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.
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.
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 are highly multiplexed optical analytical systems for improved nucleic acid sequencing. The systems include a plurality of highly multiplexed optical chips, at least one optical source, and a plurality of optical delivery devices for illuminating an array of nanoscale rection regions on each of the optical chips. In use, the reaction regions contain fluorescent nucleic acid sequencing reagents and are arranged to report nucleic acid sequence information to optical detectors associated with the multiplexed optical chips in real time. The systems enable a massive increase in the scale of nucleic acid sequencing reactions capable of being performed within a single instrument without a corresponding increase in size, complexity, or cost of the instrument.
G02B 6/12 - Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type of the integrated circuit kind
C12Q 1/6874 - Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation [SBH]
G01N 21/00 - Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
G01N 21/17 - Systems in which incident light is modified in accordance with the properties of the material investigated
G01N 21/63 - Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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
5.
RAPID PRECIPITATION-DRIVEN KILOBASE SIZE SELECTION OF HMW DNA
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.
B01J 20/10 - Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
C12Q 1/66 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
C23C 8/06 - Solid state diffusion of only non-metal elements into metallic material surfaces; Chemical surface treatment of metallic material by reaction of the surface with a reactive gas, leaving reaction products of surface material in the coating, e.g. conversion coatings, passivation of metals using gases
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.
Modified template switching oligonucleotides (TSOs), compositions containing modified TSOs, and methods for employing modified TSOs to synthesize cDNA from RNA templates, where the cDNA includes an adapter region at the 3' end, are provided. The modified TSOs include at least one 2'-fluoro-ribonucleotide in the 3' annealing region and provide for improved conversion of RNA into full-length cDNA, resulting in increased yield and complexity as compared to non-modified TSOs and thereby finding use in generating cDNA from samples having low RNA input.
The present disclosure provides, inter alia, methods, compositions, and computer implemented processes for resolving long and highly similar, but non-identical, genomic regions to improve assembly quality, especially for polyploid genomes. Aspects of the disclosure are draw to using exact string matching of homopolymer-collapsed sequence reads to determine whether two sequences overlap and thus represent the same genomic region (e.g., the same haplotype in polyploid genomes) or whether the sequences represent different genomic regions.
G16B 15/00 - ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
G16B 40/00 - ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
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.
A method for identifying a nucleotide in a template nucleic acid by (a) providing a plurality of primer-template nucleic acid hybrids, wherein the primers have an extendable 3' end; (b) contacting the plurality with: (i) blocked nucleotides to produce a first subset of the primer-template nucleic acid hybrids that include a blocked nucleotide at the 3' end, and (ii) a ternary complex inhibitor to produce a second subset of the primer-template nucleic acid hybrids that include a ternary complex inhibitor; (c) forming ternary complexes that each include a polymerase, a primer-template nucleic acid hybrid of the first subset, and a cognate nucleotide; and (d) detecting the ternary complexes, thereby identifying a nucleotide in the template nucleic acid.
Methods, compositions, kits and apparatuses that include a fluid, the fluid containing a ternary complex and Li+, wherein the ternary complex includes a primed template nucleic acid, a polymerase, and a nucleotide cognate for the next correct base for the primed template nucleic acid molecule. As an alternative or addition to Li+, the fluid can contain betaine or a metal ion that inhibits polymerase catalysis such as Ca2+. In addition to Li+, the fluid can contain polyethylenimine (PEI) with or without betaine.
A method of determining nucleic acid sequences can include steps of (a) obtaining signal data from a nucleic acid sequencing procedure carried out on an array of nucleic acid features; (b) extracting signals from each nucleic acid feature to produce multiple extracted signal traces that each correlate signal characteristics with sequencing cycle for a particular nucleotide type at a particular nucleic acid feature; (c) comparing the series of signals for different nucleotide types at each of the features to distinguish a candidate base call from background signals for each cycle at each feature; (d) applying a baseline adjustment to each series of signals based on the extracted background signals; and (e) comparing the adjusted signal traces for different nucleotide types at each of the features, thereby distinguishing adjusted signals having characteristics of a base call from adjusted background signals for each cycle at each feature.
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.
An apparatus can include a vessel, a reference surface, a preload, a scan actuator, and a transmitter. The reference surface can form a structural loop with a detector. The preload can be configured to urge the vessel to contact an area on the reference surface. The scan actuator can be configured to slide the vessel along the reference surface in a scan dimension. The transmitter can be configured to direct signal from the vessel to a detector and/or direct energy from an energy source to the vessel, when the vessel is urged by the preload to contact the reference surface.
Disclosed herein are finished products, methods, compositions and kits for derivatizing plastic (e.g., "polymer") surfaces in a manner that renders the surfaces appropriate for various downstream applications. For example, flow cells incorporating modified plastic surfaces provide greatly enhanced stability for retention of attached chemical species such as polypeptides and nucleic acids.
B01J 20/28 - Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
B01D 15/02 - Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor with moving adsorbents
B01J 20/289 - Phases chemically bonded to a substrate, e.g. to silica or to polymers bonded via a spacer
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 b ase call.
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.
B01J 19/00 - Chemical, physical or physico-chemical processes in general; Their relevant apparatus
C12Q 1/6876 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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
18.
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.
A method for separating a target allele from a mixture of nucleic acids by (a) providing a mixture of nucleic acids in fluidic contact with a stabilized ternary complex that is attached to a solid support, wherein the stabilized ternary complex includes a polymerase, primed nucleic acid template, and next correct nucleotide, wherein the template has a target allele, wherein the next correct nucleotide is a cognate nucleotide for the target allele, and wherein the stabilized ternary complex is attached to the solid support via a linkage between the polymerase and the solid support or via a linkage between the next correct nucleotide and the solid support; and (b) separating the solid support from the mixture of nucleic acids, thereby separating the target allele from the mixture of nucleic acids.
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.
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.
Provided are engineered DNA polymerases exhibiting modified functionality, and polynucleotides encoding same. Modified features include: (1) reduced catalytic activity in the presence of magnesium ions and/or (2) reduced affinity for primed template nucleic acid molecules in the absence of cognate nucleotide, and an ability to discriminate between cognate and non-cognate nucleotides under low salt conditions. Sequencing By BindingTM procedures employing the engineered polymerases have certain advantages. The engineered polymerases can have other uses as well.
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.
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.
Provided are methods and systems for detecting formation of nucleotide-specific ternary complexes comprising a DNA polymerase, a nucleic acid, and a nucleotide complementary to the templated base of the primed template nucleic acid. The methods and systems facilitate determination of the next correct nucleotide without requiring chemical incorporation of the nucleotide into the primer. These results can even be achieved in procedures employing unlabeled, native nucleotides.
Provided are sequencing-by-binding methods of detecting cognate nucleotides using a crippled DNA polymerizing enzyme that possesses the ability to bind the next correct nucleotide downstream of a primer in a template-dependent fashion, but does not possess the activity needed to promote phosphodiester bond formation. Use of the crippled DNA polymerase permits interrogation of one nucleotide at a time, without incorporation of any nucleotide. Labeled nucleotides, such as fluorescently labeled nucleotides, can be used in conjunction with the crippled DNA polymerase to establish cognate nucleotide identity in a rapid manner.
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.
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.
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.
G02B 6/12 - Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type of the integrated circuit kind
A multiprocessor pipeline architecture that converts signals from sequencing sample acquisition into sequence data, comprising: a custom coprocessor card configured to directly receive a stream of serialized sensor data generated by an image sensor, wherein the sensor data represents frame-by-frame intensity values for pixels comprising the image sensor; a first coprocessor that continually receives the stream of serialized sensor data and transposes the frame-by-frame intensity values into reaction cell chunks; a buffer that repeatedly receives the reaction cell chunks and stores in contiguous memory locations the reaction cell chunks for each respective reaction cell over a larger predetermined time window to create larger reaction cell chunks; and a plurality of second coprocessors that retrieve the larger reaction cell chunks from the buffer and convert, in parallel, the pixel intensity values into base-by-base sequence data.
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
32.
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.
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
C12Q 1/6837 - Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
C40B 30/04 - Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
C40B 40/00 - Libraries per se, e.g. arrays, mixtures
C40B 40/06 - Libraries containing nucleotides or polynucleotides, or derivatives thereof
C40B 70/00 - Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or barcodes
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.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
C40B 30/04 - Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
An analytical assembly within a unified device structure for integration into an analytical system. The analytical assembly is scalable and includes a plurality of analytical devices, each of which includes a reaction cell, an optical sensor, and at least one optical element positioned in optical communication with both the reaction cell and the sensor and which delivers optical signals from the cell to the sensor. Additional elements are optionally integrated into the analytical assembly. Methods for forming and operating the analytical system are also disclosed.
G01N 21/63 - Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
G08C 23/04 - Non-electric signal transmission systems, e.g. optical systems using light waves, e.g. infrared
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.
Apparatus, systems and methods for use in analyzing discrete reactions at ultra high multiplex with reduced optical noise, and increased system flexibility. Apparatus include substrates having integrated optical components that increase multiplex capability by one or more of increasing density of reaction regions, improving transmission of light to or collection of light from discrete reactions regions. Integrated optical components include reflective optical elements which re-direct illumination light and light emitted from the discrete regions to more efficiently collect emitted light. Particularly preferred applications include single molecule reaction analysis, such as polymerase mediated template dependent nucleic acid synthesis and sequence determination.
G02B 6/12 - Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type of the integrated circuit kind
G01N 21/00 - Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
37.
SUBSTRATES AND OPTICAL SYSTEMS AND METHODS OF USE THEREOF
This specification provides substrates for use in various applications, including single-molecule analytical reactions. Methods for propagating optical energy within a substrate are provided. Devices comprising waveguide substrates and dielectric omnidirectional reflectors 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 21/17 - Systems in which incident light is modified in accordance with the properties of the material investigated
B82Y 20/00 - Nanooptics, e.g. quantum optics or photonic crystals
C12M 1/34 - Measuring or testing with condition measuring or sensing means, e.g. colony counters
G01N 21/00 - Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
G01N 21/63 - Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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 guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type of the integrated circuit kind
C12Q 1/00 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
38.
SUBSTRATES AND OPTICAL SYSTEMS AND METHODS OF USE THEREOF
This invention provides substrates for use in various applications, including single-molecule analytical reactions. Methods for propagating optical energy within a substrate are provided. Devices comprising waveguide substrates and dielectric omnidirectional reflectors 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 21/00 - Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
C12M 1/34 - Measuring or testing with condition measuring or sensing means, e.g. colony counters
G01N 33/483 - Physical analysis of biological material
C12Q 1/00 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
39.
GENERATION OF MODIFIED POLYMERASES FOR IMPROVED ACCURACY IN SINGLE MOLECULE SEQUENCING
Provided are compositions comprising modified recombinant polymerases that exhibit branching fractions that are less than the branching fractions of the polymerases from which they were derived, or branching fractions that are less than about 25% for a phosphate-labeled nucleotide analog. Also provided are compositions comprising modified recombinant polymerases that exhibit closed polymerase/DNA complexes with increased stability relative to the parental polymerases. Also provided are compositions comprising modified recombinant polymerases that exhibit decreased rate constants relative to the parental polymerases. Provided are methods for generating polymerases with the aforementioned phenotypes. Provided are methods of using such polymerases to make a DNA or to sequence a DNA template.
Compositions and methods for nucleic acid sequencing include template constructs that comprise double stranded portions in a partially or completely contiguous constructs, to provide for redundant sequence determination through one or both of sequencing sense and antisense strands, and iteratively sequencing the entire construct multiple times. Additional sequence components are also optionally included within such template constructs. Methods are also provided for the use and preparation of these constructs as well as sequencing compositions for their application.
Compositions for nucleic acid sequencing are provided that include template constructs that comprise double stranded portions in partially or completely contiguous constructs, to provide for redundant sequence determination through one or both of sequencing sense and antisense strands, and iteratively sequencing the entire construct multiple times. Additional sequence components are also optionally included within such template constructs. Methods are also provided for the use and preparation of these constructs as well as sequencing compositions for their application.
Labeled reactant compositions, and particularly labeled nucleic acid reaction compositions, that include structural components that maintain potentially damaging labeling components sufficiently distal from the reactant portion of the molecule such that damaging effects of the label group on other reaction components, such as enzymes, are reduced, minimized and/or eliminated.
C12Q 1/00 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
G01N 33/58 - Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
44.
ALTERNATE LABELING STRATEGIES FOR SINGLE MOLECULE SEQUENCING
Systems and methods of enhancing fluorescent labeling strategies as well as systems and methods of using non-fluorescent and/or non-optic labeling strategies, e.g., as with single molecule sequencing using ZMWs, are described.
C12Q 1/00 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
C12M 1/34 - Measuring or testing with condition measuring or sensing means, e.g. colony counters
C12M 1/40 - Apparatus specially designed for the use of free, immobilised, or carrier-bound enzymes, e.g. apparatus containing a fluidised bed of immobilised enzymes
Computer implemented methods, and systems performing such methods for processing signal data from analytical operations and systems, and particularly in processing signal data from sequence-by- incorporation processes to identify nucleotide sequences of template nucleic acids and larger nucleic acid molecules, e.g., genomes or fragments thereof.
Mitigative and remedial approaches to reduction of autofluorescence background noise are applied in analytical systems that rely upon sensitive measurement of fluorescent signals from arrays of fluorescent signal sources. Such systems are for particular use in fluorescence based sequencing by incorporation systems that rely upon small numbers or individual fluorescent molecules in detecting incorporation of nucleotides in primer extension reactions. Systems and methods for analyzing highly multiplexed sample arrays using highly multiplexed, high-density optical systems to illuminate high- density sample arrays and/or provide detection and preferably confocal detection off signals emanating from such high-density arrays. Systems and methods are applied in a variety of different analytical operations, including analysis of biological and biochemical reactions, including nucleic acid synthesis and derivation of sequence information from such synthesis.
Substrates, systems and methods for analyzing materials that include waveguide arrays disposed upon or within the substrate such that evanescent fields emanating from the waveguides illuminate materials disposed upon or proximal to the surface of the substrate, permitting analysis of such materials. The substrates, systems and methods are used in a variety of analytical operations, including, inter alia, nucleic acid analysis, including hybridization and sequencing analyses, cellular analyses and other molecular analyses.
G01N 21/63 - Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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
C40B 60/00 - Apparatus specially adapted for use in combinatorial chemistry or with libraries
Substrates, including zero mode waveguide substrates that have been fabricated to provide additional f.upsilon..pi.clionai elements and/or components including increased volumes for positioning of active surfaces and or components for the mitigation of negative electrochemical properties of the underlying substrates.
G02B 6/10 - Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type
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 33/48 - Biological material, e.g. blood, urine; Haemocytometers
B82Y 15/00 - Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
49.
ARTICLES HAVING LOCALIZED MOLECULES DISPOSED THEREON AND METHODS OF PRODUCING SAME
Methods of producing substrates having selected active chemical regions by employing elements of the substrates in assisting the localization of active chemical groups in desired regions of the substrate. The methods may include optical, chemical and/or mechanical processes for the deposition, removal, activation and/or deactivation of chemical groups in selected regions of the substrate to provide selective active regions of the substrate.
Methods and systems for real-time monitoring of optical signals from arrays of signal sources, and particularly optical signal sources that have spectrally different signal components. Systems include signal source arrays in optical communication with optical trains that direct excitation radiation to and emitted signals from such arrays and image the signals onto detector arrays, from which such signals may be subjected to additional processing.
Devices, systems and methods of using same where hybrid substrate materials are provided with a substantially uniform surface to provide uniformity of properties, including interaction with their environments. Uniform surfaces are applied as coatings over, e.g., hybrid metal/silica, metal/polymer, metal/metal surfaces to mask different chemical properties of differing regions of the surface and to afford a protective surface for the hybrid structure.
C23C 16/00 - Chemical coating by decomposition of gaseous compounds, without leaving reaction products of surface material in the coating, i.e. chemical vapour deposition (CVD) processes
53.
METHODS AND SYSTEMS FOR MONITORING MULTIPLE OPTICAL SIGNALS FROM A SINGLE SOURCE
Methods and systems for monitoring a plurality of different optical signals from a single source of such signals, where each such different optical signal is spatially separated from other such signals and directed to different detectors or locations upon a single detector, which direction is generally accomplished through the use of a small number of optical components and/or manipulations.
G01D 5/28 - Mechanical means for transferring the output of a sensing member; Means 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 converting; Transducers not specially adapted for a specific variable using optical means, i.e. using infrared, visible or ultraviolet light with deflection of beams of light, e.g. for direct optical indication
G01N 21/75 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
G02B 26/08 - Optical devices or arrangements for the control of light using movable or deformable optical elements for controlling the direction of light
The present invention relates to optical confinements, method of preparing and methods of using them for analyzing molecules and/or monitoring chemical reactions. The apparatus and methods embodied in the present invention are particularly useful for high-throughput and low-cost single-molecular analysis.
B82Y 5/00 - Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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
C12M 1/40 - Apparatus specially designed for the use of free, immobilised, or carrier-bound enzymes, e.g. apparatus containing a fluidised bed of immobilised enzymes
C40B 60/00 - Apparatus specially adapted for use in combinatorial chemistry or with libraries
G01N 21/00 - Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
G01N 21/75 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
G02B 6/12 - Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type of the integrated circuit kind
patents
