge at physiological pH, are provided. Methods of producing such proteins are also provided, as are methods of immobilizing, sequencing, and making nucleic acids employing such proteins.

Abstract: Arrays of integrated analytical devices and their methods for production are provided. The arrays are useful in the analysis of highly multiplexed optical reactions in large numbers at high densities, including biochemical reactions, such as nucleic acid sequencing reactions. The devices allow the highly sensitive discrimination of optical signals using features such as spectra, amplitude, and time resolution, or combinations thereof. The devices include an integrated diffractive beam shaping element that provides for the spatial separation of light emitted from the optical reactions.

Abstract: A circuit comprising a substrate with sectors on the substrate is provided, each sector comprising clock and data lines, a controller in electrical communication with the clock and data lines, a counter bias line, an amplifier input line and nano-electronic measurement devices on the substrate. A source of each device is coupled to the counter bias line and a drain of each device is coupled to the amplifier input line to obtain an electrical signal on the drain, the identity of which is determined by electrical interaction between the device and a charge label. Each device drain is gated by a corresponding switch between an on state, in which the drain is connected to the amplifier input line, and an off state, in which the drain is isolated from the amplifier input line. The controller controls switch states responsive to clock signal line pulses and data input line data.

Abstract: Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions, and/or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing. Such properties can include enhanced metal ion coordination, reduced exonuclease activity, reduced reaction rates at one or more steps of the polymerase kinetic cycle, decreased branching fraction, altered cofactor selectivity, increased yield, increased thermostability, increased accuracy, increased speed, increased readlength, and the like. Also provided are nucleic acids which encode the polymerases with the aforementioned phenotypes, as well as methods of using such polymerases to make a DNA or to sequence a DNA template.

Abstract: Compositions, methods, and systems are provided for fluorescent polymerase enzyme substrates comprising protein shields for improving enzyme photostability in single molecule real time sequencing. Fluorescent polymerase enzyme substrates of the invention have a protein shield between the fluorescent dye moieties and nucleotide moieties of the polymerase enzyme substrate. The polymerase enzyme substrates have a nucleotide component and a dye component, each attached to a protein. The attachments can be covalent. The protein can, for example, prevent the direct interaction of the fluorescent dye moiety with the enzyme when carrying out nucleotide synthesis, preventing photodamage to the enzyme. The polymerase enzyme substrates of the invention can have multiple dyes and multiple nucleotide moieties.

Abstract: A method for determining the presence of an allele, including (a) binding a polymerase to a double stranded nucleic acid that includes a primer hybridized to a template, the template including a first allele of a locus; (b) adding a nucleotide to the primer via catalytic activity of the polymerase, thereby producing an extended nucleic acid; (c) dissociating the polymerase from the extended nucleic acid; (d) detecting dissociation of the polymerase from the extended nucleic acid; and (e) comparing the dissociation of the polymerase from the extended nucleic acid to dissociation of the polymerase from a second double stranded nucleic acid, the second double stranded nucleic acid including a primer hybridized to the same position of the locus as the primer of the extended nucleic acid.