Abstract: The Application relates to methods, and systems for nucleotide sequencing comprising producing polymerase reactions that comprise both catalytic and non-catalytic divalent metal ions. Polymerase/template/primer complexes are immobilized on a substrate. The complexes are exposed to a solution comprising a non-catalytic metal and nucleotides labeled with a detectable label on a portion of the nucleotide that is released upon incorporation. The cognate nucleotide is sequestered in the active site of the polymerase, unable to proceed to incorporation. After observing the sequestered cognate nucleotide, the complex is exposed to a catalytic metal, resulting in the incorporation of the bound cognate nucleotide and consequent release of the label resulting in a single-base extended primer.

Abstract: Compositions, systems and methods employing nucleic acid binding proteins for use in the regulation and/or modulation of nucleic acid based reactions, including transcription, translation, modification, digestion, and hybridization reactions. Such compositions are employed in controlling a variety of different reaction types involving nucleic acids.

Abstract: Provided are compositions comprising modified DNA polymerases that exhibit improved photostability compared to the parental polymerases from which they were derived. Provided are methods for generating enzymes, such as DNA polymerases, with the aforementioned phenotype. Provided are methods of using polymerases with increased resistance to photodamage to make a DNA or to sequence a DNA template.

Abstract: Compositions, methods, and systems are provided for nucleotide analogs comprising protein shields for improving enzyme photostability in single molecule real time sequencing. Nucleotide analogs of the invention have a protein shield between the dye moieties and nucleotide moieties of the analog. The nucleotide analogs have two avidin proteins connected by a central dye component, and each avidin protein is further connected to a nucleotide component. The protein can prevent the direct interaction of the dye moiety with the enzyme carrying out nucleotide synthesis preventing photodamage to the enzyme. The nucleotide analogs of the invention can have multiple dyes and multiple nucleotide moieties.

Abstract: Provided are compositions comprising modified DNA polymerases that exhibit improved photostability compared to the parental polymerases from which they were derived. Provided are methods for generating enzymes, such as DNA polymerases, with the aforementioned phenotype. Provided are methods of using polymerases with increased resistance to photodamage to make a DNA or to sequence a DNA template.

Abstract: Compositions, kits, methods and systems for single molecule nucleotide sequencing comprising producing polymerase reactions having lithium that control the median pulse width for incorporated nucleotides are disclosed. The levels of lithium are used to control pulse width while allowing other sequencing parameters to remain within a desirable range.

Abstract: Compositions and methods are provided for nucleotide analogs comprising protein shields for improving enzyme photostability in single molecule real time sequencing. Nucleotide analogs of the invention have a protein shield between the dye moieties and nucleotide moieties of the analog. The protein prevents the direct interaction of the dye moiety with the enzyme carrying out nucleotide synthesis preventing photodamage to the enzyme. The nucleotide analogs of the invention can have multiple dyes and multiple nucleotide moieties.

Abstract: Provided are methods for sequencing a nucleic acid with a sequencing enzyme, e.g., a polymerase or exonuclease. The sequencing enzyme can optionally be exchanged with a second sequencing enzyme, which continues the sequencing of the nucleic acid. In certain embodiments, a template is fixed to a surface through a template localizing moiety. The template localizing moiety can optionally anneal with the nucleic acid and/or associate with the sequencing enzyme. Also provided are compositions comprising a nucleic acid and a first sequencing enzyme, which can sequence the nucleic acid and optionally exchange with a second sequencing enzyme present in the composition. Compositions in which a template localizing moiety is immobilized on a surface are provided. Also provided are methods for using data from analytical reactions wherein two different enzymes are employed, e.g., at a same or different reaction regions.

Abstract: Compositions, kits, methods and systems for single molecule nucleotide sequencing comprising producing polymerase reactions having monovalent cations that control the median pulse width for incorporated nucleotides are disclosed. The levels of alkali metals such as lithium, sodium, potassium, rubidium, and cesium in the polymerization are used to control pulse width while allowing other sequencing parameters to remain within a desirable range.

Abstract: The invention relates to devices and methods for nanopore sequencing. The invention includes arrays of nanopores having incorporated electronic circuits, for example, in CMOS. In some cases, the arrays of nanopores comprise resistive openings for isolating the electronic signals for improved sequencing. Methods for controlling translocation of through the nanopore are disclosed.

Abstract: Real time electronic sequencing methods, devices, and systems are described. Arrays of nanoscale electronic elements comprising capacitive devices with one or two electrodes, or arrays of nanoFET devices are used to provide sequence information about a template nucleic acid in a polymerase-template complex bound proximate to the nanoscale electronic elements or to the substrate proximate to the nanoscale electronic element. A sequencing reaction mixture comprising nucleotide analogs having impedance labels, capacitive labels, or conductivity labels is introduced to the array of nanoscale electronic elements comprising capacitive devices or nanoFETs under conditions of polymerase mediated nucleic acid synthesis. The time sequence of incorporation of nucleotide analogs is determined by identifying the types of labels of the nucleotide analogs that are incorporated into the growing strand using measured impedance, conductivity, or capacitance.

Abstract: Multi-biotinylated reactants are provided which can be used in divalent complexes for various applications such as colocalization, labeling, immobilization, and purification. Methods for constructing, purifying, and using the bis-biotinylated reactants are also provided. In certain embodiments, two bis-biotinylated reactants are bound to a single streptavidin tetramer to provide a complex having a 1:1 stoichiometry with respect to the bis-biotinylated reactants.

Abstract: Provided are methods for sequencing a nucleic acid with a sequencing enzyme, e.g., a polymerase or exonuclease. The sequencing enzyme can optionally be exchanged with a second sequencing enzyme, which continues the sequencing of the nucleic acid. In certain embodiments, a template is fixed to a surface through a template localizing moiety. The template localizing moiety can optionally anneal with the nucleic acid and/or associate with the sequencing enzyme. Also provided are compositions comprising a nucleic acid and a first sequencing enzyme, which can sequence the nucleic acid and optionally exchange with a second sequencing enzyme present in the composition. Compositions in which a template localizing moiety is immobilized on a surface are provided. Also provided are methods for using data from analytical reactions wherein two different enzymes are employed, e.g., at a same or different reaction regions.

Abstract: Systems and methods are provided for single-molecule sequencing of template nucleic acids in which the signal from a label attached to a polymerase enzyme is used to monitor conformational changes in the polymerase which occur while labeled nucleotides or nucleotide analogs are added to a growing nucleic acid chain which is complementary to the template nucleic acid. The signal indicative of the conformational state of the enzyme is used to determine with higher confidence when true nucleotide or nucleotide analog incorporation events occur, allowing for the improved quality of base calls and sequence determination.

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 include increased resistance to photodamage, and can also 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: The application relates to methods, and systems for nucleotide sequencing comprising producing polymerase reactions that comprise both catalytic and non-catalytic divalent metal ions. Polymerase/template/primer complexes are immobilized on a substrate. The complexes are exposed to a solution comprising a non-catalytic metal and nucleotides labeled with a detectable label on a portion of the nucleotide that is released upon incorporation. The cognate nucleotide is sequestered in the active site of the polymerase, unable to proceed to incorporation. After observing the sequestered cognate nucleotide, the complex is exposed to a catalytic metal, resulting in the incorporation of the bound cognate nucleotide and consequent release of the label resulting in a single-base extended primer.

Abstract: The Application relates to compositions, kits, methods, and systems for nucleotide sequencing comprising producing polymerase reactions that comprise both catalytic and non-catalytic divalent metal ions. Effective ratios and amounts of catalytic and non-catalytic divalent metal ions are described.

Abstract: Compositions and methods are provided for nucleotide analogs comprising protein shields for improving enzyme photostability in single molecule real time sequencing. Nucleotide analogs of the invention have a protein shield between the dye moieties and nucleotide moieties of the analog. The protein prevents the direct interaction of the dye moiety with the enzyme carrying out nucleotide synthesis preventing photodamage to the enzyme. The nucleotide analogs of the invention can have multiple dyes and multiple nucleotide moieties.

Abstract: Provided are methods and compositions for the production of linear single-stranded nucleic acids, which can be used as templates in high-throughput sequencing systems. Also provided are methods and compositions for the production of closed single-stranded nucleic acid loops, which can be used as templates in high-throughput sequencing systems.

Abstract: Provided are methods for enhanced sequencing of nucleic acid templates. Also provided are reaction conditions that increase branching fractions during polymerization reactions. Also provided are compositions comprising modified recombinant polymerases that exhibit branching fractions that are higher than the branching fractions of the polymerases from which they were derived. Provided are compositions comprising modified recombinant polymerases that exhibit delayed translocation relative to the polymerases from which they were derived. Also provided are compositions comprising modified recombinant polymerases that exhibit increased nucleotide or nucleotide analog residence time at an active site of the polymerase. Provided are methods for generating polymerases with the aforementioned phenotypes and methods of using such polymerases to sequence a DNA template or make a DNA.