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.
G01N 21/77 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
G01N 21/78 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
G02B 6/12 - Light guidesStructural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type of the integrated circuit kind
G02B 6/132 - Integrated optical circuits characterised by the manufacturing method by deposition of thin films
G02B 6/136 - Integrated optical circuits characterised by the manufacturing method by etching
H10F 39/00 - Integrated devices, or assemblies of multiple devices, comprising at least one element covered by group , e.g. radiation detectors comprising photodiode arrays
2.
Recombinant Polymerases for Incorporation of Protein Shield Nucleotide Analogs
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.
Methods, compositions, and systems for distributing nucleic acids into array regions are provided. The methods, compositions, and systems utilize nucleic acid condensing agents to increase efficiency of distribution of the nucleic acids into the array regions. Various methods for facilitating distribution of the nucleic acids to the array regions are provided.
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.
G01N 21/77 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
G02B 6/024 - Optical fibres with cladding with polarisation-maintaining properties
G02B 6/10 - Light guidesStructural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type
G02B 6/12 - Light guidesStructural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type of the integrated circuit kind
G02B 6/122 - Basic optical elements, e.g. light-guiding paths
G02B 23/24 - Instruments for viewing the inside of hollow bodies, e.g. fibrescopes
G02F 1/365 - Non-linear optics in an optical waveguide structure
5.
PROCESS FOR COGNATE NUCLEOTIDE DETECTION IN A NUCLEIC ACID SEQUENCING WORKFLOW
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.
C07H 21/02 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
C07H 99/00 - Subject matter not provided for in other groups of this subclass
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.
C07H 19/207 - Purine radicals with the saccharide radical being esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine-adenine dinucleotide or nicotinamide-adenine dinucleotide
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
C09B 23/06 - Methine or polymethine dyes, e.g. cyanine dyes characterised by the methine chain containing an odd number of CH groups three CH groups, e.g. carbocyanines
C09B 23/08 - Methine or polymethine dyes, e.g. cyanine dyes characterised by the methine chain containing an odd number of CH groups more than three CH groups, e.g. polycarbocyanines
C09B 69/00 - Dyes not provided for by a single group of this subclass
C12Q 1/6876 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
7.
HIGH SPEED SCANNING SYSTEM WITH ACCELERATION TRACKING
Disclosed herein is a high throughput optical scanning device and methods of use. The optical scanning device and methods of use provided herein can allow high throughput scanning of a continuously moving object with a high resolution despite fluctuations in stage velocity. This can aid in high throughput scanning of a substrate, such as a biological chip comprising fluorophores. Also provided herein are improved optical relay systems and scanning optics.
G02B 27/64 - Imaging systems using optical elements for stabilisation of the lateral and angular position of the image
G01D 5/347 - Mechanical means for transferring the output of a sensing memberMeans for converting the output of a sensing member to another variable where the form or nature of the sensing member does not constrain the means for convertingTransducers not specially adapted for a specific variable using optical means, i.e. using infrared, visible or ultraviolet light with attenuation or whole or partial obturation of beams of light the beams of light being detected by photocells using displacement encoding scales
H04N 3/08 - Scanning details of television systemsCombination thereof with generation of supply voltages by optical-mechanical means only having a moving reflector
An analytical device including an optically opaque cladding, a sequencing layer including a substrate disposed below the cladding, and a waveguide assembly for receiving optical illumination and introducing illumination into the device. The illumination may be received from a top, a side edge, and a bottom of the device. The waveguide assembly may include a nanoscale aperture disposed in the substrate and extending through the cladding. The aperture defines a reaction cell for receiving a set of reactants. In various aspects, the device includes a sensor element and the illumination pathway is through the sensor element. Waveguides and illumination devices, such as plasmonic illumination devices, are also disclosed. Methods for forming and operating the devices are also disclosed.
G01N 21/75 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
G01N 21/77 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
G01N 33/543 - ImmunoassayBiospecific binding assayMaterials therefor with an insoluble carrier for immobilising immunochemicals
G02B 6/122 - Basic optical elements, e.g. light-guiding paths
9.
METHODS AND COMPOSITIONS FOR CAPPING NUCLEIC ACIDS
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.
Optics collection and detection systems are provided for measuring optical signals from an array of optical sources over time. Methods of using the optics collection and detection systems are also described.
G01N 21/75 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
G01N 21/77 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
G01N 33/543 - ImmunoassayBiospecific binding assayMaterials therefor with an insoluble carrier for immobilising immunochemicals
G02B 6/122 - Basic optical elements, e.g. light-guiding paths
Disclosed herein are methods and systems for detection and discrimination of optical signals from a densely packed substrate. There have broad applications for biomolecule detection near or below the diffraction limit of optical systems, including in improving the efficiency and accuracy or polynucleotide sequencing applications.
Methods, compositions, and systems are provided for the detection of 5-methylcytosine modifications in nucleic acid, particularly DNA, samples. A template or plurality of templates is sequenced using single molecule real time sequencing. Feature vectors are produced using specific features. In some aspects, a feature vector having a reduced feature set is provided and these feature vectors are input into a deep learning model.
This invention provides analytical devices for use in various applications, including detection of single-molecule analytical reactions. Methods for propagating optical energy within a substrate are provided. Devices comprising waveguide substrates, dielectric omnidirectional reflectors, and optical couplers are provided. Waveguide substrates with improved uniformity of optical energy intensity across one or more waveguides and enhanced waveguide illumination efficiency within an analytic detection region of the arrays are provided.
G01N 21/77 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
G01N 33/543 - ImmunoassayBiospecific binding assayMaterials therefor with an insoluble carrier for immobilising immunochemicals
15.
Labeled nucleotide analogs, reaction mixtures, and methods and systems for sequencing
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.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
C07H 19/10 - Pyrimidine radicals with the saccharide radical being esterified by phosphoric or polyphosphoric acids
C07H 19/207 - Purine radicals with the saccharide radical being esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine-adenine dinucleotide or nicotinamide-adenine dinucleotide
Disclosed herein are methods and systems for detection and discrimination of optical signals from a densely packed substrate. These have broad applications for biomolecule detection near or below the diffraction limit of optical systems, including in improving the efficiency and accuracy of polynucleotide sequencing applications.
Disclosed herein are methods and systems for detection and discrimination of optical signals from a densely packed substrate. These have broad applications for biomolecule detection near or below the diffraction limit of optical systems, including in improving the efficiency and accuracy of polynucleotide sequencing applications.
C12Q 1/6874 - Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation [SBH]
C12Q 1/689 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Reaction mixtures are provided having at least a first reactant and a second reactant that produce signals in response to excitation illumination. The signals produced by the reactants have peaks at the same wavelengths, but have distinct signal intensities. In some embodiments, the reactants are FRET-labeled.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
C07H 19/207 - Purine radicals with the saccharide radical being esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine-adenine dinucleotide or nicotinamide-adenine dinucleotide
C12Q 1/6818 - Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
G01N 33/542 - ImmunoassayBiospecific binding assayMaterials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
G01N 33/58 - Chemical analysis of biological material, e.g. blood, urineTesting involving biospecific ligand binding methodsImmunological testing involving labelled substances
19.
RECOMBINANT POLYMERASES FOR INCORPORATION OF PROTEIN SHIELD NUCLEOTIDE ANALOGS
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.
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.
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.
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.
Methods and systems for single molecule sequencing using concatemers of copies of sense and antisense strands. Concatemers are provided, for example, by carrying out rolling circle amplification on a circular molecule having sense and antisense regions to produce repeated copies of the sense and antisense regions connected by linking regions. The circular molecules can be produced by ligating hairpin adapters to each end of a double-stranded nucleic acid having a sense and antisense strand. The ligations can be carried out, for example using blunt end ligation. In some cases, a single molecule consensus sequence for a single template molecule is obtained. A single read from each template molecule can be obtained by comparing the sequence information of the sense and antisense regions.
C12Q 1/689 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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.
C12Q 1/6848 - Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
C07K 14/36 - Peptides having more than 20 amino acidsGastrinsSomatostatinsMelanotropinsDerivatives thereof from bacteria from ActinomycesPeptides having more than 20 amino acidsGastrinsSomatostatinsMelanotropinsDerivatives thereof from bacteria from Streptomyces (G)
Integrated target waveguide devices and optical analytical systems comprising such devices are provided. The target devices include an optical coupler that is optically coupled to an integrated waveguide and that is configured to receive optical input from an optical source through free space, particularly through a low numerical aperture interface. The devices and systems 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 provide for the efficient and reliable coupling of optical excitation energy from an optical source to the optical reactions. Optical signals emitted from the reactions can thus be measured with high sensitivity and discrimination. The devices and systems are well suited for miniaturization and high throughput.
G01N 21/77 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
G02B 6/124 - Geodesic lenses or integrated gratings
G02B 6/12 - Light guidesStructural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type of the integrated circuit kind
G02B 6/42 - Coupling light guides with opto-electronic elements
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.
C07H 19/10 - Pyrimidine radicals with the saccharide radical being esterified by phosphoric or polyphosphoric acids
C07H 19/207 - Purine radicals with the saccharide radical being esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine-adenine dinucleotide or nicotinamide-adenine dinucleotide
The invention provides a novel class of cyanine dyes that are functionalized with sulfonic acid groups and a linker moiety that facilitates their conjugation to other species and substituent groups which increase the water-solubility, and optimize the optical properties of the dyes. Also provided are conjugates of the dyes, methods of using the dyes and their conjugates and kits including the dyes and their conjugates.
C07D 209/14 - Radicals substituted by nitrogen atoms, not forming part of a nitro radical
C07D 401/14 - Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
C07D 405/14 - Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
C07H 19/10 - Pyrimidine radicals with the saccharide radical being esterified by phosphoric or polyphosphoric acids
C07K 5/09 - Tripeptides the side chain of the first amino acid containing more amino groups than carboxyl groups, or derivatives thereof, e.g. Lys, Arg
C07K 5/11 - Tetrapeptides the side chain of the first amino acid containing more amino groups than carboxyl groups, or derivatives thereof, e.g. Lys, Arg
C09B 23/01 - Methine or polymethine dyes, e.g. cyanine dyes characterised by the methine chain
C09B 23/06 - Methine or polymethine dyes, e.g. cyanine dyes characterised by the methine chain containing an odd number of CH groups three CH groups, e.g. carbocyanines
C09B 23/08 - Methine or polymethine dyes, e.g. cyanine dyes characterised by the methine chain containing an odd number of CH groups more than three CH groups, e.g. polycarbocyanines
C09B 23/10 - Methine or polymethine dyes, e.g. cyanine dyes characterised by the methine chain containing an even number of CH groups
C09B 23/16 - Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing hetero atoms
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.
This invention provides devices for use in various analytical applications including single-molecule analytical reactions. Methods for detecting analytes optically by propagating optical energy by waveguides within a substrate are provided. Analytical devices are provided which have both shallow and deep waveguides in which illumination light is transported through the deep waveguides and coupled into the shallow waveguides. The shallow waveguides provide evanescent field illumination to analytes, such as single-molecule analytes, within nanometer scale wells. Integrated devices including integrated detectors such as CMOS detectors are included.
C12Q 1/6874 - Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation [SBH]
G01N 21/77 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
G01N 33/543 - ImmunoassayBiospecific binding assayMaterials therefor with an insoluble carrier for immobilising immunochemicals
30.
DIGITAL ANALYSIS OF MOLECULAR ANALYTES USING SINGLE MOLECULE DETECTION
Methods and systems are provided for small molecule analyte detection using digital signals, key encryption, and communications protocols. The methods provide detection of a large numbers of proteins, peptides, RNA molecules, and DNA molecules in a single optical or electrical detection assay within a large dynamic range.
Disclosed herein are methods and systems for detection and discrimination of optical signals from a densely packed substrate. These have broad applications for biomolecule detection near or below the diffraction limit of optical systems, including in improving the efficiency and accuracy of polynucleotide sequencing applications.
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.
C07H 19/207 - Purine radicals with the saccharide radical being esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine-adenine dinucleotide or nicotinamide-adenine dinucleotide
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
C09B 23/06 - Methine or polymethine dyes, e.g. cyanine dyes characterised by the methine chain containing an odd number of CH groups three CH groups, e.g. carbocyanines
C09B 23/08 - Methine or polymethine dyes, e.g. cyanine dyes characterised by the methine chain containing an odd number of CH groups more than three CH groups, e.g. polycarbocyanines
C09B 69/00 - Dyes not provided for by a single group of this subclass
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.
Methods, compositions, and systems are provided that allow for reliable sequencing of the initial sequence region of a sequence of interest. The methods of the invention allow for more reliable barcoding of subpopulations of nucleic acids to be sequenced.
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.
Disclosed herein are methods of detecting at least one target biomolecule in at least one single cell comprising lysing the single cell or cells and performing a cell identification assay and target identification assay. Also disclosed herein are methods for preparing a sample for undergoing single cell analysis, wherein the single cell analysis comprises performing a cell identification assay and a target identification assay.
Disclosed herein are methods and systems for detection and discrimination of optical signals from a densely packed substrate. These have broad applications for biomolecule detection near or below the diffraction limit of optical systems, including in improving the efficiency and accuracy of polynucleotide sequencing applications.
Disclosed herein are methods and systems for detection and discrimination of optical signals from a densely packed substrate. These methods and systems may have broad applications for biomolecule detection near or below the diffraction limit of optical systems, including in improving the efficiency and accuracy of polynucleotide sequencing applications.
The invention relates to methods and compositions for the detection and quantification of nucleotide sequence variants, such as genetic polymorphisms, with decreased error and increased sensitivity, including single molecule detection. Detection of genetic polymorphisms, including single nucleotide polymorphisms (SNPs), is highly useful for the study of physiology, disease, phylogeny and forensics. Current methods for the detection and identification of nucleic acid sequence variants, such as genetic polymorphisms, lack the sensitivity to accurately detect low incidence mutations, sequence variants or alleles. Detection techniques for highly multiplexed single molecule identification and quantification of analytes using optical systems are disclosed. Analytes include, but are not limited to, nucleic acid, such as DNA and RNA molecules, with and without modifications. Techniques described herein include use of specific and non-specific probes complementary to nucleic acids of interest for detailed characterization of nucleotide sequence variants and highly multiplexed single molecule identification and quantification.
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.
G01N 21/77 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
G02B 6/024 - Optical fibres with cladding with polarisation-maintaining properties
G02B 6/10 - Light guidesStructural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type
G02B 6/12 - Light guidesStructural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type of the integrated circuit kind
G02B 6/122 - Basic optical elements, e.g. light-guiding paths
G02F 1/365 - Non-linear optics in an optical waveguide structure
B82Y 15/00 - Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
B82Y 20/00 - Nanooptics, e.g. quantum optics or photonic crystals
G02B 23/24 - Instruments for viewing the inside of hollow bodies, e.g. fibrescopes
41.
METHODS AND COMPOSITIONS FOR LOADING OF POLYMERASE COMPLEXES
The present disclosure provides methods, compositions, and systems for distributing polymerase compositions into array regions. In particular, the described methods, compositions, and systems utilize density differentials and/or additives to increase efficiency in the distribution of polymerase compositions to a surface as compared to methods utilizing only diffusion control.
Methods, compositions, and systems for distributing nucleic acids into array regions are provided. The methods, compositions, and systems utilize nucleic acid condensing agents to increase efficiency of distribution of the nucleic acids into the array regions. Various methods for facilitating distribution of the nucleic acids to the array regions are provided.
Disclosed herein is a high throughput optical scanning device and methods of use. The optical scanning device and methods of use provided herein can allow high throughput scanning of a continuously moving object with a high resolution despite fluctuations in stage velocity. This can aid in high throughput scanning of a substrate, such as a biological chip comprising fluorophores. Also provided herein are improved optical relay systems and scanning optics.
H04N 5/232 - Devices for controlling television cameras, e.g. remote control
G01D 5/347 - Mechanical means for transferring the output of a sensing memberMeans for converting the output of a sensing member to another variable where the form or nature of the sensing member does not constrain the means for convertingTransducers not specially adapted for a specific variable using optical means, i.e. using infrared, visible or ultraviolet light with attenuation or whole or partial obturation of beams of light the beams of light being detected by photocells using displacement encoding scales
G02B 27/64 - Imaging systems using optical elements for stabilisation of the lateral and angular position of the image
H04N 3/08 - Scanning details of television systemsCombination thereof with generation of supply voltages by optical-mechanical means only having a moving reflector
Disclosed herein are methods and systems for detection and discrimination of optical signals from a densely packed substrate. There have broad applications for biomolecule detection near or below the diffraction limit of optical systems, including in improving the efficiency and accuracy or polynucleotide sequencing applications.
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.
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
B01J 20/10 - Solid sorbent compositions or filter aid compositionsSorbents for chromatographyProcesses for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
46.
Labeled nucleotide analogs, reaction mixtures, and methods and systems for sequencing
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.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
C07H 19/10 - Pyrimidine radicals with the saccharide radical being esterified by phosphoric or polyphosphoric acids
C07H 19/207 - Purine radicals with the saccharide radical being esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine-adenine dinucleotide or nicotinamide-adenine dinucleotide
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.
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
C07K 14/36 - Peptides having more than 20 amino acidsGastrinsSomatostatinsMelanotropinsDerivatives thereof from bacteria from ActinomycesPeptides having more than 20 amino acidsGastrinsSomatostatinsMelanotropinsDerivatives thereof from bacteria from Streptomyces (G)
C09B 23/08 - Methine or polymethine dyes, e.g. cyanine dyes characterised by the methine chain containing an odd number of CH groups more than three CH groups, e.g. polycarbocyanines
C09B 69/10 - Polymeric dyesReaction products of dyes with monomers or with macromolecular compounds
A method for determining sequences from sense and antisense strands of a nucleic acid, including (a) providing a nucleic acid cluster attached to a solid support, wherein the nucleic acid cluster includes a sense strand and an antisense strand of a concatemer, the concatemer including multiple copies of a sequence unit, the sequence unit including a target sequence and a primer binding site; (b) hybridizing a primer to a primer binding site in the antisense strand; (c) extending the primer along the antisense strand to determine the sequence from at least a portion of the target sequence in the antisense strand; (d) hybridizing a second primer to a primer binding site in the sense strand; and (e) extending the second primer along the sense strand to determine the sequence from at least a portion of the target sequence in the sense strand.
Provided herein is a method of performing a nucleic acid sequencing reaction using a sequencing system comprising a reagent cartridge coupled to a detection apparatus, and a pump coupled to the reagent cartridge. The reagent cartridge includes a plurality of reagent reservoirs and a main channel. The detection apparatus includes a flow cell that is coupled to the reagent cartridge when the reagent cartridge interacts transiently with the detection apparatus.
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.
G01N 21/77 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
G01N 21/78 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
G02B 6/132 - Integrated optical circuits characterised by the manufacturing method by deposition of thin films
G02B 6/136 - Integrated optical circuits characterised by the manufacturing method by etching
G02B 6/12 - Light guidesStructural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type of the integrated circuit kind
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.
Disclosed herein are methods and systems for detection and discrimination of optical signals from a densely packed substrate. These may have broad applications for biomolecule detection near or below the diffraction limit of optical systems, including in improving the efficiency and accuracy of polynucleotide sequencing applications.
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.
Protected fluorescent reagent compounds and their methods of synthesis are provided. The compounds 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 compounds contain fluorescent dye elements, that allow the compounds to be detected with high sensitivity at desirable wavelengths, binding elements, that allow the compounds to be recognized specifically by target biomolecules, and protective shield elements, that decrease undesirable contacts between the fluorescent dye elements and the bound target biomolecules and that therefore decrease photodamage of the bound target biomolecules by the fluorescent dye elements.
C07H 19/10 - Pyrimidine radicals with the saccharide radical being esterified by phosphoric or polyphosphoric acids
C07F 9/6558 - Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
C07F 9/6561 - Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
C09B 23/06 - Methine or polymethine dyes, e.g. cyanine dyes characterised by the methine chain containing an odd number of CH groups three CH groups, e.g. carbocyanines
C09B 23/08 - Methine or polymethine dyes, e.g. cyanine dyes characterised by the methine chain containing an odd number of CH groups more than three CH groups, e.g. polycarbocyanines
C09B 69/10 - Polymeric dyesReaction products of dyes with monomers or with macromolecular compounds
Provided herein include a method for modifying polymerase-nucleic acid complexes, including (a) providing a plurality of surface-immobilized polymerase-nucleic acid complexes in a vessel, wherein the nucleic acid includes a primed-template nucleic acid, wherein at least a subset of the surface-immobilized polymerase-nucleic acid complexes include ternary complexes further including nucleotides; and (b) washing the surface with an aqueous solution including a diol, sulfoxide or polyol, thereby removing the nucleotides from the vessel and retaining the surface-immobilized polymerase-nucleic acid complexes in the vessel.
Optics collection and detection systems are provided for measuring optical signals from an array of optical sources over time. Methods of using the optics collection and detection systems are also described.
G01N 21/75 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
G01N 21/77 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
G01N 33/543 - ImmunoassayBiospecific binding assayMaterials therefor with an insoluble carrier for immobilising immunochemicals
G02B 6/122 - Basic optical elements, e.g. light-guiding paths
57.
METHODS AND COMPOSITIONS FOR DELIVERY OF MOLECULES AND COMPLEXES TO REACTION SITES
The present invention provides methods, compositions, and systems for distributing molecules and complexes into reaction sites. In particular, the methods, compositions, and systems of the present invention result in an active loading of molecules and complexes into reaction sites with improved efficiency over loading by passive diffusion methods alone.
A method of determining a nucleic acid sequence that includes steps of: (a) contacting a primed template nucleic acid with a series of mixtures for forming ternary complexes, wherein each of the mixtures includes a polymerase and nucleotide cognates for at least two different base types suspected of being present at the next template position of the template nucleic acid; (b) monitoring the next template position for ternary complexes formed by the series of mixtures, wherein a signal state indicates presence or absence of ternary complex formed at the next template position by each individual mixture, thereby determining a series of signal states that encodes a base call for the next template position; and (c) decoding the series of signal states to distinguish a correct base call for the next template position from an error in the base call.
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.
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 integrated illumination elements and 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.
G01N 21/77 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
Provided herein are systems, devices, and methods for improved optical waveguide transmission and alignment in an analytical system. Waveguides in optical analytical systems can exhibit variable and increasing back reflection of single-wavelength illumination over time, thus limiting their effectiveness and reliability. The systems are also subject to optical interference under conditions that have been used to overcome the back reflection. Novel systems and approaches using broadband illumination light with multiple longitudinal modes have been developed to improve optical transmission and analysis in these systems. Novel systems and approaches for the alignment of a target waveguide device and an optical source are also disclosed.
G02B 6/12 - Light guidesStructural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type of the integrated circuit kind
G01N 21/77 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
G02B 6/122 - Basic optical elements, e.g. light-guiding paths
G02B 6/124 - Geodesic lenses or integrated gratings
G02B 6/42 - Coupling light guides with opto-electronic elements
G02B 27/09 - Beam shaping, e.g. changing the cross-sectioned area, not otherwise provided for
G02B 6/34 - Optical coupling means utilising prism or grating
62.
Recombinant polymerases with increased phototolerance
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.
A flow cell that includes (a) a gasket interposed between a first substrate and a second substrate, wherein the gasket, the first substrate and the second substrate are impermeable to aqueous liquid and liquid adhesive, wherein the gasket has a footprint on the first substrate that delineates a channel for containing the aqueous liquid; (b) a via in the gasket, the via containing a solidified liquid adhesive that bonds the first substrate to the second substrate, wherein the solidified liquid adhesive in the via is separated from the channel by the gasket; and (c) a channel port connecting the channel to the exterior of the flow cell, wherein the channel port is permeable to the aqueous liquid.
Provided herein are methods of purifying a sample containing nucleic acids to obtain isolated nucleic acids of a desired size range and methods of sequencing nucleic acids of a desired size range. The methods include a) combining a nucleic acid-containing sample with a precipitation buffer in a container to provide a precipitation mixture in which the precipitation buffer comprises water, a buffer, a salt, and polyvinyl pyrrolidinone (PVP) and/or Ficoll. The methods also include precipitating the nucleic acids in the precipitation mixture to provide a precipitated nucleic acid portion and a remaining sample portion. The precipitated nucleic acid portion predominantly comprises nucleic acid molecules above a selected size cutoff value and the remaining sample portion predominantly comprises nucleic acid molecules below the selected size cutoff value. The methods also include separating the precipitated nucleic acid portion from the remaining sample portion. Related compositions and kits are also provided herein.
Arrays of integrated analytical devices 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. In particular, the arrays provide increased efficiency of optical collection and decreased background signal as the lateral dimensions of the unit cell of devices within the array are decreased, for example as they are decreased to 2 μm, or even less.
The present invention provides methods and compositions for carrying out nucleic acid sequencing, particularly paired-end sequencing. The methods use concatemeric sequencing templates that can be produced by rolling circle amplification of asymmetric circular nucleic acids having a central double-stranded region comprising a target nucleic acid sequence that is connected at each end to form a circular construct.
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.
C09B 69/10 - Polymeric dyesReaction products of dyes with monomers or with macromolecular compounds
C07H 19/10 - Pyrimidine radicals with the saccharide radical being esterified by phosphoric or polyphosphoric acids
C07H 19/207 - Purine radicals with the saccharide radical being esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine-adenine dinucleotide or nicotinamide-adenine dinucleotide
Disclosed herein are methods and systems for storing data and/or information on nucleic acid molecules, storing the nucleic acid molecules, and retrieving the data and/or information. These methods and systems have broad applications for data storage, including in improving the efficiency and accuracy of retrieving data.
A method for identifying a nucleotide in a primed template nucleic acid, including the steps of (a) providing a vessel having a primed template nucleic acid, polymerase and a nucleotide cognate of a first base type; (b) examining the vessel for a stabilized ternary complex including the polymerase and the nucleotide cognate of the first base type bound at a base position of the primed template nucleic acid; (c) delivering a nucleotide cognate of a second base type to the vessel, whereby the vessel retains the primed template nucleic acid and the polymerase from step (b); (d) examining the vessel for a stabilized ternary complex including the polymerase and the nucleotide cognate of the second base type bound at the base position of the primed template nucleic acid; and (e) identifying the type of nucleotide at the base position of the primed template nucleic acid.
The present invention provides methods, compositions, and systems for distributing molecules and complexes into reaction sites. In particular, the methods, compositions, and systems of the present invention result in loading of polymerase enzyme complexes into a predetermined number of reaction sites, including nanoscale wells.
Provided include methods, compositions, kits, and systems for replenishing a rolling circle amplification (RCA) reaction in a vessel. The RCA reaction can be initiated by contacting a nucleic acid template and a primer with a loading buffer comprising a DNA polymerase and polymerase extension agents including a divalent metal cation and a polyelectrolyte, followed by replenishing with an amplification buffer to continue the nucleic acid amplification through primer extension. The amplification buffer is different in composition from the loading buffer and does not comprise any DNA polymerase.
C12Q 1/6848 - Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
C12Q 1/6853 - Nucleic acid amplification reactions using modified primers or templates
72.
SINGLE-MOLECULE SEEDING AND AMPLIFICATION ON A SURFACE
Provided includes methods, compositions and systems for single molecule seeding and amplification on a flow cell. In some embodiments, nucleic acids are isothermally seeded and amplified on a flow cell comprising multiple binding areas (e.g., pads), resulting in an ensemble of substantially the same amplified molecules on each of the binding areas.
A method for determining sequences from sense and antisense strands of a nucleic acid, including (a) providing a nucleic acid cluster attached to a solid support, wherein the nucleic acid cluster includes a sense strand and an antisense strand of a concatemer, the concatemer including multiple copies of a sequence unit, the sequence unit including a target sequence and a primer binding site; (b) hybridizing a primer to a primer binding site in the antisense strand; (c) extending the primer along the antisense strand to determine the sequence from at least a portion of the target sequence in the antisense strand; (d) hybridizing a second primer to a primer binding site in the sense strand; and (e) extending the second primer along the sense strand to determine the sequence from at least a portion of the target sequence in the sense strand.
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.
Disclosed herein include methods of specifying sites (e.g., sites for colony formation) on a surface (e.g., a planar surface) and generating a flow cell having the sites specified on a surface. Also disclosed are methods of performing sequencing (e.g., sequencing-by-synthesis and sequencing-by-binding) using the flow cell generated and processing (e.g., aligning, orienting, sorting, and assessing quality) images of the flow cell captured during sequencing.
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.
C07H 19/207 - Purine radicals with the saccharide radical being esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine-adenine dinucleotide or nicotinamide-adenine dinucleotide
G01N 33/542 - ImmunoassayBiospecific binding assayMaterials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
G01N 33/58 - Chemical analysis of biological material, e.g. blood, urineTesting involving biospecific ligand binding methodsImmunological testing involving labelled substances
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.
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.
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.
Methods are provided for detecting a single compound analyte immobilized to a solid substrate by serially contacting and removing different probes to the same analyte.
H03M 13/15 - Cyclic codes, i.e. cyclic shifts of codewords produce other codewords, e.g. codes defined by a generator polynomial, Bose-Chaudhuri-Hocquenghem [BCH] codes
G16B 25/00 - ICT specially adapted for hybridisationICT specially adapted for gene or protein expression
G16B 40/00 - ICT specially adapted for biostatisticsICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
82.
Digital analysis of molecular analytes using electrical methods
Electrical detection methods are used to identify and further characterize single-molecule target analytes such as proteins and nucleic acids. A composition including a probe region and a tail region is contacted with a target analyte. The probe region specifically binds to the target analyte. The tail region is coupled to the probe region, and includes a nucleic acid template for polynucleotide synthesis. When conditions are such that polynucleotide synthesis occurs along the tail region, one hydrogen ion is released for every nucleotide that is incorporated into the tail region. A transistor such as an ISFET detects and measures changes in ion concentration, and these measurements can be used to identify the tail region and thus characterize the corresponding target analyte.
C12Q 1/6804 - Nucleic acid analysis using immunogens
C40B 20/04 - Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
83.
High speed scanning systems for super resolution imaging
Disclosed herein is a high throughput optical scanning system to generate super resolution images and methods of use. The optical scanning device and methods of use provided herein can allow high throughput scanning of a continuously moving object with a high resolution despite fluctuations in stage velocity. This can aid in high throughput scanning of a substrate, such as a biological chip comprising fluorophores. Also provided herein are improved optical relay systems and scanning optics.
G02B 27/58 - Optics for apodization or superresolutionOptical synthetic aperture systems
G06T 3/4053 - Scaling of whole images or parts thereof, e.g. expanding or contracting based on super-resolution, i.e. the output image resolution being higher than the sensor resolution
G06T 7/70 - Determining position or orientation of objects or cameras
H04N 23/55 - Optical parts specially adapted for electronic image sensorsMounting thereof
H04N 23/56 - Cameras or camera modules comprising electronic image sensorsControl thereof provided with illuminating means
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.
G02B 6/024 - Optical fibres with cladding with polarisation-maintaining properties
F21V 8/00 - Use of light guides, e.g. fibre optic devices, in lighting devices or systems
G01N 21/77 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
G02B 6/12 - Light guidesStructural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type of the integrated circuit kind
G02F 1/365 - Non-linear optics in an optical waveguide structure
G02B 6/10 - Light guidesStructural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type
G02B 6/122 - Basic optical elements, e.g. light-guiding paths
B82Y 15/00 - Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
B82Y 20/00 - Nanooptics, e.g. quantum optics or photonic crystals
G02B 23/24 - Instruments for viewing the inside of hollow bodies, e.g. fibrescopes
Sets of compounds bearing detectably different groups of labels are provided. Typically, different compounds bear different numbers of a single type of label and are thus distinguishable by the amplitude of signal produced by the label. The compounds are assembled from label components and protein cores to facilitate modular production of the compounds. In compounds containing two or more proteins, the proteins are typically covalently linked. Useful sets of compounds include sets of labeled nucleotide analogs, particularly dye-label nucleotide analogs that include tetravalent biotin-binding protein cores.
Methods, devices, and systems for performing intermittent detection during analytical reactions are provided. Such methods facilitate collection of reaction data from disparate reaction times. Further, such methods are useful for reducing photo-induced damage of one or more reactants in an illuminated analytical reaction at a given reaction time. In preferred embodiments, the reaction mixture is subjected to at least one illuminated and non-illuminated period and allowed to proceed such that the time in which the reaction mixture is illuminated is less than a photo-induced damage threshold period.
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.
Disclosed herein, inter alia, are methods for modifying a nucleotide, for example including reacting a nucleotide having a 3′-O-oxime moiety such as
Disclosed herein, inter alia, are methods for modifying a nucleotide, for example including reacting a nucleotide having a 3′-O-oxime moiety such as
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
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
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.
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.
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.
Real time electronic sequencing devices, chips, and systems are described. Arrays of nanoFET devices are used to provide sequence information about a template nucleic acid in a polymerase-template complex bound to the nanoFET. The nanoFET devices typically have a source, a drain and a gate comprising a nanowire. A single polymerase enzyme complex comprising a polymerase enzyme complexed with the template nucleic acid is bound to the gate. The polymerase is bound to the gate non-covalently through a polymeric binding agent that has two strands, each strand interacting with the nanowire such that the polymerase is in a central location between the strands with the polymeric binding agent extending away from the polymerase complex along the nanowire in both directions.
B82Y 10/00 - Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
B82Y 15/00 - Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
C12Q 1/6874 - Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation [SBH]
G01N 27/414 - Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
G11C 19/28 - Digital stores in which the information is moved stepwise, e.g. shift registers using semiconductor elements
H01L 29/06 - Semiconductor bodies characterised by the shapes, relative sizes, or dispositions of the semiconductor regions
H01L 29/16 - Semiconductor bodies characterised by the materials of which they are formed including, apart from doping materials or other impurities, only elements of Group IV of the Periodic System in uncombined form
92.
Hierarchical silica lamella for magnetic nucleic acid extractions
Disclosed herein is a novel method to fabricate magnetic silica nanomembranes using thin polymer cores based on silica deposition and self-wrinkling induced by thermal shrinkage. These micro- and nano-scale structures have vastly enlarged the specific area of silica, thus the magnetic silica nanomembranes can be used for solid phase extraction of nucleic acids. The magnetic silica nanomembranes are suitable for nucleic acid purification and isolation and demonstrated better performance than commercial particles in terms of nucleic acid recovery yield and integrity. In addition, the magnetic silica nanomembranes may have high nucleic acid capacity due to significantly enlarged specific surface area of silica. Methods of use and devices comprising the magnetic silica nanomembranes are also provided herein.
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.
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.
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.
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
G01N 33/58 - Chemical analysis of biological material, e.g. blood, urineTesting involving biospecific ligand binding methodsImmunological testing involving labelled substances
C07K 14/36 - Peptides having more than 20 amino acidsGastrinsSomatostatinsMelanotropinsDerivatives thereof from bacteria from ActinomycesPeptides having more than 20 amino acidsGastrinsSomatostatinsMelanotropinsDerivatives thereof from bacteria from Streptomyces (G)
C12Q 1/6874 - Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation [SBH]
C09B 23/08 - Methine or polymethine dyes, e.g. cyanine dyes characterised by the methine chain containing an odd number of CH groups more than three CH groups, e.g. polycarbocyanines
C09B 69/10 - Polymeric dyesReaction products of dyes with monomers or with macromolecular compounds
An analytical device including an optically opaque cladding, a sequencing layer including a substrate disposed below the cladding, and a waveguide assembly for receiving optical illumination and introducing illumination into the device. The illumination may be received from a top, a side edge, and a bottom of the device. The waveguide assembly may include a nanoscale aperture disposed in the substrate and extending through the cladding. The aperture defines a reaction cell for receiving a set of reactants. In various aspects, the device includes a sensor element and the illumination pathway is through the sensor element. Waveguides and illumination devices, such as plasmonic illumination devices, are also disclosed. Methods for forming and operating the devices are also disclosed.
G01N 21/75 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
G01N 21/77 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
G01N 33/543 - ImmunoassayBiospecific binding assayMaterials therefor with an insoluble carrier for immobilising immunochemicals
G02B 6/122 - Basic optical elements, e.g. light-guiding paths
97.
Process for cognate nucleotide detection in a nucleic acid sequencing workflow
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.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
C07H 21/02 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
C07H 99/00 - Subject matter not provided for in other groups of this subclass
C12Q 1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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.
C12Q 1/6853 - Nucleic acid amplification reactions using modified primers or templates
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
C12Q 1/6827 - Hybridisation assays for detection of mutation or polymorphism
C12Q 1/6883 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
C12Q 1/6872 - Methods for sequencing involving mass spectrometry
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.
G01N 21/78 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
G01N 21/77 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
G02B 6/132 - Integrated optical circuits characterised by the manufacturing method by deposition of thin films
G02B 6/136 - Integrated optical circuits characterised by the manufacturing method by etching
G02B 6/12 - Light guidesStructural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type of the integrated circuit kind
100.
Digital analysis of molecular analytes using electrical methods
Electrical detection methods are used to identify and further characterize single-molecule target analytes such as proteins and nucleic acids. A composition including a probe region and a tail region is contacted with a target analyte. The probe region specifically binds to the target analyte. The tail region is coupled to the probe region, and includes a nucleic acid template for polynucleotide synthesis. When conditions are such that polynucleotide synthesis occurs along the tail region, one hydrogen ion is released for every nucleotide that is incorporated into the tail region. A transistor such as an ISFET detects and measures changes in ion concentration, and these measurements can be used to identify the tail region and thus characterize the corresponding target analyte.
C12Q 1/6804 - Nucleic acid analysis using immunogens
C40B 20/04 - Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
patents
