[go: up one dir, main page]

US20060062531A1 - Fabrication of optical confinements - Google Patents

Fabrication of optical confinements Download PDF

Info

Publication number
US20060062531A1
US20060062531A1 US11/229,376 US22937605A US2006062531A1 US 20060062531 A1 US20060062531 A1 US 20060062531A1 US 22937605 A US22937605 A US 22937605A US 2006062531 A1 US2006062531 A1 US 2006062531A1
Authority
US
United States
Prior art keywords
array
optical
confinements
cladding
confinement
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US11/229,376
Other languages
English (en)
Inventor
Stephen Turner
Jonas Korlach
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pacific Biosciences of California Inc
Original Assignee
Pacific Biosciences of California Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US10/944,106 external-priority patent/US7170050B2/en
Application filed by Pacific Biosciences of California Inc filed Critical Pacific Biosciences of California Inc
Priority to US11/229,376 priority Critical patent/US20060062531A1/en
Assigned to PACIFIC BIOSCIENCES OF CALIFORNIA, INC. reassignment PACIFIC BIOSCIENCES OF CALIFORNIA, INC. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: KORLACH, JONAS, TURNER, STEPHEN
Assigned to PACIFIC BIOSCIENCES OF CALIFORNIA, INC. reassignment PACIFIC BIOSCIENCES OF CALIFORNIA, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KORLACH, JONAS, TURNER, STEPHEN
Publication of US20060062531A1 publication Critical patent/US20060062531A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/028—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations having reaction cells in the form of microtitration plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B6/00—Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings
    • G02B6/10—Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type
    • G02B6/12—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
    • G02B6/13—Integrated optical circuits characterised by the manufacturing method
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0654—Lenses; Optical fibres
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0896—Nanoscaled
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing

Definitions

  • FCS Fluorescence Correlation Spectroscopy
  • the detection volume of traditional FCS is approximately 0.5 femtoliters (or 0.5 ⁇ 10 ⁇ 15 liters), and is achieved through the use of a high numerical aperture microscope objective lens to tightly focus a laser beam.
  • This detection volume single molecules can be observed in solutions at concentrations of up to approximately one nanomolar. This concentration range is unacceptably low for most biochemical reactions, which have reaction constants that are typically in or above the micromolar range. At lower concentrations, these reactions either do not proceed acceptably fast, or behave in a qualitatively different fashion than is useful in most analyses. To observe single molecules at higher, more relevant concentrations, the observation volume would typically need to be reduced to far smaller dimensions.
  • a principal aspect of the present invention is the design of optical devices and methods for characterizing molecules and/or monitoring chemical reactions.
  • the devices and methods of the present invention are particularly suited for single-molecule analysis.
  • the present invention provides an array of optical confinements having a surface density exceeding 4 ⁇ 10 4 confinements per mm 2 , preferably exceeding 10 5 confinements per mm 2 .
  • the individual confinement in the array provide an effective observation volume that is less than one nanoliter (10X ⁇ 9 liter), less than one picoliter, or less than one femtoliter, preferably on the order of zeptoliter.
  • each of the individual confinement provides an effective observation volume that is less than 1000 zeptoliters, 100 zeptoliters, 80 zeptoliters, or less than 50 zeptoliters, or even less than 10 zeptoliters.
  • each of the individual confinement yields an effective observation volume that permits resolution of individual molecules present at a concentration that is higher than one nanomolar, or higher than 100 nanomolar, or on the order of micromolar range. In certain preferred aspects, each of the individual confinement yields an effective observation volume that permits resolution of individual molecules present at a physiologically relevant concentration, e.g., at a concentration higher than about 1 micromolar, or higher than 50 micromolar range or even higher than 100 micromolar.
  • the array may comprise a zero-mode waveguide or other nanoscale optical structure.
  • the array of optical confinements may further comprise another array of confinements that does not yield the above-described effective observation volume or does not permit resolution of individual molecules.
  • the array of optical confinements may be coupled with or integrated into a microtiter plate, where a separate array of optical confinements may be disposed within each of several different wells on a multiwell reaction plate.
  • the array of optical confinement may comprise at least about 2 ⁇ 10 5 optical confinement, or at least about 10 6 , or at least about 10 7 optical confinements.
  • the present invention provides a method of creating a plurality of optical confinements having the aforementioned characteristics.
  • the method involves the steps of (a) providing a substrate; and (b) forming an array of optical confinements having a surface density exceeding 4 ⁇ 10 4 confinements per mm 2 , wherein the individual confinement comprises a zero-mode waveguide comprising: a cladding surrounding a core, wherein said cladding is configured to preclude propagation of electromagnetic energy of a wavelength longer than a cutoff wavelength longitudinally through the core of the zero-mode waveguide; and (c) illuminating the array with an electromagnetic radiation of a frequency less than the cutoff frequency, thereby creating the plurality of optical confinements.
  • the present invention provides a method of creating an optical observation volume that permits resolution of individual molecules.
  • the method involves providing a zero-mode waveguide that comprises a cladding surrounding a core, wherein said cladding is configured to preclude propagation of electromagnetic energy of a frequency less than a cutoff frequency longitudinally through the core of the zero-mode waveguide, wherein upon illuminating the zero-mode waveguide with an electromagnetic radiation of a frequency less than the cutoff frequency, the zero-mode waveguide yields an effective observation volume that permits resolution of individual molecules.
  • the effective observation volume is less than one nanoliter (10 ⁇ 9 liter), less than one picoliter, or less than one femtoliter, preferably on the order of zeptoliters.
  • the method yields an effective observation volume that permits resolution of individual molecules present at a concentration that is higher than one nanomolar, more often higher than 100 nanomolar, and preferably on the order of micromolar range.
  • individual molecules present at a concentration higher than about 5 micromolar, or higher than 7.5 micromolar, or even higher than 50 micromolar range can be resolved by the method of the present invention.
  • the present invention also provides a method of detecting interactions among a plurality of molecules.
  • the method comprises the steps of (a) placing the plurality of molecules in close proximity to an array of zero-mode waveguides, wherein individual waveguides in the array are separated by a distance sufficient to yield detectable intensities of diffractive scattering at multiple diffracted orders upon illuminating the array with an incident wavelength; (b) illuminating the array of zero-mode waveguides with an incident wavelength; and (c) detecting a change in the intensities of diffractive scattering of the incident wavelength at the multiple diffracted orders, thereby detecting the interactions among a plurality of molecules.
  • the present invention also provides a method of reducing diffractive scattering upon illuminating an array of optical confinements with an incident wavelength, wherein the array comprises at least a first optical confinement and a second optical confinement, said method comprising: forming the array of optical confinements wherein the optical confinement is separated from the second optical confinement by a distance such that upon illumination with the incident wavelength, intensity of diffractive scattering resulting from the first optical confinement at a given angle is less than that if the first optical confinement were illuminated with the same incident wavelength in the absence of the optical confinement.
  • the aforementioned optical confinements are zero mode waveguides.
  • the present invention also includes a method of detecting a biological analyte using an array of optical confinements having a density on a substrate exceeding 4 ⁇ 10 4 confinements per mm 2 or any other density described herein or equivalents thereof.
  • the method typically involves illuminating at least one optical confinement within the array that is suspected to contain the analyte with an incident light beam.
  • the invention also provides a method of using of an array of optical confinements having a density on a substrate exceeding 4 ⁇ 10 4 confinements per mm 2 any other density described herein or equivalents thereof for performing multiple chemical reactions.
  • the method comprises the steps of placing the plurality of reaction samples comprising labeled reactants into the optical confinements in the array, wherein a separate reaction sample is placed into a different confinement in the array; subjecting the array to conditions suitable for formation of products of the chemical reactions; and detecting the formation of the products with said optical system.
  • the invention provides a method of sequencing a plurality of target nucleic acid molecules.
  • the method typically involves (a) providing an array of optical confinements having a density on a substrate exceeding 4 ⁇ 10 4 confinements per mm 2 , or any other density described herein or equivalents thereof, wherein said optical confinements provide an effective observation volume that permits observation of individual molecules; and an optical system operatively coupled to the optical confinements that detects signals from the effective observation volume of said confinement; (b) mixing in the optical confinements the plurality of target nucleic acid molecules, primers complementary to the target nucleic acid molecules, polymerization enzymes, and more than one type of nucleotides or nucleotide analogs to be incorporated into a plurality of nascent nucleotide strands, each strand being complementary to a respective target nucleic acid molecule; (c) subjecting the mixture of step (b) to a polymerization reaction under conditions suitable for formation of the nascent nucleotide strands by template-
  • the present invention also provides an apparatus comprising an array of waveguides on a solid support having a fill fraction greater than about 0.0001, wherein said waveguides are suitable for holding a biological reagent, and wherein waveguides provide an effective observation volume that permits observation of individual molecules present in said biological reagent; and an optical system that detects said individual molecules in said waveguides, by e.g., detecting signals from the effective observation volume.
  • the array has a fill fraction greater than about 0.001.
  • the array has a fill fraction greater than about 0.01, in some instances greater than 0.1, or within the range about 0.001 to about 0.1.
  • the present invention also provides various methods of using such high fill fraction array.
  • the present invention provides a method of detecting a biological analyte. The method comprises optically capturing the analyte within an optical confinement that is created by (a) providing an array of waveguides having a fill fraction greater than about 0.0001; and (b) illuminating at least one waveguide within the array that is suspected to contain the analyte with an incident light beam thereby detecting the analyte.
  • the present invention provides a method of performing multiple chemical reactions involving a plurality of reaction samples using the subject high fill fraction array.
  • the method involves (a) providing a subject high fill fraction array; (b) placing the plurality of reaction samples comprising labeled reactants into the waveguides in the array, wherein a separate reaction sample is placed into a different waveguide in the array; (c) subjecting the array to conditions suitable for formation of products of the chemical reactions; and (d) detecting the formation of the products with an optical system.
  • the step of detecting may comprise illuminating the different waveguides with an incident light beam and detecting an optical signal emitted from the reaction samples.
  • Applicable chemical reactions may involve protein-protein interactions, nucleic acid-protein interactions, and nucleic acid-nucleic acid interactions.
  • the present invention provides a method of sequencing a plurality of target nucleic acid molecule using a fill fraction greater than about 0.0001.
  • the present invention further provides a method of sequencing nucleic acid using an array having a high fill faction.
  • the method typically involves a) providing an array of waveguides having a fill fraction greater than about 0.0001, or 0.001, or 0.01 or even 0.1; (b) mixing in the waveguides the plurality of target nucleic acid molecules, primers complementary to the target nucleic acid molecules, polymerization enzymes, and more than one type of nucleotides or nucleotide analogs to be incorporated into a plurality of nascent nucleotide strands, each strand being complementary to a respective target nucleic acid molecule; (c) subjecting the mixture of step (b) to a polymerization reaction under conditions suitable for formation of the nascent nucleotide strands by template-directed polymerization of the nucleotides or nucleotide analogs; (d) illuminating the waveguides with an incident light beam; and (e) identifying the nucleotides or the
  • the method comprises (a) subjecting a target nucleic acid molecule to a template-directed polymerization reaction to yield a nascent nucleic acid strand that is complementary to the target nucleic acid molecule in the presence of a plurality of types of nucleotides or nucleotide analogs, and a polymerization enzyme exhibiting strand-displacement activity; and (b) registering a time sequence of incorporation of nucleotides or nucleotide analogs into the nascent nucleotide strand.
  • the target nucleic acid molecule is a circular nucleic acid, or is a linear or circular template strand synthesized from a circular nucleic acid sequence such that the synthesized strand includes multiple repeated copies of the original circular strand, and is thus is subject to the sequencing operations of the invention.
  • the target nucleic acid molecule is sequenced multiple times, e.g., more than once, or more than twice by the polymerization enzyme.
  • the polymerization enzyme is a DNA polymerase, such as a modified or unmodified ⁇ 29 polymerase.
  • a solid support having a surface wherein the surface has a polymerization enzyme array attached to it, wherein members of the array comprise individually and optically resolved polymerization enzymes possessing strand-displacement activities.
  • a zero mode waveguide comprising a first molecular complex immobilized therein, said molecular complex comprising a polymerization enzyme complexed with a target nucleic acid, wherein the polymerization enzyme processes a sequence of nucleotides in said target nucleic acid multiple times via template-dependent replication of the target nucleic acid.
  • the present invention is a method of fabricating an array of optical confinements that exhibits a minimal intensity of diffractive scattering of an incident wavelength.
  • the method comprises providing a substrate; and forming the array of optical confinements on the substrate such that individual confinements in the array are separated from each other at a distance less than one half of the wavelength.
  • the present invention includes a method of fabricating an optical confinement the method comprises a cladding surrounding a core, comprising: (a) providing a substrate coated with a layer of photoresist; (b) patterning said layer of photoresist to define boundaries of said core; (c) removing said layer of photoresist surrounding said defined boundaries so that a sufficient amount of photoresist remains to occupy said core; (d) depositing a layer of cladding material over said remaining photoresist and said substrate; (e) removing at least a portion of said cladding material deposited over said remaining photoresist; and (f) removing said photoresist of step (e) to form said core surrounded by said cladding of said optical confinement.
  • the photoresist is negative and said patterning step employs a positive pattern. In another aspect, the photoresist is positive and said patterning step employs a negative pattern.
  • the removing step can be effected by a technique selected from the group consisting of etching, mechanical polishing, ion milling, and solvent dissolution.
  • the layer of cladding material can be deposited by a thermal evaporation method or vapor deposition.
  • FIG. 1 depicts a top view of an array of illustrative optical confinements, here zero-mode waveguides arranged in a square format.
  • FIG. 2 depicts a top view of an array of illustrative optical confinements, here zero-mode waveguides arranged in a non-square format.
  • FIG. 3 depicts a top view of an illustrative 2-dimentional array with an illustrative angle and two different unit vector lengths.
  • FIG. 4 depicts a top view of an illustrative regular disposition of ZMWs.
  • FIG. 5 depicts an array of arrays, in which a subarray 71 is part of a super array 72 .
  • FIG. 6 illustrates a process of negative tone fabrication.
  • FIG. 7 illustrates an array of ZMWs optically linked to an optical system.
  • FIG. 8 depicts a scanning electron micrographs of ZMW structures fabricated by positive tone resist (left panels) or negative tone resist (right panels).
  • the grain structure of the polycrystalline film is visible in the image as flecks, and the ZMWs as dark round structures.
  • FIG. 9 depicts a single-molecule DNA sequence pattern recognition in ZMWs using artificial pre-formed replication forks.
  • FIG. 10 depicts a coated ZMW 101 that is bound to a substrate 105 .
  • the ZMW comprises a sidewall 102 , a coating 103 on the upper surface, and a metal film 104 .
  • FIG. 11 depicts one alignment strategy and optical setup.
  • FIG. 12 depicts an alternative optical confinement made of porous film 91 on a substrate 93 .
  • 92 represents the pores in the film.
  • FIGS. 13 A-B depict an alignment detection system and the associated components.
  • FIG. 14 depicts several exemplary photocleavable blockers and the applicable wavelength applied to cleave the blocking groups.
  • FIG. 15 depicts an exemplary reversible extension terminator in which the photocleavable blocker is conjugated to a detectable label (e.g., fluorescent label).
  • a detectable label e.g., fluorescent label
  • FIG. 16 depicts an exemplary profile of fluorescent bursts corresponding to the time sequence of incorporation of two types of labeled nucleotides or nucleotide analogs in single-molecule sequencing reaction using the subject optical confinement.
  • Luminescence refers to the emission of light from a substance for any reason other than a rise in its temperature.
  • atoms or molecules emit photons of electromagnetic energy (e.g., light) when then move from an “excited state” to a lower energy state (usually the ground state); this process is often referred to as “decay”.
  • electromagnetic energy e.g., light
  • the luminescence process is referred to as “photoluminescence”.
  • the exciting cause is an electron
  • the luminescence process is referred to as “electroluminescence”. More specifically, electroluminescence results from the direct injection and removal of electrons to form an electron-hole pair, and subsequent recombination of the electron-hole pair to emit a photon.
  • Luminescence which results from a chemical reaction is usually referred to as “chemiluminescence”.
  • Luminescence produced by a living organism is usually referred to as “bioluminescence”.
  • photoluminescence is the result of a spin allowed transition (e.g., a single-singlet transition, triplet-triplet transition)
  • the photoluminescence process is usually referred to as “fluorescence”.
  • fluorescence Typically, fluorescence emissions do not persist after the exciting cause is removed as a result of short-lived excited states which may rapidly relax through such spin allowed transitions.
  • photoluminescence is the result of a spin forbidden transition (e.g., a triplet-singlet transition)
  • the photoluminescence process is usually referred to as “phosphorescence”.
  • phosphorescence emissions persist long after the exciting cause is removed as a result of long-lived excited states which may relax only through such spin-forbidden transitions.
  • a “luminescent label” or “luminescent signal” may have any one of the above-described properties.
  • electromagnetic radiation refers to electromagnetic waves of energy including, for example, in an ascending order of frequency (or alternatively, in a descending order of wavelength), infrared radiation, visible light, ultraviolet (UV) light, X-rays, and gamma rays.
  • an “effective observation volume” typically refers to that volume that is observable by the detection means employed for a given application.
  • an effective observation volume typically refers to that volume that is observable by the detection means employed for a given application.
  • fluorescence based detection it is that volume which is exposed to excitation radiation and/or from which emission radiation is gathered by an adjacent optical train/detector.
  • an effective observation volume is dictated by the propagation of excitation radiation into the waveguide core, and particularly that volume that is exposed to light that is at least 1%, and preferably at least 10% of the original intensity of excitation radiation entering the waveguide core.
  • a “primer” is a short polynucleotide, generally with a free 3′ OH group, that binds to a target nucleic acid (or template) potentially present in a sample of interest by hybridizing with the target nucleic acid, and thereafter promoting polymerization of a polynucleotide complementary to the target.
  • operatively linked to or “operatively coupled to” are used interchangeably herein. They refer to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • nucleotide generally refers to a molecule comprising a base, sugar and one or more anionic groups, preferably phosphates.
  • the molecule may comprise one, two, three, four, five or more phosphates groups and/or other groups such as sulfate.
  • the term also encompasses nucleotide analogs that are structurally analogous to naturally occurring nucleotides and are capable of acting substantially like nucleotides, for example exhibiting base complementarity with one or more of the bases that occur in DNA or RNA, and/or being capable of base-complementary incorporation in synthesizing nucleotide strand by a polymerization enzyme.
  • polynucleotide refers to a polymeric form of “nucleotides” of any length.
  • a “type of nucleotide” refers to a set of nucleotides that share a common characteristic that is to be detected. For instance, the types of nucleotides can be classified into four categories: A, T, C, and G for DNA, or A, U, C and G for RNA. In some embodiments, each type of nucleotides used in the a reaction will be labeled with a unique label that is distinguishable from the rest.
  • optical confinement refers to an area in which the reactants for an intended reaction within the confinement are confined and resolved by optical means.
  • a “polynucleotide probe” refers to a polynucleotide used for detecting or identifying its corresponding target polynucleotide in a hybridization reaction.
  • hybridize refers to the ability of the polynucleotide to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues in a hybridization reaction.
  • the hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.
  • the hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.
  • Hybridization can be performed under conditions of different “stringency”. Relevant conditions include temperature, ionic strength, time of incubation, the presence of additional solutes in the reaction mixture such as form amide, and the washing procedure. Higher stringency conditions are those conditions, such as higher temperature and lower sodium ion concentration, which require higher minimum complementarity between hybridizing elements for a stable hybridization complex to form.
  • a low stringency hybridization reaction is carried out at about 40° C. in 10 ⁇ SSC or a solution of equivalent ionic strength/temperature.
  • a moderate stringency hybridization is typically performed at about 50° C. in 6 ⁇ SSC, and a high stringency hybridization reaction is generally performed at about 60° C. in 1 ⁇ SSC.
  • a double-stranded polynucleotide can be “complementary” or “homologous” to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second.
  • “Complementarity” or “homology” is quantifiable in terms of the proportion of bases in opposing strands that are expected to form hydrogen bonding with each other, according to generally accepted base pairing rules.
  • One aspect of the present invention is the design of optical devices and methods for characterizing molecules and/or monitoring chemical reactions.
  • the optical devices of the present invention allow multiplexing of large numbers of single-molecule analyses under physiologically relevant conditions.
  • the present invention provides a high density array of optical confinements having a surface density exceeding 4 ⁇ 10 4 confinements per mm 2 , preferably exceeding 10 5 , wherein the individual confinement in the array provides an effective observation volume on the order of zeptoliters.
  • the array may contain at least about 2 ⁇ 10 5 , at least about 10 6 , or at least about 10 7 optical confinements.
  • the individual confinement in the array provides an effective observation volume less than about 1000 zeptoliters, more preferably less than about 900, more preferably less than about 80, even more preferably less than about 10 zeptoliters. Where desired, an effective observation volume less than 1 zeptoliter can be provided.
  • the individual confinement yields an effective observation volume that permits resolution of individual molecules present at a physiologically relevant concentration.
  • the physiologically relevant concentrations for most biochemical reactions range from micro-molar to millimolar because most of the enzymes have their Michaelis constants in these ranges.
  • preferred array of optical confinements has an effective observation volume for detecting individual molecules present at a concentration higher than about 1 micromolar ( ⁇ M), or more preferably higher than 50 ⁇ M, or even higher than 100 ⁇ M.
  • the array may comprise zero-mode waveguides or alternative nanoscale optical structures.
  • alternative structures include but are not limited to porous films with reflective index media, and confinements using index matching solids.
  • zero-mode waveguide refers to an optical guide in which the majority of incident radiation is attenuated, preferably more than 80%, more preferably more than 90%, even more preferably more than 99% of the incident radiation is attenuated.
  • the rapid decay of incident electromagnetic radiation at the entrance of such guide provides an extremely small observation volume effective to detect single molecules, even when they are present at a concentration as high as in the micromolar range.
  • the zero-mode waveguide of the present invention typically comprises a cladding surrounding a core (i.e., partially or fully), wherein the cladding is configured to preclude propagation of electromagnetic energy of a wavelength higher than the cutoff wavelength longitudinally through the core of the zero-mode waveguide.
  • the cladding is typically made of materials that prevent any significant penetration of the electric and the magnetic fields of an electromagnetic radiation. Suitable materials for fabricating the cladding include but are not limited to alloys, metals, and semi-conducting materials, and any combination thereof. Alloys include any of the numerous substances having metallic properties but comprising two or more elements of which at lest one is a metal. Alloys may vary in the content or the amount of the respective elements-whether metallic or non metallic.
  • Preferred alloys generally improve some desirable characteristics of the material over a pure elemental material. Characteristics that can be improved through the use of mixtures of materials include, chemical resistance, thermal conductivity, electrical conductivity, reflectivity, grain size, coefficient of thermal expansion, brittleness, temperature tolerance, conductivity, and/or reduce grain size of the cladding.
  • alloys suitable for the present invention may involve mixtures where one component is present at fractions as low as 0.0001%. In other instances, alloys with large fractions of more than one compound will be desirable.
  • One embodiment of the ZMW uses aluminum as the cladding of the ZMW structure. As an example of how alloys can be beneficial to a ZMW structure, it is useful to consider different alloys of aluminum in how they would affect a ZMW. In the art of metalurgy, numerous materials are alloyed with aluminum.
  • Non-limiting examples of materials suitable to alloy with aluminum are antimony, arsenic, beryllium, bismuth, boron, cadmium, calcium, carbon, cerium, chromium, cobalt, copper, gallium, hydrogen, indium, iron, lead, lithium, magnesium, manganese, mercury, molybdenum, nickel, niobium, phosphorous, silicon, vanadium, zinc and others.
  • the introduction of boron to aluminum is known to increase the conductivity of aluminum.
  • An increase in conductivity of the metal film may improve the performance by decreasing the penetration depth thereby decreasing the observation volume.
  • a preferred embodiment includes an alloy of aluminum that is more than 0.0001% of a dopant.
  • a more preferred embodiment includes an alloy of aluminum that is more than 0.005% of a dopant.
  • a still more preferred embodiment includes an allow of aluminum that is more than 0.1% of a dopant.
  • a preferred embodiment of the device includes a metal film that is less than 1% arsenic.
  • a more preferred embodiment of the device includes a metal films that is less than 0.1% arsenic.
  • a still more preferred embodiment includes a metal film that is less than 0.001% arsenic.
  • a still more preferred embodiment includes a metal film that is less than 0.00001% arsenic.
  • An additional preferred embodiment includes a metal film that is less than 1% lead.
  • a still more preferred embodiment includes a metal film that is less than 0.1% lead.
  • a still more preferred embodiment includes a metal film that is less than 0.01% lead.
  • a still more preferred embodiment includes a metal film that is less than 0.001% lead.
  • a still more preferred embodiment includes a film that is less than 0.00001% lead.
  • impurities that tend to reduce the conductivity, thereby worsening the confinement, will be undesirable.
  • vanadium is known in the art of metallurgy to reduce the conductivity of aluminum.
  • a preferred embodiment includes a metal film that is less than 0.1% vanadium.
  • a still more preferred embodiment includes a metal film that is less than 0.01% vanadium.
  • a still more preferred embodiment includes a film that is less than 0.001% vanadium.
  • Semi-conducting materials suitable for fabricating the cladding are generally opaque, and they include silicon, silicates, silicon nitride, gallium phosphide, gallium arsenide, or any combinations thereof.
  • the cladding of the subject zero-mode waveguide may be coated with materials to improve the surface quality. For instance, coating may enhance the durability of the cladding material. In addition, coating is particularly desirable if the reactants contained in the core are prone to interact or adhere to the cladding material.
  • coating materials are available in the art. Some of the materials may covalently adhere to the surface, others may attach to the surface via non-covalent interactions.
  • Non-limiting examples of coating materials include aluminum oxide film, silanization reagent such as dimethychlorosilane, dimethydichlorosilane, hexamethyldisilazane or trimethylchlorosilane, polymaleimide, and siliconizing reagents such as silicon oxide, AquasilTM, and SurfasilTM.
  • An illustrative coated ZMW ( 101 ) is shown in FIG. 10 .
  • the ZMW ( 101 ) is bound to a substrate 105 .
  • the ZMW comprises a sidewall 102 , a coating 103 on the upper surface, and a metal film 104 .
  • the confinement may be advantageous to construct the confinement from metal compositions that are inhomogeneous combinations of more than one material. For example, for certain applications, it may be beneficial to provide a composition that comprises more than one layer, each layer having a different composition, or composition that varies within a layer. This can have beneficial effects on several aspects of the performance of the confinement, including but not limited to the nature of the optical confinement, the structural strength and behavior of the device, the characteristics of the surface chemistry of the device or the like. In one embodiment the confinement comprises two layers in which one of the layers serves to enhance the adhesion of the second layer to a substrate.
  • the composition of the cladding film varies as a function of the axial position relative to the confinement, so as to provide different optical performance than would be obtained from a layer of uniform composition.
  • the film comprises a composition that has a larger value of skin depth close to the surface of the substrate, and comprises a composition that has a smaller value of skin depth further from the surface of the substrate, so that the nature of the confinement is to be more uniform in shape near the surface and then tapering off more quickly a larger distances away from the substrate.
  • the thicknesses of two different layers comprising the cladding of the confinement are chosen so that a specific optical condition is achieved at the substrate of the device, such as constructive or destructive interference.
  • the internal cavity (i.e., the core) surrounded by the cladding may adopt a convenient size, shape or volume so long as propagating modes of electromagnetic radiation in the guide is effectively prevented.
  • the core typically has a lateral dimension less than the cutoff wavelength ( ⁇ c ).
  • ⁇ c is approximately 1.7 ⁇ d.
  • the cross sectional area of the core may be circular, elliptical, oval, conical, rectangular, triangular, polyhedral, or in any other shape.
  • the various shapes can have particular suitability for certain applications. For instance, elongated cross-sections can be useful to provide enhanced access to molecules with mechanical persistence or stiffness, such as DNA.
  • Cross sections ranging from extended slots to ovals of various aspect ratio will significant increase the accessibility of the persistent molecule to the detection zone of the structure, without excessive compromise in the axial attenuation of radiation.
  • uniform cross sectional area is preferred, the cross sectional area may vary at any given depth of the guide if desired.
  • Preferred average cross sectional areas range from 100 nm 2 to 10,000 nm 2 .
  • the core is non-cylindrical.
  • a non-cylindrical core comprises an opening on the upper surface and a base at the bottom surface that is entirely surrounded by the cladding, wherein the opening is narrower in lateral dimension than the base.
  • This configuration significantly restricts the diffusion of reactants, and hence increases the average residence time in the observation volume.
  • Such configuration is particularly useful for measuring the association rate constant (on-rate) of a chemical reaction.
  • the core comprises an opening that is wider in lateral dimension than the base. Such configuration allows easier access to large molecules that impose a steric or entropic hindrance to entering the structure if the open end of the zero mode waveguide was as small as the base needed to be for optical performance reasons. Examples include the accessibility for long strand polyelectrolytes such as DNA molecules that are subject to entropic forces opposing entry into small openings.
  • the zero-mode waveguides embodied in the present invention have a relatively high fill fraction ratio, typically above 0.0001, preferably above 0.001, more preferably above 0.01, and even more preferably above 0.1.
  • fill fraction of a pattern refers to the ratio of the area occupied by the foreground of the pattern to the total area occupied by the pattern (foreground and background, together).
  • the terms “fill fraction ratio” and “fill faction” are used interchangeably.
  • the foreground is considered to be the area occupied by the core of the zero-mode waveguide
  • the background is the area between the zero-mode waveguide (e.g., the aluminum film that forms the cladding in certain designs).
  • the zero-mode waveguides with high fill fraction ratios are particularly useful for performing homogenous assays.
  • the fill fraction can be calculated by summing the total areas of all of the zero-mode waveguides in the array and dividing by the total available area including both the zero-mode waveguides and the spaces between them. For example, if a zero-mode waveguide has a diameter of 50 nm, then the area of this zero-mode waveguide is one fourth of 7,850 square nanometers or 1962.5 nm 2 . If these zero-mode waveguides are in a square array separated by 100 nm, the total available area is 10,000 square nanometers for each zero-mode waveguide. Therefore, the array has a fill fraction of one fourth of 78% or 19.6%, which would provide nearly four orders of magnitude higher signal strength in a surface binding assay than a zero-mode waveguide having a fill fraction on the order of 0.01%.
  • a bioassay such as an ELISA or other molecular binding bioassay
  • one limitation is the inability to operate “homogeneously”, or in a mode where solutions may be added to a mixture but nothing removed. This complicates highly multiplexed assays, as provisions for both adding and removing material from a large number of wells is significantly more complex than the provisions for simply adding materials.
  • the removal of materials is necessary, because the fluorescent (or other) markers that remain free in solution at the end of the assay would interfere with the ability to detect markers bound to the reaction surface. Techniques to overcome this have been devised to exploit the short range of radioactive emissions from certain radioisotopes, but these techniques have inherent difficulties associated with personnel safety and waste disposal.
  • TIR total internal reflection confinement
  • confocal detection Other methods for confining the sensitivity of the assay to the surface have been devised, such as total internal reflection confinement (TIR), and confocal detection.
  • TIR total internal reflection confinement
  • confocal detection confocal detection.
  • the zero-mode waveguide photonic structure allows a simpler and less expensive optical system configuration than either of these techniques, and vastly outperforms both from the perspective of confinement of sensitivity to the surface.
  • the fill fraction is important in bioassays, because the effective probe area is limited to the surface area of the bottoms of the zero-mode waveguide in the detection region.
  • the amount of signal detectable in such an assay will be directly proportional to the available area, and having a larger fraction of the available surface occupied by zero-mode waveguides will thus increase the signal strength of measurements of such assays.
  • a high fill fraction structure would be generally useful in any surface sensitivity application, not limited to the ELISA assay.
  • the cutoff wavelength is the wavelength above which the waveguide is essentially incapable of propagating electromagnetic energy along the waveguide under the illumination geometry used. Given the geometry of the core, and the properties of the cladding material, as well as the wavelength of the incident electromagnetic radiation, one skilled in the art can readily derive the cutoff wavelength by solving the Maxwell's equations (see, e.g., John D. Jackson, CLASSICAL ELECTRODYNAMICS, second edition, John Willey and Sons). The choice of the incident wavelength will depend on the particular application in which the subject array is to be employed. In certain aspects, the incident wavelength may be selected from a range of about 10 nm to about 1 mm.
  • the incident wavelength is typically selected from the range of about 380 nm to about 800 nm.
  • Polarized (linearly or preferably circularly polarized) or unpolarized incident radiation is generally employed to illuminate the array in order to create a desired observation volume.
  • the present invention provides an alternative optical confinement termed external reflection confinement (ERC).
  • ERC external reflection confinement
  • the low index medium is the electromagnetic radiation carrier
  • the high index (and opaque) medium is the reflector.
  • the roles of the refractive indices are reversed as compared to the IRC situation.
  • ERC generally requires some kind of means to provide the analyte (i.e., the molecules under investigation) in the opaque phase.
  • IRC relies on reflection of an electromagnetic radiation incident on an interface between high index of refraction and low index of refraction.
  • light is incident above the critical angle of total internal reflection (known in the art)
  • all of the incident electromagnetic radiation is reflected and none is transmitted into the low index phase.
  • a thin region of evanescent radiation is established proximal to the interface on the low index side.
  • This radiation field is typically an exponentially decaying field with an attenuation length in the range from about 100 nm to about 200 nm, depending on the angle of incidence and the indices of refraction of the two phases.
  • the low index phase is a solution containing an analyte
  • the evanescent radiation can be used to probe the analyte in the solution with a high degree of surface sensitivity.
  • the carrier of the propagating electromagnetic radiation is a transparent low index film
  • the analyte-bearing medium is a high-index metallic opaque film.
  • most of the radiation is reflected irrespective of the angle of incidence, and non-reflected light is rapidly attenuated according to the skin depth of the metal.
  • means is provided to convey the analyte within the metal phase. Theses means can take the form of a nanocapillary tube constructed within the metal layer. When sufficiently small, the presence of such a tube will have little effect on the distribution of energy in the two media, but can be amply large enough to convey biomolecules. To be small enough, any defects in the metal film must be small compared with the wavelength of the illumination.
  • optical confinement can be fabricated starting with a high index transparent susbtrate such as sapphire, spin coat 200 nm of PMMA (polymethyl methacrylate) resist resin. Exposure to electron beam lithography will render isolated spots soluble according to the pattern applied. After development, the device will have nano-scale holes in the PMMA layer and are ready to be used in a TIR setup. Axial confinement is unaffected by the PMMA layer, as it has nearly the same index of refraction as the solution containing the analyte, but the solution is physically prevented from approaching near the surface except where the holes are situated, providing a degree of lateral confinement given by the diameter of the holes.
  • PMMA polymethyl methacrylate
  • the optical confinements can be provided with an optical system capable of detecting and/or monitoring interactions between reactants at the single-molecule level.
  • Such optical system achieves these functions by first generating and transmitting an incident wavelength to the reactants contained in the confinements, followed by collecting and analyzing the optical signals from the reactants.
  • Such systems typically employ an optical train that directs signals from an array of confinements onto different locations of an array-based detector to simultaneously detect multiple different optical signals from each of multiple different confinements.
  • the optical trains typically include optical gratings or wedge prisms to simultaneously direct and separate signals having differing spectral characteristics from each confinement in an array to different locations on an array based detector, e.g., a CCD.
  • the optical system applicable for the present invention comprises at least two elements, namely an excitation source and a photon detector.
  • the excitation source generates and transmits incident light used to optically excite the reactants contained in the optical confinement.
  • the source of the incident light can be a laser, laser diode, a light-emitting diode (LED), a ultra-violet light bulb, and/or a white light source.
  • more than one source can be employed simultaneously.
  • the use of multiple sources is particularly desirable in applications that employ multiple different reagent compounds having differing excitation spectra, consequently allowing detection of more than one fluorescent signal to track the interactions of more than one or one type of molecules simultaneously.
  • a wide variety of photon detectors are available in the art.
  • Representative detectors include but are not limited to optical reader, high-efficiency photon detection system, photodiode (e.g. avalanche photo diodes (APD)), camera, charge couple device (CCD), electron-multiplying charge-coupled device (EMCCD), intensified charge coupled device (ICCD), and confocal microscope equipped with any of the foregoing detectors.
  • the subject arrays of optical confinements contain various alignment aides or keys to facilitate a proper spatial placement of the optical confinement and the excitation sources, the photon detectors, or the optical transmission element as described below.
  • the subject optical system may also include an optical transmission element whose function can be manifold. First, it collects and/or directs the incident wavelength to the optical confinement containing the reactants. Second, it transmits and/or directs the optical signals emitted from the reactants inside the optical confinement to the photon detector. Third, it may select and/or modify the optical properties of the incident wavelengths or the emitted wavelengths from the reactants. Illustrative examples of such element are diffraction gratings, arrayed waveguide gratings (AWG), optic fibers, optical switches, mirrors, lenses (including microlens and nanolens), collimators.
  • AMG arrayed waveguide gratings
  • the optical transmission element can be planar waveguides in optical communication with the arrayed optical confinements.
  • a planar waveguides can be operatively coupled to an array of zero-mode waveguides to directly channel incident wavelengths to the respective cores of the zero-mode waveguides so as to minimize the loss of wave energy.
  • the planar channel can be included as a detachable unit located at the base of array substrate, or it can be bonded to the substrate as an integral part of the array.
  • optical transmission element suitable for use in the present invention encompasses a variety of optical devices that channel light from one location to another in either an altered or unaltered state.
  • optical transmission devices include optical fibers, diffraction gratings, arrayed waveguide gratings (AWG), optical switches, mirrors, (including dichroic mirrors), lenses (including microlens and nanolens), collimators, filters, prisms, and any other devices that guide the transmission of light through proper refractive indices and geometries.
  • the optical confinement of the present invention is operatively coupled to a photon detector.
  • the arrayed optical confinement is operatively coupled to a respective and separate photon detector.
  • the confinement and the respective detector can be spatially aligned (e.g., 1:1 mapping) to permit an efficient collection of optical signals from the waveguide.
  • a particularly preferred setup comprises an array of zero-mode waveguides, wherein each of the individual waveguides is operatively coupled to a respective microlens or a nanolens, preferably spatially aligned to optimize the signal collection efficiency.
  • a combination of an objective lens, a spectral filter set or prism for resolving signals of different wavelengths, and an imaging lens can be used in an optical train, to direct optical signals from each confinement to an array detector, e.g., a CCD, and concurrently separate signals from each different confinement into multiple constituent signal elements, e.g., different wavelength spectra, that correspond to different reaction events occurring within each confinement.
  • an array detector e.g., a CCD
  • FIG. 7 An exemplary optical setup is shown in FIG. 7 , in which an array of ZMWs is optically linked to an optical system.
  • This system comprises a ZMW array film ( 81 ), a glass cover slip ( 82 ) through which light transmits and further converges through set of integral lenses ( 83 ) made of a material having a different index of refraction than that of the glass.
  • 84 shows a ZMW structure
  • 85 indicates a ray of light being focused onto the ZMW by the integral lenses such as the embedded microlens.
  • FIG. 11 depicts one alignment strategy and optical system.
  • the system comprises a photodetector 131 , an optional lens 132 for collecting light, a ZMW 133 having a metal film 134 coupled to a substrate 135 , and an objective lens 136 that is aligned with the incident light beam 137 .
  • FIG. 13 depicts an exemplary alignment detection system and the associated components.
  • the illustrative system 13 A comprises an optical confinement such as a zero-mode waveguide 111 having a metal film 113 coupled to a substrate 114 .
  • the zero-mode waveguide 111 typically contains signal generating molecules 112 , and is optically linked to the associated components including an objective lens 115 , a beam splitter/dichroic cube 117 , optically a telen lens 120 (used in infinity corrected systems), and a photodetector 122 (e.g., a quadrant photodetector).
  • 116 depicts rays propagating though system.
  • 118 depicts the incident illumination rays.
  • 119 depicts the return rays moving towards the detector 122 .
  • FIG. 13B depicts a front view of the quadrant photodiode. Shown in the center of the figure is a beam mis-aligned on the center of the quadrant detector. The four voltages generated by the four quadrants can be processed to determine the degree and direction of mis-alignment of the beam and thus the optical confinement such as ZMW 111 .
  • the subject arrays may comprise a single row or a plurality of rows of optical confinements on the surface of a substrate, where a plurality of lanes are present, for example, usually at least 2, more commonly more than 10, and more commonly more than 100.
  • the subject array of optical confinements may align horizontally or diagonally long the x-axis or the y-axis of the substrate.
  • the individual confinements can be arrayed in any format across or over the surface of the substrate, such as in rows and columns so as to form a grid, or to form a circular, elliptical, oval, conical, rectangular, triangular, or polyhedral pattern. To minimize the nearest-neighbor distance between adjacent optical confinements, a hexagonal array is preferred.
  • the array of optical confinements may be incorporated into a structure that provides for ease of analysis, high throughput, or other advantages, such as in a microtiter plate and the like.
  • Such setup is also referred to herein as an “array of arrays.”
  • the subject arrays can be incorporated into another array such as microtiter or multi-well plate wherein each micro well of the plate contains a subject array of optical confinements.
  • multi-well plates comprise multiple reaction vessels or wells, e.g., in a 48 well, 96 well, 384 well or 1536 well format. In such cases, the wells are typically disposed on 18 mm, 9 mm, 4.5 mm, or 2.25 mm centers, respectively.
  • FIG. 5 An illustrative array of arrays is depicted in FIG. 5 in which a subarray 71 is part of a super array 72 .
  • Arrays can also be arranged in lattices.
  • FIG. 4 depicts a top view of an illustrative regular disposition of ZMWs. In this configuration, there is a lattice defined by the parameters d 1 , d 2 , and the angle 53 .
  • a complex unit cell that comprises a plurality of ZMWs in an arrangement that is defined by a list of angles and distances with one angle and one distance for each element of the unit cell.
  • 52 represents the first lattice distance
  • 53 represents the lattice angle
  • 54 represents the second lattice distance
  • 55 represents the unit cell first distance
  • 56 represents unit cell first angle. While this figure shows an array with a unit cell of two components, the unit cell can have any plurality of elements.
  • the subject arrays comprise a plurality of optical confinements.
  • the arrays have at least about 20 ⁇ 10 4 distinct optical confinements, preferably at least about 20 ⁇ 10 6 distinct confinements, and more preferably at least about 20 ⁇ 10 8 confinements.
  • the density of the spots on the solid surface in certain embodiments is at least above 4 ⁇ 10 4 confinements per mm 2 , and usually at least about 8 ⁇ 10 4 , at least about 1.2 ⁇ 10 5 , or at least about 4 ⁇ 10 6 confinements per mm 2 , but does not exceed 4 ⁇ 10 12 confinements per mm 2 , and usually does not exceed about 4 ⁇ 10 10 confinements per mm 2 .
  • the overall size of the array generally ranges from a few nanometers to a few millimeters in thickness, and from a few millimeters to 50 centimeters in width or length.
  • Preferred arrays have an overall size of about few hundred microns in thickness and may have any width or length depending on the number of optical confinements desired.
  • the array of optical confinements e.g. zero-mode waveguides
  • the array comprises a representative zero-mode waveguide 21 , separated from an adjacent waveguide by a distance “d” (22 represents the inter-zero mode waveguide spacing).
  • the array of optical confinements, e.g. zero-mode waveguides are arranged in a non-square format.
  • the array comprises a representative zero-mode waveguide 31 , separated from an adjacent waveguide by a distance “d” ( 32 represents the inter-zero mode waveguide spacing).
  • 33 shows the angle formed between any three adjacent ZMWs (e.g., 60 degrees).
  • FIG. 3 depicts a top view of another illustrative 2-dimentional array.
  • the adjacent optical confinements are separated in one dimension by a distance of “d 1 ” and in another dimension by a distance of “d 2 ”, with a unit vector angle 43 .
  • the spacing between the individual confinements can be adjusted to support the particular application in which the subject array is to be employed. For instance, if the intended application requires a dark-field illumination of the array without or with a low level of diffractive scattering of incident wavelength from the optical confinements, then the individual confinements are typically placed close to each other relative to the incident wavelength.
  • the present invention provides an array of zero-mode waveguides comprising at least a first and at least a second zero-mode waveguide, wherein the first zero-mode waveguide is separated from the second zero-mode waveguide by a distance such that upon illumination with an incident wavelength, intensity of diffractive scattering observed from the first zero-mode waveguide at a given angle is less than that if the first zero-mode waveguide were illuminated with the same incident wavelength in the absence of the second zero-mode waveguide. Diffractive scattering can be reduced or significantly eliminated if an array comprises zero-mode waveguides spaced in a regular spaced lattice where the separation of zero-mode waveguides from their nearest neighbors is less than half the wavelength of the incident wavelength.
  • the structure behaves as a zero-order grating.
  • Such gratings are incapable of scattering incident light despite having a large number of elements that by themselves would scatter very effectively.
  • This arrangement is highly desirable for illumination approaches such as dark field illumination, where surface scattering would cause excitation radiation to be collected by the objective lens, thus increasing background noise.
  • Useful wavelengths for illumination range from 250 nm up to 8 microns, meaning that an array of zero-mode waveguides with a spacing of less than 4000 nm would still be useful for application in this manner. A spacing of less than 2000 nm is more preferable, while a spacing of less than 1000 nm is even more preferable in this respect.
  • Some configurations with spacing larger than one half of the wavelength can have the same advantage if the illumination is applied asymmetrically, or if the collection cone angle is configured to be less than 90 degrees. In addition to the benefit of reduced diffractive scattering, narrow spacing between the individual confinements decreases the illumination area and thus lowers the power demand.
  • Arrays having the optical confinements spaced far apart relative to the incident wavelength also have desirable properties. While the angle-dependent scattering raises the background signal that could be disadvantageous for certain applications, it provides a means particularly suited for characterizing the size and shape of the optical confinements. It also readily permits ensemble bulk measurements of molecule interactions, involving especially unlabelled molecules. Arrays suited for such applications generally contain individual confinements separated by more than one wavelength of the incident radiation, usually more than 1.5 times the incident wavelength, but usually does not exceed 150 times the incident wavelength.
  • kits containing the optical confinement arrays of this invention include those that allow characterizing molecules and/or monitoring chemical reactions at a single-molecule level.
  • Each kit usually comprises the devices and reagents which render such characterization and/or monitoring procedure possible.
  • the contents and packaging of the kit will differ.
  • the kit typically comprises: (a) an array of optical confinements, preferably zero-mode waveguides of the present invention, that permits resolution of individual molecules or the reaction of individual molecules, such as those that are present at a concentration higher than about 1 micromolar; (b) sequencing reagents typically including polymerases, aqueous buffers, salts, primers, and nucleotides or nucleotide analogs. Where desired a, ‘control’ nucleic acids of known sequence can be included to monitor the accuracy or progress of the reaction.
  • the reagents can be supplied in a solid form, immobilized form, and/or dissolved/suspended in a liquid buffer suitable for inventory storage, and later for exchange or addition into the reaction medium when the test is performed. Suitable individual packaging is normally provided.
  • the kit can optionally provide additional components that are useful in the procedure. These optional components include, but are not limited to, buffers, capture reagents, developing reagents, labels, reacting surfaces, control samples, instructions, and interpretive information. Diagnostic or prognostic procedures using the kits of this invention can be performed by clinical laboratories, experimental laboratories, practitioners, or private individuals.
  • the array of the present invention can be manufactured using nanofabrication techniques provided by the present invention, as well as those known in the fields of Integrated Circuit (IC) and Micro-Electro-Mechanical System (MEMS).
  • IC Integrated Circuit
  • MEMS Micro-Electro-Mechanical System
  • the fabrication process typically proceeds with selecting an array substrate, followed by using appropriate IC processing methods and/or MEMS micromachining techniques to construct and integrate the optical confinement and other associated components.
  • the array of optical confinements is present on a rigid substrate.
  • flexible materials can be employed.
  • a rigid support does not readily bend.
  • solid materials which are not rigid supports with respect to the present invention include membranes, flexible metal or plastic films, and the like.
  • the rigid substrates of the subject arrays are sufficient to provide physical support and structure to optical confinements present thereon or therein under the assay conditions in which the array is employed, particularly under high throughput handling conditions.
  • the substrates upon which the subject patterns of arrays are disposed may take a variety of configurations ranging from simple to complex, depending on the intended use of the array.
  • the substrate could have an overall slide or plate configuration, such as a rectangular or disc configuration, where an overall rectangular configuration, as found in standard microtiter plates and microscope slides, is preferred.
  • the thickness of the rigid substrates will be at least about 0.01 mm and may be as great as 1 cm or more, but will usually not exceed about 5 cm. Both the length and the width of rigid substrate will vary depending on the size of the array of optical confinements that are to be fabricated thereon or therein.
  • the substrates of the subject arrays may be fabricated from a variety of materials.
  • the materials from which the substrate is fabricated is preferably transparent to visible and/or UV light. Suitable materials include glass, semiconductors (e.g., silicate, silicon, silicates, silicon nitride, silicon dioxide, quartz, fused silica, and gallium arsenide), plastics, and other organic polymeric materials.
  • semiconductors e.g
  • the substrate of the subject arrays comprise at least one surface on which a pattern of optical confinements is present, where the surface may be smooth or substantially planar, or have irregularities, such as depressions or elevations.
  • the surface may be modified with one or more different layers of compounds that serve to modulate the properties of the surface in a desirable manner. Modification layers of interest include: inorganic and organic layers such as metals, metal oxides, polymers, small organic molecules, functional moieties such as avidin/biotin and the like.
  • the choice of methods for applying the coating materials will depend on the type of coating materials that is used. In general, coating is carried out by directly applying the materials to the zero-mode waveguide followed by washing the excessive unbound coating material from the surface.
  • coating materials may be deposited using other conventional techniques, such as chemical vapor deposition (CVD), sputtering, spin coating, in situ synthesis, and the like. Certain coating materials can be cross-linked to the surface via heating, radiation, and/or by chemical reactions. In preferred aspects, suitable coating materials are coupled to substrate surfaces either covalently or through ionic or hydrophobic/hydrophilic interactions. In the case of silica based substrates, for example, silane chemistries are particularly suited for covalently attaching coating materials to surfaces, e.g., coupling groups, specific binding moieties, and the like. Such chemistries are well known to those of ordinary skill in the art and can be practiced without undue experimentation.
  • Fabrication of the subject array substrates can be performed according to the methods described as follows or other standard techniques of IC-processing and/or MEMS micromachining.
  • the standard techniques known in the art include but are not limited to electron-beam lithography, photolithography, chemical vapor or physical vapor deposition, dry or wet etching, ion implantation, plasma etching, bonding, and electroplating. Additional fabrication prosesses are detailed in the U.S. Patent Application Publication No. 2003/0174992, the content of which is incorporated by reference in its entirety.
  • the present invention provides a negative tone fabrication process, which provides for the creation of optical confinements having more uniform and consistent dimensions than conventional positive tone fabrication processes that can yield optical confinements of varying dimensions.
  • Table 1 A comparison of the two fabrication processes is shown in Table 1 below.
  • a negative resist is applied to the substrate.
  • a resist is negative if it is rendered insoluble by application of some agent, wherein the case of photoresists or e-beam resists, the agent is optical energy or electron beam energy, respectively.
  • a positive tone resist can be used with a negative pattern.
  • a negative tone pattern is characterized by the application of the agent in all areas except the location of the optical confinement, e.g., zero-mode waveguide, contrasted with a positive tone image in which the agent is confined only to the optical confinement area. In either case, after development of the resist, resist remains only in the areas where the optical confinement is intended to lie. It is useful in many cases to use means to achieve an undercut sidewall profile of these remaining resist features.
  • undercut sidewalls for example, in electron beam lithography.
  • one method is to apply to layers of electron beam resist to the surface sequentially, the upper film having a higher sensitivity to the energy delivered to it by the electron beam. Because the beam has a tendency to spread, a larger area of the upper film will be rendered insoluble than in the lower layer, resulting in an overhang beneath the upper layer as desired.
  • the metal film comprising the optical confinement can be applied by one of several methods, including metal evaporation, molecular beam epitaxy and others.
  • the resist profile is undercut as discussed above, the metal that is deposited in the regions still occupied by the resist will rest on top of the resist rather than resting on the device surface.
  • the resist layer is subsequently removed by any of several techniques including solvent dissolution either with or without ultrasonication or other mechanical agitation, reactive plasma etching, vaporization or others.
  • the metal which rested on the resist features is removed as the resist is removed (“lifted off”), while the resist resting directly on the substrate remains to form the walls of the optical confinement.
  • the advantage of this process is that the size of the optical confinement is determined by the size of the resist feature, and does not rely on the fidelity of reactive ion etch pattern transfer mechanisms, which can be highly variable for metal films, especially aluminum a desirable metal for these devices.
  • the positive tone process is subject to the inherent variation in resist feature sizes plus the variation due to pattern transfer, while the negative tone process is subject to the first variability but not the second.
  • Metal thin film techniques suffer from much less lateral variation, and so the overall accuracy is better.
  • This method also does not rely on the availability of a suitable etch for the metal in question, allowing the application of the process to a much wider selection of metals than the positive tone process.
  • FIG. 6 is a schematic presentation of an illustrative negative tone process to make zero-mode waveguides.
  • the substrate 11 is first coated with a layer of negative resist 12 .
  • the substrate can be coated with a second resist layer 13 .
  • Exposure of the resist to the same pattern electron beam lithography tool used in the positive tone process generates the opposite pattern as previously observed, namely one of a periodic array of small pillars of remaining resist, and empty gaps between the pillars 15 .
  • the final zero-mode waveguide structures are created by coating this pattern with a thin metal layer such as an aluminum layer 17 , and then dissolving the underlying negative resist pillars 18 .
  • FIG. 8 depicts a scanning electron micrographs of ZMW structures fabricated by positive tone resist (left panels) or negative tone resist (right panels). The grain structure of the polycrystalline film is visible in the image as flecks, and the ZMWs as dark round structures.
  • a variant negative tone process is termed nanocasting.
  • the steps of nanocasting are similar except that the use of bi-layer resist is avoided.
  • the process first involves depositing on the surface of a substrate (in this case a single-layer resist would be used). The electron beam exposure and development follow, leaving a cylindrical feature for each dot in the exposure pattern.
  • This process is inherently three dimensional, in that a negative replica of the exterior surface of the three-dimensional resist feature is reproduced in the interior surface of the metal films that forms the optical confinement walls.
  • the undercut resist profile and the various methods used to produce this are not necessary, as in the negative tone process, they are used specifically to prevent contact of the deposited film with the sides of the resist feature.
  • the deposited film faithfully reproduces the exterior surface of the resist feature, so an undercut figure would only be used if a non-cylindrical confinement is desired.
  • the sidewalls will be very thin near the top of the resist feature, which in some instances can be a cylindrical pillar.
  • This weak point can be subject to direct mechanical disruption allowing the removal of the metal above the resist feature and hence the ZMW location.
  • An isotropic etch, either solution phase or plasma can be used to further thin the film until this weak point separates, achieving the same effect.
  • the metal deposition step has a low degree of anisotropy (such as sputtering or electroplating)
  • the resist material can be exposed through chemical mechanical polishing, or ion milling.
  • the resist material is then removed by solvent dissolution, or reactive ion etching. This completes the fabrication steps, provided the appropriate pattern is applied and the other parameters are correctly chosen.
  • the subject devices including optical confinements and associated optical systems provide a effective means for analyzing molecules and monitoring chemical reactions in real time.
  • the subject device and detection/monitoring methods may be used in a wide variety of circumstances including analysis of biochemical and biological reactions for diagnostic and research applications.
  • the present invention is applied in the elucidation of nucleic acid sequences for research applications, and particularly in sequencing individual human genomes as part of preventive medicine, rapid hypothesis testing for genotype-phenotype associations, in vitro and in situ gene-expression profiling at all stages in the development of a multi-cellular organism, determining comprehensive mutation sets for individual clones and profiling in various diseases or disease stages.
  • Other applications include measuring enzyme kinetics, and identifying specific interactions between target molecules and candidate modulators of the target molecule. Further applications involve profiling cell receptor diversity, identifying known and new pathogens, exploring diversity towards agricultural, environmental and therapeutic goals.
  • the subject devices and methods allow high-throughput single-molecule analysis.
  • Single-molecule analysis provides several compelling advantages over conventional approaches to studying biological events.
  • the analysis provides information on individual molecules whose properties are hidden in the statistically averaged information that is recorded by ordinary ensemble measurement techniques.
  • the analysis can be multiplexed, it is conducive to high-throughput implementation, requires smaller amounts of reagent(s), and takes advantage of the high bandwidth of optical systems such as modern avalanche photodiodes for extremely rapid data collection.
  • single-molecule counting automatically generates a degree of immunity to illumination and light collection fluctuations, single-molecule analysis can provide greater accuracy in measuring quantities of material than bulk fluorescence or light-scattering techniques. As such, single-molecule analysis greatly improves the efficiency and accuracy in genotying, gene expression profiling, DNA sequencing, nucleotide polymorphism detection, pathogen detection, protein expression profiling, and drug screening.
  • the present invention provides a method of simultaneously sequencing a plurality of target nucleic acids.
  • the method generally involves (a) providing an array of optical confinements of the present invention; (b) mixing in the confinements a plurality of target nucleic acid molecules, primers complementary to the target nucleic acid molecules, polymerization enzymes, and more than one type of nucleotides or nucleotide analogs to be incorporated into a plurality of nascent nucleotide strands each being complimentary to a respective target nucleus and molecules; (c) subjecting the mixture to a polymerization reaction under conditions suitable for formation of the nascent nucleotide strands by template-directed polymerization; (d) illuminating the waveguides with an incident light beam; and (e) identifying the nucleotides or the nucleotide analogs incorporated into each na
  • the subject sequencing methods can be used to determine the nucelic acid of any nucleic acid molecule, including double-stranded or single-stranded, linear or circular nucleic acids (e.g., circular DNA), single stranded DNA hairpins, DNA/RNA hybrids, RNA with a recognition site for binding of the polymerase, or RNA hairpins.
  • the methods of the present invention are suitable for sequencing complex nucleic acid structures, such as 5′ or 3′ non-translation sequences, tandem repeats, exons or introns, chromosomal segments, whole chromosomes or genomes.
  • the temporal order of base additions during the polymerization reaction is identified on a single molecule of nucleic acid. Such identifying step takes place while the template-directed extension of primer or polymerization is taking place within the optical confinement.
  • single-molecule sequencing is performed in a homogenous assay that does not require transfer, separation, or washing away any reactant or by-product (e.g. fluorophore cleaved from a nucleotide) after each base addition event. In certain aspects of the homogenous assay, single-molecule sequencing is performed without adding reactants to the mixture prior to reading the next base sequence.
  • a polymerase enzyme is provided anchored within the effective observation volume within an optical confinement. Template dependent synthesis of a complementary strand is then carried out while observing the volume, and using labeled nucleotide analogs that are capable of being sequentially incorporated into the growing strand without interruption, e.g., for deprotection, etc.
  • nucleotide analogs bearing a label on a non-incorporated phosphate group or derivative, e.g., the beta, gamma, delta, etc. phosphate of a nucleotide polyphosphate which is cleaved from the analog during incorporation are used in such methods.
  • nucleotide analogs provide an advantage of being sequentially incorporated into the growing nucleic acid strand, and having their labeling groups removed in the incorporation process so as to not provide increasing signal noise during synthesis that would result if such labels remained associated with the synthesized strand.
  • incorporation event provides for prolonged presence of the labeled analogs within the observation volume (as compared to random diffusion of non-incorporated analogs into the observation volume), the signal associated within incorporation is readily identifiable.
  • a template nucleic acid is used that provides for the redundant or iterative reading/synthesis of tandem repeats of a particular sequence segment of interest.
  • the systems of the invention typically provide for redundancy in numerous ways, to correct for any errors that may arise in template dependant synthesis by the polymerase enzyme.
  • the methods of the invention focus on single molecules, redundant processes are employed to assure that mis-incorporation events by a polymerase are corrected for in data analysis.
  • such redundancy is supplied by utilizing arrays of multiple different confinements that are being applied to a given sequence of interest, e.g., in a single well of a multi-well plate.
  • the invention also provides for the iterative sequencing of a given sequence segment (or a copy thereof) multiple times within a single confinement.
  • iterative sequencing may be accomplished by providing the sequence segment of interest in a circular template format, so that the polymerase processes around the circular template (allowing the elucidation of the sequence of such template) multiple times.
  • a similar result is accomplished by using a template-dependant circular template bearing the sequence segment of interest.
  • synthesis product will typically include, in a single linear strand, multiple copies of the circular template, again, providing for iterative sequencing of the sequence segment of interest. Further, redundancy is additionally accomplished by circularizing this linear, multi-copy template and iteratively sequencing multiple copies, multiple times.
  • a similar result is obtained by performing concatmerization of amplicons generated in a single-molecule amplification strategy, several of which are known to those skilled in the art. These strategies can employ dilution to the single molecule level, or isolation of molecules in small micelles in a two-phase emulsion during amplification. The concatmerized strand is then sequenced as a single template, and redundant information is generated from a single molecule in this fashion.
  • a similar result is obtained by using a long double stranded template with nicks and/or gaps at multiple locations along it.
  • the molecule can then be caused to initiate single molecule sequencing at several locations along the strand, each location comprising a confinement that independently sequences the strand. Because the several confinements are acting on the same strand, the result is that the same template is sequenced several times providing redundant information from a single molecule.
  • a reaction mixture comprising the target nucleic acid(s) of interest, primers complementary to the target nucleic acids, polymerization enzymes, and more than one type of nucleotides or nucleotide analogs, is applied to an array of optical confinements.
  • each optical confinement receives only one target nucleic acid molecule that is to be sequenced. This can be achieved by diluting a minute amount of target nucleic acids in a large volume of solution containing the rest of the reactants required for the sequencing process.
  • a non-cylindrical waveguide whose the opening is narrower in lateral dimension than the base, can be used to restrict the entry of multiple target nucleic acids.
  • the target nucleic acid can be immobilized to the inner surface of the optical confinement by a number of ways.
  • the target nucleic acid can be immobilized onto an optical confinement by attaching (1) a primer or (2) a single-stranded target nucleic acid or (3) double-stranded or partially double-stranded target nucleic acid molecule.
  • the target nucleic acid molecule is hybridized to the attached oligonucleotide primer, (2) an oligonucleotide primer is hybridized to the immobilized target nucleic acid molecule to form a primed target nucleic acid molecule complex, or (3) a recognition site for the polymerase is created on the double-stranded or partially double-stranded target nucleic acid (e.g., through interaction with accessory proteins, such as a primase).
  • a nucleic acid polymerizing enzyme on the primed target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at the site of polymerization.
  • the polymerization enzyme is first attached to a surface of the subject optical confinement within the effective observation volume of the confinement, and in a position suitable for the target nucleic acid molecule complex to move relative to the polymerization enzyme.
  • Non-limiting exemplary binding moieties for attaching either nucleic acids or polymerases to a solid support include streptavidin or avidin/biotin linkages, carbamate linkages, ester linkages, amide, thiolester, (N)-functionalized thiourea, functionalized maleimide, amino, disulfide, amide, hydrazone linkages, and among others.
  • Antibodies that specifically bind to the target nucleic acids or polymerases can also be employed as the binding moieties.
  • a silyl moiety can be attached to a nucleic acid directly to a substrate such as glass using methods known in the art.
  • the polymerases may be modified to contain one or more epitopes such as Myc, HA (derived from influenza virus hemagglutinin), poly-histadines, and/or FLAG, for which specific antibodies are available commercially.
  • the polymerases can be modified to contain heterologous domains such as glutathione S-transferase (GST), maltose-binding protein (MBP), specific binding peptide regions (see e.g., U.S. Pat. Nos. 5,723,584, 5,874,239 and 5,932,433), or the Fc portion of an immunoglobulin.
  • GST glutathione S-transferase
  • MBP maltose-binding protein
  • specific binding peptide regions see e.g., U.S. Pat. Nos. 5,723,584, 5,874,239 and 5,932,433
  • the respective binding agents for these domains namely glutathione, maltose, and antibodies directed to the Fc portion
  • binding moieties or agents of either the polymerases or nucleic acids they immobilize can be applied to the support by conventional chemical techniques which are well known in the art. In general, these procedures can involve standard chemical surface modifications of a support, incubation of the support at different temperature levels in different media comprising the binding moieties or agents, and possible subsequent steps of washing and cleaning.
  • nucleotides utilized in accordance with the single-molecule sequencing method are conjugated with detectable labels so that a photon detector can detect and distinguish their presence within the subject optical confinements.
  • Preferred labels are luminescent labels, and especially fluorescent or chromogenic labels.
  • fluorophores suitable for the present sequencing method include but are not limited to 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Couma
  • rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives. Additional fluorophores applicable for the subject sequencing methods
  • the labels can be attached to the phosphate backbone, on the base, on the ribose unit, or a combination thereof.
  • Preferred labels are those that do not substantially impede the continuous addition of nucleotides in a sequencing reaction.
  • Such labels include those linked to the alpha phosphate, the beta phosphate, the terminal phosphate, or the delta or more distal phosphates in tetra, penta or hexa phosphate nucleotides, or the base unit of a nucleotide.
  • Nucleotides comprising labeled terminal phosphates are particularly preferred because no additional means is required to remove the label in the sequencing procedure.
  • the bond cleavage in the nucleotide occurs between the alpha and the beta phosphate, causing the beta and terminal phosphate (e.g., the gamma phosphate as in dNTP) to be released from the site of polymerization.
  • the label attached to the terminal phosphate is separated from the nascent strand once the nucleotide is being incorporated.
  • terminal-phosphate-linked nucleotides may comprise three or more phosphates, typically about three to about six phosphates, preferably about three to about five phosphates.
  • Table 1 lists numerous examples of nucleotides with labeled terminal phosphates. Many other terminal-phosphate-linked nucleotides have been developed and are detailed in U.S. patent application No. 2003/0124576, which is incorporated herein by reference in its entirety.
  • Nucleotides comprising modified phosphate backbones can also be used.
  • the modified component can be a phosphordiamidate, methylphosphonate, alkyl phosphotriester, formacetal, phosphorodithioate, phosphothioate, phosphoramidothioate, phosphoramidate, or an analog thereof.
  • the nucleotides or nucleotide analogs used in the present invention are reversible extension terminators comprising reversible blocking groups.
  • the blocking group on a reversible extension terminator is linked to a detectable label.
  • the blocking group and the detectable label are located on different positions of a nucleotide.
  • the blocking group is also a label.
  • An illustrative reversible extension terminator comprises a labeled ribose unit at the 3′ end.
  • Each label on the ribose unit typically acts as a reversible blocking group that must be removed before the next nucleotide addition event can take place during a polymerization reaction.
  • Preferred 3′-ribose labels comprise photo-removable functional groups that can be deprotected upon exposure to a light beam at a suitable wavelength.
  • the reversible blocking group is located at the 2′ or the 4′ position of the ribose unit of a nucleotide.
  • the reversible blocking group is linked to or conjugated to the base (adenine, thymine, cytosine, guanine, or uracil) a nucleotide.
  • Non-limiting examples of reversible blocking groups, and especially photocleavable blocking groups include but are not limited to those molecules depicted in FIGS. 14 and 15 and those described in the co-pending application Ser. No. 60/649,009, which is incorporated herein by reference in its entirety.
  • the wavelength used to cleave the photocleavable blocking groups will depend on the choice of the blocking group.
  • the wavelength may range from about 320 nm to about 800 nm.
  • the wavelength for cleaving the blocking group is about the same as the wavelength used to detect the label. In other embodiments, the wavelength for cleaving the blocking group is different from the wavelength used to detect the label.
  • composition is prepared by treating a mixture comprising labeled and unlabeled nucleotides or nucleotide analogs with an agent that specifically modifies unlabeled or incorrectly labeled nucleotides or nucleotide analogs to reduce their ability to be used in a hybridization or sequencing assay.
  • the agent used specifically modifies unlabeled or incorrectly labeled nucleotides analogs to render them incapable of being used in a hybridization or sequencing assay.
  • the nucleotides can be modified so that they no longer contain structures generally needed for the Watson Crick base pairing in a hybridization or template-directed sequencing assay.
  • base units of the nucleotides are modified.
  • phosphate groups, preferably terminal phosphate groups, of the nucleotides or nucleotide analogs are modified to yield molecules that are incorporated to a lesser extent into a nascent nucleic acid strand during a template-directed polymerization reaction.
  • the terminal phosphate groups of a nucleotide or nucleotide analogs are modified to yield molecules that cannot or that substantially cannot be incorporated into a nascent nucleic acid strand during a template-directed polymerization reaction.
  • the agents can comprise one or more enzymes.
  • a variety of enzymes known in the art are suitable for modifying the nucleotides or nucleotide analogs, e.g. by cleaving or altering the configuration of the sugar, base, or phosphates, so as to disrupt the specific Watson Crick base pairing.
  • Exemplary agents include but are not limited to guanine or adenine P-ribosyl transferase, purine nucleoside phosphorylase, AMP nuleosidase, nucleoside deoxyribosyl transferase for purines, and orotate P-ribosyl transferase, thymidine phosphorylase, thymidine or uridine nucleosidase, uridine phosphorylase, pyrimidine nucleoside phosphorylase nucleoside deoxyribosyl transferase.
  • Enzymes applicable for modifying the terminal phosphate groups of nucleotides or nucleotide analogs include a wide array of phosphatases.
  • An example of such enzyme is Shrimp Alkaline Phosphatase (SAP) that can remove the gamma and beta phosphates from a deoxynucleoside triphosphate (dNTP).
  • SAP Shrimp Alkaline Phosphatase
  • dNTP deoxynucleoside triphosphate
  • the enzyme can convert specifically unlabeled dNTP into a nucleoside monophosphate dNMP which is generally incapable of being utilized by a polymerase enzyme in a template-directed sequencing reaction. It has been shown, that this phosphatase selectively modify nucleotides that are not labeled, e.g. at the terminal phosphate.
  • the SAP will preferentially act on unlabeled nucleotides, leaving a larger proportion of labeled nucleotides available for incorporation in a sequencing reaction.
  • Suitable phosphatases that can be used include but are not limited to calf intestinal alkaline phosphatases, and/or phosphatases of other mammals, crustaceans, and other animals. Examples of phosphatases that may be useful practicing the present invention can be found in U.S. 20040203097, U.S. 20040157306, U.S. 20040132155; and U.S. 20040110180.
  • Any other naturally occurring or synthetic phosphatases or phosphatases made by recombinant DNA technology can also be used so long as they specifically or preferentially convert unlabeled nucleotides or analogs (as compared to labeled nucleotides), to molecules that are substantially incapable of being utilized by a polymerization enzyme.
  • Directed molecular evolution can also be used to enhance and extend the activity of related enzymes to yield the desired property described above.
  • a wide variety of mutagenesis techniques, both in silicon and in situ, are available in the art.
  • mutagenesis or screening assay for generating such enzymes can involve a first test for abrogation of polymerization in the system with unlabeled nucleotides, and a second screen checking for the retention of polymerization activity in the presence of labeled nucleotides. Both of these screens can be performed in the context of a highly multiplexed parallel assay. Enzymes showing some beneficial specificity can be retained, mutated by some method, and then re-screened. Methods such as these have been shown to produce many orders of magnitude improvement in specificity and performance.
  • Enzymes capable of selectively or preferentially modifying a subset of unlabeled nucleotides can also be employed.
  • creatine kinase enzyme is specific for the removal of a phosphate from adenoside triphosphate, and will not act on other bases.
  • Other enzymes that selectively or preferentially act on one or more types of unlabeled nucleotides can also be used.
  • nucleotide modifying enzymes described above can be used to pre-treat the nucleotides or nucleotide analogs, or can be used in the hybridization and/or sequencing reaction mixture, e.g., along with other hybridization or sequencing reagents.
  • the reaction conditions under which the modification of the nucleotides takes place will vary depending on the choice of the modifying enzymes.
  • the conditions may be set within the following parameters: pH is between 4.0 and 12.0, more preferably between pH 6.0 and 10.0, more preferably between 7.0 and 9.0, more preferably less than 8, more preferably between 7 and 8, and most preferably pH 7.5 and 8.5, preferably controlled by a buffer.
  • the buffer can be Tris-based preferably at pH 7.5 to pH 8.5.
  • Other buffers may be used such as, but not limited to: organic buffers such as MOPS, HEPES, TRICINE, etc., or inorganic buffers such as phosphate or acetate.
  • Buffers or other agents may be added to control the pH of the solution thereby increasing the stability of the enzymes.
  • reducing agent such as but not limited to dithiotreitol (DTT) or 2-mercaptoethanol may be added to limit enzyme oxidation that might adversely affect stability of the enzymes.
  • DTT dithiotreitol
  • 2-mercaptoethanol may be added to limit enzyme oxidation that might adversely affect stability of the enzymes.
  • the enzymes can be heat-inactivated by raising the reaction temperature to at least about 65° C., preferably between about 65° C. to about 80° C.
  • the enzymes can be depleted from the reaction mixture by, e.g., centrifugation through a filter (e.g., Millipore) that has a molecular weight cutoff smaller than the size of the enzyme.
  • the mixture generally comprises less than about 30%, preferably less than about 20%, more preferably less than about 10%, more preferably less than about 5%, more preferably less than about 1%, more preferably less than about 0.5%, or more preferably less than about 0.1%, and even more preferably less than 0.01% of unlabeled nucleotides or unlabeled nucleotide analogs.
  • This enriched mixture of labeled nucleotides or nucleotide analogs is particularly useful for high-resolution detection of the labeled nucleotides in a single-molecule sequence reaction.
  • the result of the foregoing treatment is a process for synthesis of nucleic acids, preferably for elucidating a template sequence using substantially only nucleotides, e.g., substantially complete replacement of native nucleotides with nucleotide analogs, and particularly labeled analogs.
  • nucleotides for the subject sequencing methods allows detection via fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • an excited fluorophore the donor
  • a light absorbing molecule the acceptor
  • Nucleotides of this type can comprise a donor fluorophore attached to the base, ribose or preferably the phosphate backbone (e.g., attached to the terminal phosphate), and an acceptor fluorophore attached to the base, ribose or the phosphate backbone where the donor is not attached.
  • the donor fluorophore is attached to the terminal phosphate, and an acceptor fluorophore is linked to the base or the ribose unit of the nucleotide.
  • a fluorescent signal can be detected which can be caused by the release of poly-phosphate that is no longer quenched.
  • the donor fluorophore can be present in a nucleotide, and the acceptor is located in the polymerase, or vice versa.
  • the fluorophore in the polymerase can be provided by a green fluorescent protein (GFP) or a mutant thereof that has a different emission and/or absorption spectrum relative to the wildtype green fluorescent protein.
  • GFP green fluorescent protein
  • the GFP mutant H9-40 (Tsien et al., Ann. Rev. Biochem. 67: 509 (1998)) which is excited at 399 nm and emits at 511 nm, may serve as a donor fluorophore for use with BODIPY, fluorescein, rhodamine green and Oregon green.
  • tetramethylrhodamine, LissamineTM, Texas Read and napthofluorescein can be used as acceptor fluorophores with this GFP mutant.
  • Other representative donors and acceptors capable of fluorescence energy transfer include, but are not limited to, 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine and derivatives: acridine, acridine isothiocyanate; 5-(2′-aminoethyl)aminonap-hthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphth-alimide-3,5 disulfonate; N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; Brilliant Yellow; coumarin and derivatives: coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanine dyes; cyano
  • both donor and acceptor fluorophores may be present upon each nucleotide analog, where the donor provides a substantially uniform excitation spectrum, but donates energy to an acceptor that provides an emission spectrum that is different for each type of analog, e.g., A, T, G, or C.
  • Such configurations provide an ability to utilize a single excitation source for multiple different emission profiles, reducing energy input requirements for the systems utilized.
  • xanthene dyes including fluoresceins and rhodamine dyes can be used as donor and acceptor pairs. Many of these dyes contain modified substituents on their phenyl moieties which can be used as the site for bonding to the terminal phosphate or the base of a nucleotide.
  • acceptors acting as quenchers capable of quenching a wide range of wavelengths of fluorescence can be used. Representative examples of such quenchers include 4-(4′-dimethylaminophenylaz-o)-benzoic acid (DABCYL), dinitrophenyl (DNP) and trinitrophenyl (TNP).
  • the polymerization enzymes suitable for the present invention can be any nucleic acid polymerases that are capable of catalyzing template-directed polymerization with reasonable synthesis fidelity.
  • the polymerases can be DNA polymerases or RNA polymerases, a thermostable polymerase or a thermally degradable polymerase wildtype or modified.
  • thermostable polymerases include polymerases from Thermus aquaticus, Thermus caldophilus, Thermusfiliformis, Bacillus caldotenax, Bacillus stearothermophus, Thermus thermophilus, Pyrococcus woesei, Pyrococcus furiosus, Thermococcus litoralis , and Thermotoga maritima .
  • Useful thermodegradable polymersases include E. coli DNA polymerase, the Klenow fragment of E. coli DNA polymerase, T4 DNA polymerase, T7 DNA polymerase.
  • polymerization enzymes that can be used to determine the sequence of nucleic acid molecules include E. coli T7, T3, SP6 RNA polymerases and AMV, M-MLV and HIV reverse transcriptases.
  • the polymerase can be bound to the primed target nucleic acid sequence at a primed single-stranded nucleic acid, an origin of replication, a nick or gap in a double-stranded nucleic acid, a secondary structure in a single-stranded nucleic acid, a binding site created by an accessory protein, or a primed single-stranded nucleic acid.
  • the polymerization enzymes exhibit enhanced efficiency as compared to the wildtype enzymes for incorporating unconventional or modified nucleotides, e.g., nucleotides linked with fluorophores.
  • Recombinant DNA techniques can be used to modify the wildtype enzymes. Such techniques typically involve the construction of an expression vector or a library of expression vector, a culture of transformed host cells under such condition such that expression will occur. Selection of the polymerases that are capable of incorporating unconventional or modified nucleotides can be carried out using any conventional sequencing methods as well as the sequencing methods disclosed herein.
  • sequencing is carried out with polymerases exhibiting a high degree of processivity, i.e., the ability to synthesize long stretches of nucleic acid by maintaining a stable nucleic acid/enzyme complex.
  • a processive polymerase can typically synthesize a nascent strand over about 10 kilo bases. With the aid of accessory enzymes (e.g., helicases/primases), some processive polymerases can synthesize even over 50 kilobases.
  • T7 DNA polymerase complexed with helicase/primase can synthesize several 100 kilobases of nucleotides while maintaining a stable complex with the target nucleic acid (Kelman et al., “Processivity of DNA Polymerases: Two Mechanisms, One Goal” Structure 6: 121-125 (1998)).
  • sequencing is performed with polymerases capable of rolling circle replication, i.e., capable of replicating circular DNA templates including but not limited to plasmids and bacteriophage DNA.
  • a preferred rolling circle polymerase exhibits strand-displacement activity, and preferably has reduced or essentially no 5′ to 3′ exonuclease activity. Strand displacement results in the synthesis of tandem copies of a circular DNA template, thus allowing re-sequencing the same DNA template more than once. Re-sequencing the same DNA template greatly enhances the chances to detect any errors made by the polymerase, because the same errors unlikely would be repeated by the polymerase and the same error certainly would not be exponentially amplified as in a polymerase chain reaction.
  • Non-limiting examples of rolling circle polymerases suitable for the present invention include but are not limited to T5 DNA polymerase (Chatterjee et al., Gene 97:13-19 (1991)), and T4 DNA polymerase holoenzyme (Kaboord and Benkovic, Curr. Biol. 5:149-157 (1995)), phage M2 DNA polymerase (Matsumoto et al., Gene 84:247 (1989)), phage PRD1 DNA polymerase (Jung et al., Proc. Natl. Aced. Sci. USA 84:8287 (1987), and Zhu and Ito, Biochim. Biophys. Acta. 1219:267-276 (1994)), Klenow fragment of DNA polymerase I (Jacobsen et al., Eur. J. Biochem. 45:623-627 (1974)).
  • a preferred class of rolling circle polymerases utilizes protein priming as a way of initiating replication.
  • Exemplary polymerases of this class are modified and unmodified DNA polymerase, chosen or derived from the phages ⁇ 29, PRD1, Cp-1, Cp-5, Cp-7, ⁇ 15, ⁇ 1, ⁇ 21, ⁇ 25, BS 32 L17, PZE, PZA, Nf, M2Y (or M2), PR4, PR5, PR722, B103, SF5, GA-1, and related members of the Podoviridae family.
  • the wildtype bacteriophage ⁇ 29 genome consists of a linear double-stranded DNA (dsDNA) of 19,285 base pairs, having a terminal protein (TP) covalently linked to each 5′end.
  • dsDNA linear double-stranded DNA
  • TP terminal protein
  • a histone-like viral protein forms a nucleoprotein complex with the origins of replication that likely contributes to the unwinding of the double helix at both DNA ends (Serrano et al., The EMBO Journal 16(9): 2519-2527 (1997)).
  • the DNA polymerase catalyses the addition of the first dAMP to the hydroxyl group provided by the TP.
  • This protein-primed event occurs opposite to the second 3′ nucleotide of the template, and the initiation product (TP-dAMP) slides back one position in the DNA to recover the terminal nucleotide After initiation, the same DNA polymerase replicates one of the DNA strands while displacing the other.
  • TP-dAMP initiation product
  • the high processivity and strand displacement ability of ⁇ 29 DNA polymerase makes it possible to complete replication of the ⁇ 29 TP-containing genome (TP-DNA) in the absence of any helicase or accessory processivity factors (reviewed by Serrano et al., The EMBO Journal 16(9): 2519-2527 (1997)).
  • Modified ⁇ 29 DNA polymerases having reduced 5′ to 3′ exonuclease activity have also been described (U.S. Pat. Nos. 5,198,543 and 5,001,050, both being incorporated herein). These polymerases are particularly desirable for sequencing as the 5′ to 3′ exonucleases, if present excessively, may degrade the nascent strand being synthesized.
  • Strand displacement can be enhanced through the use of a variety of accessory proteins. They include but are not limited to helicases (Siegel et al., J. Biol. Chem. 267:13629-13635 (1992)), herpes simplex viral protein ICP8 (Skaliter and Lehman, Proc. Natl, Acad. Sci. USA 91(22):10665-10669 (1994)), single-stranded DNA binding proteins (Rigler and Romano, J. Biol. Chem. 270:8910-8919 (1995)), adenovirus DNA-binding protein (Zijderveld and van der Vliet, J. Virology 68(2):1158-1164 (1994)), and BMRF1 polymerase accessory subunit (Tsurumi et al., J. Virology 67(12):7648-7653 (1993)).
  • helicases Siegel et al., J. Biol. Chem. 267:13629-13635 (1992
  • the sequence reaction involves a single complex of strand-displacement polymerization enzyme and a circular target DNA, which is immobilized to an optical confinement.
  • the strand-displacement polymerization enzyme directs the synthesis of a nascent strand and a time sequence of incorporating the various types of labeled nucleotides or nucleotide analogs into the nascent strand is registered.
  • the strand-displacement polymerase is allowed to synthesize multiple tandem repeats of the target DNA, and thus effecting re-sequencing the same circular DNA target multiple times.
  • This multiple rounds of or redundant sequencing can take place under an isothermal condition and/or at ambient temperature.
  • sequencing can be carried out at the speed of at least 1 base per second, preferably at least 10 bases per second, more preferably at least 100 bases per second. It has been reported that polymerases can polymerize 1,000 bases per second in vivo and 750 bases per second in vitro (see, e.g. Kelman et al., “Processivity of DNA Polymerases: Two Mechanisms, One Goal,” Structure 6: 121-125 (1998); Carter et al., “The Role of Exonuclease and Beta Protein of Phage Lambda in Genetic Recombination. II. Substrate Specificity and the Mode of Action of Lambda Exonuclease,” J. Biol. Chem.
  • the sequencing procedures of the present invention are performed under any conditions such that template-directed polymerization can take place using a polymerization enzyme.
  • the substrates of the polymerization enzyme namely the various types of nucleotides present in the sequence reaction, are adjusted to a physiologically relevant concentration.
  • the nucleotides used in the sequencing reaction are present at a concentration about Michaelis constant of the polymerization enzyme. Such concentration typically ranges from about 1 micromolar to about 50 micromolar or about 100 micromolar.
  • the sequencing procedures can also be accomplished using less than four labels employed. With three labels, the sequence can be deduced from sequencing a nucleic acid strand (1) if the fourth base can be detected as a constant dark time delay between the signals of the other labels, or (2) unequivocally by sequencing both nucleic acid strands, because in this case one obtains a positive fluorescence signal from each base pair.
  • Another possible scheme that utilizes two labels is to have one base labeled with one fluorophore and the other three bases with another fluorophore. In this case, the other three bases do not give a sequence, but merely a number of bases that occur between the particular base being identified by the other fluorophore.
  • the sequencing procedures can be performed under an isothermal condition, at ambient temperature, or under thermal cycling condition.
  • the choice of buffers, pH and the like is within the skill of practitioners in the art, and hence is not detailed herein.
  • the subject sequencing method requires the imaging of individual molecules confined in an optical confinement.
  • the polymerase and/or the nucleotides are labeled with fluorophores that emit a distinguishable optical signal when a particular type of nucleotide is incorporated into the nascent strand.
  • the sequence of the distinguishable signals is detected as the nucleotides are sequentially added to the nascent strand within the optical confinement.
  • such detection is performed without the need to transfer, separation or washing away any reactant or by-product (e.g. fluorophore cleaved from a nucleotide) after each nucleotide addition event.
  • sequence detection is performed without adding reactants to the mixture prior to reading the next base sequence nucleotide to be incorporated.
  • Imaging individual molecules confined in the subject optical confinements is performed with the aid of an optical system.
  • Such system typically comprises at least two elements, namely an excitation source and a photon detector. Numerous examples of these elements are described above.
  • the excitation source is a laser, preferably a polarized laser.
  • the choice of laser light will depend on the fluorophores attached to the different type of nucleotides and/or the polymerases. For most of the flurophorescent compounds, the required excitation light is within the range of about 300 nm to about 700 nm. For proteinaceous fluorophores such as green-flurorescent protein and mutants thereof, the excitation wavelength may range from about 488 nm to about 404 nm.
  • MPM multiphoton micropscopy
  • MPM is a form of laser-scanning microscopy that uses localized nonlinear excitation to excite fluorescence within a thin raster-scanned plane.
  • MPM as in conventional laser-scanning confocal microscopy, a laser is focused and raster-scanned across the sample. The image consists of a matrix of fluorescence intensity measurements made by digitizing the detector signal as the laser sweeps back and forth across the sample. Two-photon excitation probabilities are extremely small, and focusing increases the local intensity at the focal point.
  • a preferred MPM setup comprises MPM laser scanning microscopes and second-harmonic imaging, equipped with femtosecond mode-locked titanium sapphire lasers operating at wavelengths from about 700 to 1,000 nm. Such setup can capture more than about 100 photons per pixel in most of the conventional imaging multiphoton microscope.
  • the sequence of the distinguishable signals can also be detected by other optical systems comprising elements such as optical reader, high-efficiency photon detection system, photo multiplier tube, gate sensitive FET's, nano-tube FET's, photodiode (e.g. avalanche photo diodes (APD)), camera, charge couple device (CCD), electron-multiplying charge-coupled device (EMCCD), intensified charge coupled device (ICCD), and confocal microscope.
  • elements such as optical reader, high-efficiency photon detection system, photo multiplier tube, gate sensitive FET's, nano-tube FET's, photodiode (e.g. avalanche photo diodes (APD)), camera, charge couple device (CCD), electron-multiplying charge-coupled device (EMCCD), intensified charge coupled device (ICCD), and confocal microscope.
  • optical reader high-efficiency photon detection system
  • photo multiplier tube gate sensitive FET's
  • nano-tube FET's nano-tube FET's
  • a preferred combination comprises wide field CCD or ICCD and intensified video imaging microscopes with digital image processing capability, as well as Fluorescence Photobleaching Recovery (FPR) and Fluorescence Correlation Spectroscopy (FCS) coupled with confocal multiphoton capability and continuous data acquisition and control.
  • Such set up may further comprise modular instrument for quasi-elastic light scattering, laser DIC interferometry, correlation spectroscopy instrumentation, components of optical force microscopy, and Time Correlated Single Photon Counting (TCSPC).
  • optical systems may also comprise optical transmission elements such as diffraction gratings, arrayed waveguide gratings (AWG), optic fibers, optical switches, mirrors, lenses (including microlens and nanolens), collimators.
  • optical transmission elements such as diffraction gratings, arrayed waveguide gratings (AWG), optic fibers, optical switches, mirrors, lenses (including microlens and nanolens), collimators.
  • Other examples include optical attenuators, polarization filters (e.g., dichroic filter), wavelength filters (low-pass, band-pass, or high-pass), wave-plates, and delay lines.
  • the optical transmission element can be planar waveguides in optical communication with the arrayed optical confinements.
  • the preferred system can collect and process signals from more than 10 4 optical confinements, more than 2 ⁇ 10 4 optical confinements, or more than 10 5 optical confinements, or more than 2 ⁇ 10 5 optical confinements, or preferably more than 10 6 , or preferably more than 2 ⁇ 10 6 optical confinements, and even more preferably more than 10 7 or 2 ⁇ 10 7 optical confinements.
  • the preferred setup can monitor in real time the simultaneous and independent sequencing of nucleic acids at a speed of about 1 base per second, preferably at a speed of about 10 bases per second, more preferably at a speed of about 100 bases per second and even more preferably at 1,000 bases per second.
  • the massive parallelism coupled with the rapid sequencing reaction can provide an overall sequencing output greater than 100,000 bases per second.
  • the overall output can be scaled up to at least 1 megabase per second, preferably 10 or more megabases per second. Further by obtaining such date from multiple different sequence fragments e.g., in from two or more different reaction volumes, one can obtain independent sequences, e.g. from contiguous fragments of genomic DNA, allowing the high rate of throughput that is directly applicable to genomic sequencing.
  • the subject optical confinements and arrays of optical confinements find utility in many other chemical and biological applications where single molecule analyses are desired.
  • the subject optical confinements are applicable for any single molecule analysis involving any reagent that can be attached to the surface and for which substrates can be labeled, including, enzymes, nucleic acids, antibodies, antigens, and the like.
  • Such applications include discerning interactions involving biological molecules such as proteins, glycoproteins, nucleic acids, and lipids, as well as inorganic chemicals, or any combinations thereof. The interactions may be between nucleic acid molecules, between nucleic acid and protein, and between protein and small molecules.
  • Abnormalities in interactions involving biological molecules have long been acknowledged to account for a vast number of diseases including, numerous forms of cancer, vascular diseases, neuronal, and endocrine diseases.
  • An abnormal interaction in form of e.g., constitutive activation and premature inactivation of a signaling complex, are now known to lead to aberrant behavior of a disease cell.
  • abnormal interactions between two signaling transduction molecules such as growth factor receptors and their corresponding ligands, may result in dysfunction of cellular processes, which ultimately lead to dysregulated growth, lack of anchorage inhibition, genomic instability and/or propensity for cell metastasis.
  • a specific interaction between biological or chemical molecules typically involves a target molecule that is being investigated and a probe suspected to be able to specifically interact with the target.
  • the target and the probe are placed within an optical confinement.
  • the target-probe complex can be a protein-protein complex, a glycoprotein-protein complex (e.g., receptor and ligand complex), a protein-nucleic acid complex (e.g., transcription factor and nucleic acid complex), a protein-lipid complex, and complex of inorganic or organic small molecules.
  • each optical confinement contains only one target that is being investigated. This can be achieved by diluting a minute amount of target in a large volume of solution, such that deposition over an array of confinements results in a primary distribution, or a majority of confinements will have a single target molecule disposed there.
  • a non-cylindrical waveguide wherein the opening of the waveguide core is narrower in lateral dimension than the base, can be used to restrict the entry of multiple target proteins while permitting the entry of a number of smaller probes.
  • the target or probe can be immobilized onto the inner surface of the optical confinement by any of the methods applicable for immobilizing and depositing the polymerases described in the section above. Such methods encompass the uses of covalent and noncovalent attachments effected by a variety of binding moieties.
  • the choice of the binding moieties will depend on the nature of the target and/or the probe.
  • the binding moieties can be synthetically linked to the protein target or the probe, or made as a fusion motif or tag via a recombinant means.
  • a preferred way to immobilize the target protein or the proteinaceous probe involves the use of the streptavidin or avidin/biotin binding pair, and any other binding moieties or agents described above.
  • the reaction conditions will depend on the particular interaction that is under investigation.
  • To determine the thermal stability of the target-probe complex one may vary the reaction temperature. Stability of the target-probe complex can also be determined by varying the pH, or buffer salt concentration.
  • the interaction can be studied under physiologically relevant temperature and buffer conditions.
  • a physiologically relevant temperature ranges from approximately room temperature to approximately 37° C.
  • a physiological buffer contains a physiological concentration of salt at neutral pH ranging from about 6.5 to about 7.8, and preferably from about 7.0 to about 7.5. Adjusting the reaction conditions to discern a particular interaction in vitro between a given target and a probe is within the skill of artisans in the field, and hence is not detailed herein.
  • the target and/or the probe are generally labeled with detectable labels so that a photon detector can detect a signal indicative of their interaction.
  • Suitable labels encompass all of those labels disclosed in the Single-Molecule Sequencing section.
  • Preferred labels are luminescent labels, and especially fluorescent or chromogenic labels.
  • the target is labeled with a fluorophore whose signal is quenched upon interaction with the corresponding probe conjugated with an appropriate quencher.
  • a fluorophore whose signal is quenched upon interaction with the corresponding probe conjugated with an appropriate quencher.
  • suitable fluorophore-quencher pairs is disclosed in the section above and hence is not detailed herein.
  • a variation of this embodiment is to label the target and the probe with donor and acceptor fluorphores (or vise versa) that emit a distinguishable signal when the two molecules bind to each other.
  • donor and acceptor fluorphores or vise versa
  • a wide range of applicable donor and acceptor fluorophores is also described above. Those of skill in the art will appreciate the wide diversity of detectable labels and the combinations thereof to generate a distinguishable signal that is indicative of a specific interaction between biological molecules and/or chemical compounds.
  • the detection of the distinguishable signal indicative of a specific interaction is performed with the aid of the optical systems described herein. Any of the systems applicable for single-molecule sequencing is equally suited for detecting interactions between other biological molecules and/or chemical compounds.
  • a preferred system allows parallel data collection using arrays having a large number of optical confinements, where simultaneous and independent target-probe interactions can take place.
  • the preferred system can collect and process signals from more than 10 4 optical confinements, more than 2 ⁇ 10 4 optical confinements, more than 10 5 optical confinements, more than 2 ⁇ 10 5 optical confinements, preferably more than 10 6 , or preferably more than 2 ⁇ 10 6 optical confinements, and even more preferably more than 10 7 or 2 ⁇ 10 7 optical confinements.
  • the specific protein-protein interaction is between a cell surface receptor and its corresponding ligand.
  • Cell surface receptors are molecules anchored on or inserted into the cell plasma membrane. They constitute a large family of proteins, glycoproteins, polysaccharides and lipids, which serve not only as structural constituents of the plasma membrane, but also as regulatory elements governing a variety of biological functions.
  • the specific protein-protein interaction involves a cell surface receptor and an immunoliposome or an immunotoxin.
  • the specific protein-protein interaction may involve a cytosolic protein, a nuclear protein, a chaperon protein, or proteins anchored on other intracellular membranous structures.
  • the specific protein-protein interaction is between a target protein (e.g., an antigen) and an antibody specific for that antigen.
  • an antigen and an antibody has been explored in the context of immunoassays.
  • immunoassays There exists a variety of immunoassays in the art, but none of which permits single-molecule detection.
  • the conventional radioimmunoassay detects the interactions between a population of antigens and a population of radioactively labeled antibodies on an immunoblot.
  • Another conventional immunoassay termed ELISA Enzyme Llinked Immunoradiometric Assay
  • ELISA Enzyme Llinked Immunoradiometric Assay
  • the subject optical confinement provides an effective tool for conducting a single-molecule immunoassay. Unlike the conventional immunoassays, the specific interaction between the antigen and the antibody can be resolved at the single-molecule level. While all of the optical confinements embodied in the present invention are applicable for conducting single-molecule immunoassays, a particularly desirable system comprises an array of optical confinements with a relatively high fill fraction ratio. For example, a preferred system comprises an array of waveguides having a fill fraction greater than 0.0001, more preferably greater than about 0.001, more preferably greater than about 0.01, and even more preferably greater than 0.1.
  • the antibodies an be labeled with a suitable label selected from radioactive labels, fluorescent labels, chemiluminescent labels, enzyme tags, such as digoxigenin, ⁇ -galactosidase, urease, alkaline phosphatase or peroxidase, avidin/biotin complex, and any of the detectable labels disclosed herein.
  • a suitable label selected from radioactive labels, fluorescent labels, chemiluminescent labels, enzyme tags, such as digoxigenin, ⁇ -galactosidase, urease, alkaline phosphatase or peroxidase, avidin/biotin complex, and any of the detectable labels disclosed herein.
  • the subject immunoassays can be performed to characterize biological entities, screen for antibody therapeutics, and determine the structural conformations of a target antigen. For instance, immuoassays involving antibodies that are specific for the biological entity or specific for a by-product produced by the biological entity have been routinely used to identify the entity by forming an antibody-entity complex. Immunoassays are also employed to screen for antibodies capable of activating or down-regulating the biological activity of a target antigen of therapeutic potential. Immunoassays are also useful for determining structural conformations by using anti-idotypic antibodies capable of differentiating target proteins folded in different conformations.
  • Another important application of the aforementioned single-molecule analysis is to study enzyme kinetics, which may include determining the enzymatic turnover cycle, the dynamic behavior, folding and unfolding intermediates, and binding affinities.
  • the enzymes under investigation may be immobilized within the optical confinements or present in solutions confined within the subject optical confinements.
  • optical confinements embodied by the present invention can be employed to study enzyme kinetics.
  • the choice of a specific optical confinement will depend on the specific characteristic that is under investigation.
  • an optical confinement comprising a non-cylindrical core having an opening on the upper surface that is narrower than that of the base of the optical confinement is preferable for measuring the association rate constant (on-rate) of an enzymatic reaction.
  • This configuration significantly restricts the diffusion of reactants or substrates, and hence increases the average residence time in the observation volume.
  • an optical confinement comprising a core with an opening that is wider in lateral dimension than the base imposes impose a stearic or entropic hinderence to entering the structure, hence is useful for measuring the accessibility for large enzymes or enzymatic complexes.
  • the present invention provides a method of detecting interactions among a plurality of molecules, comprising: placing said plurality of molecules in close proximity to an array of zero-mode waveguides, wherein individual waveguides in said array are separated by a distance sufficient to yield a detectable intensity of diffractive scattering at multiple diffracted orders upon illuminating said array with an incident wavelength of light beam; illuminating said array of zero-mode waveguides with said incident wavelength; and detecting a change in said intensity of diffractive scattering of said incident wavelength at said multiple diffracted orders, thereby detecting said interactions among said plurality of molecules.
  • Arrays employed for this method typically comprises optical confinements spaced far apart relative to the incident wavelength. Such spacing of the individual optical confinements far apart relative to the illuminating radiation (e.g., half of the wavelength of the illuminating radiation) creates a larger effect on the diffractive scattering of incident light at a given angle away from the angle of specula reflection.
  • the arrays contain individual confinements separated by more than one wavelength of the incident radiation, usually more than 1.5 times the incident wavelength, but usually does not exceed 150 times the incident wavelength.
  • Arrays having the optical confinements spaced far apart relative to the incident wavelength also have desirable properties. While the angle-dependent scattering may raise the background signal that could be disadvantageous for certain applications, it provides a means particularly suited for characterizing the size and shape of the optical confinements. It also readily permits ensemble bulk measurements of molecule interactions, involving especially unlabelled molecules. Arrays suited for such applications generally contain individual confinements separated by more than one wavelength of the incident radiation, usually more than 1.5 times the incident wavelength, but usually not exceeding 150 times the incident wavelength.
  • the ensemble bulk measurement is typically performed with the aid of the optical systems described herein. Any of the setup applicable for single-molecule sequencing is equally suited for this analysis.
  • R110-dCTP (Amersham Biosciences, Piscataway, N.J.) was used as the fluorescently-tagged nucleotide analog in which the fluorophore is attached to the nucleotide via a linker to the gamma-phosphate.
  • gamma-phosphate-linked analogs are cleaved through the enzymatic activity of DNA polymerase as the attached nucleotide is incorporated into the growing DNA strand and the label is then free to diffuse out of the effective observation volume surrounding the DNA polymerase.
  • the efficient removal of the fluorophore ensures continuously low background levels and prevents significant interference with DNA polymerase activity.
  • gamma-phosphate-linked fluorophore are preferable for this application because they will enable replacement of all four bases with fluorophore-tagged analogs. Binding of a nucleotide and its subsequent incorporation into nucleic acid from a mismatch event is distinguished because the rate constants of these two processes are significantly different, and because nucleotide incorporation involves several successive steps that prevent zero delay time events.
  • the incorporation of the fluorescently labeled dCTP nucleotide was tracked during rolling-circle DNA synthesis by recording the fluorescent light bursts emitted in an individual ZMW. DNA polymerase activity was observed in many waveguides as distinct bursts of fluorescence, which lasted several minutes. The fluorescence time trace showed a characteristic double burst pattern ( FIG. 9B ), each burst corresponding to an incorporation event of a R110-dCTP analog into the DNA strand and subsequent cleavage of the fluorophore.
  • Example 2 An experiment similar to that described in Example 2, above, was performed using two different labeled nucleotide analogs.
  • the experiment can be performed using the optical setup or system and reaction mixtures detailed as follows.
  • the reference to any particular optical setup and parameter, buffer, reagent, concentration, pH, temperature, or the like, is not intended to be limiting. They are included to provide as one illustrative example of carrying out the methods of the present invention.
  • reaction mixture Approximately 10 ⁇ l of reaction mixture is used in one sequencing reaction.
  • the reaction mixture generally contains 0.5-1 mM MnCl 2 , 0.1-1 uM DNA template, 10 ⁇ M dATP, 10 ⁇ M dGTP, 10 ⁇ M SAP-treated Alexa 488-dC4P, and 10 ⁇ M SAP-treated Alexa 568-dT4P, and DNA polymerase.
  • the labeled dC4P and dT4P can also be substituted with labeled dA4P and dG4P.
  • Preparing Zero-mode waveguide Prior to the polymerization reaction, a zero-mode waveguide is typically refreshed in a plasma cleaner. A PDMS gasket covering the ZMW is placed onto the waveguide to cover the individual optical confinements. An aliquot of the reaction mixture described above except the DNA polymerase is applied without touching the waveguide surface. The diffusion background is measured. If the background (i.e., fluorescence burst from the ZMW) low and acceptable, then DNA polymerase will be applied to ZMW and immobilized thereon.
  • background i.e., fluorescence burst from the ZMW
  • the immobilization mixture typically contains 0.5 to 1 mM MnCl 2 , 0.1 to 1 uM template, 15 nM DNA polymerase, in a buffer of 25 mM Tris-HCL, pH 7.5 and 10 mM beta-mercaptoethanol.
  • the polymerase is allowed to stick to the surface of ZMW after an incubation of about 15 minutes at about 0° C.
  • the immobilization reaction mixture is then removed, replaced with the reaction mixture described above.
  • a microscope system equipped with an appropriate laser, e.g., Ar/Kr laser, that includes an optical setup for simultaneous collection and detection of signals from multiple different waveguides, and for the resolution of each of the A488 and 568 fluorophores present.
  • the system includes an objective lens and a series of dichroics/notch-off filters for separating emitted fluorescence from reflected excitation light.
  • the emitted signals are passed through a wedge filter to spatially separate the signal component of each fluorophore, and each signal is imaged onto an EMCCD camera.
  • Polymerase Activity Measurement The ZMW is placed under the microscope. Polymerization reaction is then monitored using a camera for a desired period of time, e.g., two minutes or longer, after the transillumination light is applied. The data is automatically transmitted to a computer that stores and trace the fluorescence burst of each reaction in the ZMW.
  • a circular DNA having either a block of repeating A bases followed by a block of G bases, or a series of repeating A-G bases was sequenced according to the aforementioned procedures.
  • a representative profile of the fluorescence bursts corresponding to each incorporation event of the labeled nucleotides is depicted in FIG. 16 , which indicates that real-time and single-molecule sequencing has been achieved with more than one type of labeled nucleotides.
  • Statistical analysis of pulse data from multiple separate repeats and multiple different waveguides establishes the sequence dependant detection of incorporation of labeled bases in real time.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Nanotechnology (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Optics & Photonics (AREA)
  • Biophysics (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Clinical Laboratory Science (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Microelectronics & Electronic Packaging (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Composite Materials (AREA)
  • Condensed Matter Physics & Semiconductors (AREA)
  • Materials Engineering (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Physical Or Chemical Processes And Apparatus (AREA)
US11/229,376 2004-09-17 2005-09-16 Fabrication of optical confinements Abandoned US20060062531A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US11/229,376 US20060062531A1 (en) 2004-09-17 2005-09-16 Fabrication of optical confinements

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US10/944,106 US7170050B2 (en) 2004-09-17 2004-09-17 Apparatus and methods for optical analysis of molecules
US64900905P 2005-01-31 2005-01-31
US65184605P 2005-02-09 2005-02-09
US11/229,376 US20060062531A1 (en) 2004-09-17 2005-09-16 Fabrication of optical confinements

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US10/944,106 Continuation-In-Part US7170050B2 (en) 2004-09-17 2004-09-17 Apparatus and methods for optical analysis of molecules

Publications (1)

Publication Number Publication Date
US20060062531A1 true US20060062531A1 (en) 2006-03-23

Family

ID=36073574

Family Applications (4)

Application Number Title Priority Date Filing Date
US11/228,925 Expired - Lifetime US7302146B2 (en) 2004-09-17 2005-09-16 Apparatus and method for analysis of molecules
US11/229,376 Abandoned US20060062531A1 (en) 2004-09-17 2005-09-16 Fabrication of optical confinements
US11/228,376 Expired - Lifetime US7476503B2 (en) 2004-09-17 2005-09-16 Apparatus and method for performing nucleic acid analysis
US11/228,759 Expired - Lifetime US7315019B2 (en) 2004-09-17 2005-09-16 Arrays of optical confinements and uses thereof

Family Applications Before (1)

Application Number Title Priority Date Filing Date
US11/228,925 Expired - Lifetime US7302146B2 (en) 2004-09-17 2005-09-16 Apparatus and method for analysis of molecules

Family Applications After (2)

Application Number Title Priority Date Filing Date
US11/228,376 Expired - Lifetime US7476503B2 (en) 2004-09-17 2005-09-16 Apparatus and method for performing nucleic acid analysis
US11/228,759 Expired - Lifetime US7315019B2 (en) 2004-09-17 2005-09-16 Arrays of optical confinements and uses thereof

Country Status (8)

Country Link
US (4) US7302146B2 (fr)
EP (2) EP3415641B1 (fr)
JP (3) JP2008513782A (fr)
CN (1) CN101914620B (fr)
AU (1) AU2005296200B2 (fr)
CA (1) CA2579150C (fr)
GB (1) GB2423819B (fr)
WO (1) WO2006044078A2 (fr)

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030064366A1 (en) * 2000-07-07 2003-04-03 Susan Hardin Real-time sequence determination
US20060063264A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Apparatus and method for performing nucleic acid analysis
US20070172866A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Methods for sequence determination using depolymerizing agent
US20070172819A1 (en) * 2000-12-01 2007-07-26 Hardin Susan H Enzymatic nucleic acid synthesis: compositions including pyrophosphorolysis inhibitors
US20070206189A1 (en) * 2004-09-17 2007-09-06 Stephen Turner Optical analysis of molecules
US20070250274A1 (en) * 2006-02-06 2007-10-25 Visigen Biotechnologies, Inc. Method for analyzing dynamic detectable events at the single molecule level
US20080091005A1 (en) * 2006-07-20 2008-04-17 Visigen Biotechnologies, Inc. Modified nucleotides, methods for making and using same
US20080241951A1 (en) * 2006-07-20 2008-10-02 Visigen Biotechnologies, Inc. Method and apparatus for moving stage detection of single molecular events
US20080241938A1 (en) * 2006-07-20 2008-10-02 Visigen Biotechnologies, Inc. Automated synthesis or sequencing apparatus and method for making and using same
US20090186343A1 (en) * 2003-01-28 2009-07-23 Visigen Biotechnologies, Inc. Methods for preparing modified biomolecules, modified biomolecules and methods for using same
US20120183970A1 (en) * 2009-08-06 2012-07-19 Ibis Biosciences, Inc. Non-mass determined base compositions for nucleic acid detection
US20160070034A1 (en) * 2008-06-02 2016-03-10 University Of Utah Research Foundation Localization of Near-Field Resonances in Bowtie Antennae: Influence of Adhesion Layers
US9464286B2 (en) 2002-08-12 2016-10-11 Adimab, Llc High throughput generation and affinity maturation of humanized antibody
US10189894B2 (en) 2007-09-14 2019-01-29 Adimab, Llc Rationally designed, synthetic antibody libraries and uses therefor
US10196635B2 (en) 2007-09-14 2019-02-05 Adimab, Llc Rationally designed, synthetic antibody libraries and uses therefor
US11705217B2 (en) 2008-03-28 2023-07-18 Pacific Biosciences Of California, Inc. Sequencing using concatemers of copies of sense and antisense strands
US12529164B2 (en) 2007-09-14 2026-01-20 Adimab, Llc Rationally designed, synthetic antibody libraries and uses therefor

Families Citing this family (768)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8535616B2 (en) * 2005-08-02 2013-09-17 Moxtek, Inc. Sub-wavelength metallic apertures as light enhancement devices
US7763423B2 (en) * 2005-09-30 2010-07-27 Pacific Biosciences Of California, Inc. Substrates having low density reactive groups for monitoring enzyme activity
WO2007041621A2 (fr) * 2005-10-03 2007-04-12 Xingsheng Sean Ling Sequençage assiste par hybridation et effectue avec une structure de nanopore
US20070154921A1 (en) * 2005-12-16 2007-07-05 Applera Corporation Method and System for Phase-Locked Sequencing
WO2007091280A1 (fr) * 2006-02-06 2007-08-16 Stmicroelectronics S.R.L. appareil optique et procédé pour l'inspection de sondes d'acides nucléiques par un rayonnement polarisé
US7995202B2 (en) * 2006-02-13 2011-08-09 Pacific Biosciences Of California, Inc. Methods and systems for simultaneous real-time monitoring of optical signals from multiple sources
US7715001B2 (en) * 2006-02-13 2010-05-11 Pacific Biosciences Of California, Inc. Methods and systems for simultaneous real-time monitoring of optical signals from multiple sources
US7692783B2 (en) * 2006-02-13 2010-04-06 Pacific Biosciences Of California Methods and systems for simultaneous real-time monitoring of optical signals from multiple sources
US9976192B2 (en) 2006-03-10 2018-05-22 Ldip, Llc Waveguide-based detection system with scanning light source
US8288157B2 (en) 2007-09-12 2012-10-16 Plc Diagnostics, Inc. Waveguide-based optical scanning systems
US9423397B2 (en) 2006-03-10 2016-08-23 Indx Lifecare, Inc. Waveguide-based detection system with scanning light source
US7951583B2 (en) 2006-03-10 2011-05-31 Plc Diagnostics, Inc. Optical scanning system
US9528939B2 (en) 2006-03-10 2016-12-27 Indx Lifecare, Inc. Waveguide-based optical scanning systems
US8975216B2 (en) 2006-03-30 2015-03-10 Pacific Biosciences Of California Articles having localized molecules disposed thereon and methods of producing same
JP2009539411A (ja) * 2006-06-12 2009-11-19 パシフィック バイオサイエンシーズ オブ カリフォルニア， インコーポレイテッド 分析反応を行うための基板
WO2007147110A2 (fr) * 2006-06-16 2007-12-21 Pacific Biosciences Of California, Inc. Initiation régulée d'extension d'amorce
AU2007289057C1 (en) * 2006-09-01 2014-01-16 Pacific Biosciences Of California, Inc. Substrates, systems and methods for analyzing materials
US8207509B2 (en) 2006-09-01 2012-06-26 Pacific Biosciences Of California, Inc. Substrates, systems and methods for analyzing materials
US9096953B2 (en) * 2006-09-29 2015-08-04 Intel Corporation Method for high throughput, high volume manufacturing of biomolecule micro arrays
US12135286B1 (en) * 2006-12-06 2024-11-05 Mohammad A. Mazed Optical biomodule for detection of diseases at an early onset
US11747279B2 (en) * 2006-12-06 2023-09-05 Mohammad A. Mazed Optical biomodule for detection of diseases at an early onset
US10529003B2 (en) * 2008-04-07 2020-01-07 Mohammad A. Mazed Optical biomodule for detection of diseases at an early onset
US8740670B2 (en) 2006-12-28 2014-06-03 Saint-Gobain Ceramics & Plastics, Inc. Sapphire substrates and methods of making same
KR20160137681A (ko) * 2006-12-28 2016-11-30 생-고뱅 세라믹스 앤드 플라스틱스, 인코포레이티드 사파이어 기판
KR20110124355A (ko) * 2006-12-28 2011-11-16 생-고뱅 세라믹스 앤드 플라스틱스, 인코포레이티드 사파이어 기판 및 그 제조 방법
US7956356B2 (en) * 2006-12-28 2011-06-07 Saint-Gobain Ceramics & Plastics, Inc. Sapphire substrates and methods of making same
US11940413B2 (en) 2007-02-05 2024-03-26 IsoPlexis Corporation Methods and devices for sequencing nucleic acids in smaller batches
US8551704B2 (en) 2007-02-16 2013-10-08 Pacific Biosciences Of California, Inc. Controllable strand scission of mini circle DNA
DE102007016699A1 (de) * 2007-04-04 2008-10-09 Synentec Gmbh Biochip für die Fluoreszenzanalyse von einzelnen Transportern
EP3543357A1 (fr) 2007-05-08 2019-09-25 Trustees of Boston University Fonctionnalisation chimique de nanopores à l'état solide et de réseaux de nanopores et leurs applications
EP2150806A4 (fr) * 2007-05-10 2013-01-02 Pacific Biosciences California Procédés et systèmes d'analyse de matière fluorescente à autofluorescence limitée
US20100167413A1 (en) * 2007-05-10 2010-07-01 Paul Lundquist Methods and systems for analyzing fluorescent materials with reduced autofluorescence
US7714303B2 (en) * 2007-05-10 2010-05-11 Pacific Biosciences Of California, Inc. Methods and systems for analyzing fluorescent materials with reduced authofluorescence
US20080277595A1 (en) * 2007-05-10 2008-11-13 Pacific Biosciences Of California, Inc. Highly multiplexed confocal detection systems and methods of using same
US8703422B2 (en) 2007-06-06 2014-04-22 Pacific Biosciences Of California, Inc. Methods and processes for calling bases in sequence by incorporation methods
AU2008261935B2 (en) * 2007-06-06 2013-05-02 Pacific Biosciences Of California, Inc. Methods and processes for calling bases in sequence by incorporation methods
US9151751B2 (en) * 2007-06-29 2015-10-06 Applied Biosystems, Llc Systems and methods for electronic detection with nanoFETS
EP2183388A4 (fr) * 2007-07-26 2010-09-08 Pacific Biosciences California Séquençage moléculaire redondant
US8143601B2 (en) * 2007-08-14 2012-03-27 Massachusetts Institute Of Technology Nanoscale imaging via absorption modulation
US20090118129A1 (en) * 2007-09-28 2009-05-07 Pacific Biosciences Of California, Inc. Virtual reads for readlength enhancement
US7960116B2 (en) * 2007-09-28 2011-06-14 Pacific Biosciences Of California, Inc. Nucleic acid sequencing methods and systems
US8003330B2 (en) * 2007-09-28 2011-08-23 Pacific Biosciences Of California, Inc. Error-free amplification of DNA for clonal sequencing
US8278047B2 (en) 2007-10-01 2012-10-02 Nabsys, Inc. Biopolymer sequencing by hybridization of probes to form ternary complexes and variable range alignment
WO2009073201A2 (fr) 2007-12-04 2009-06-11 Pacific Biosciences Of California, Inc. Stratégies de marquage alternées pour séquençage de molécule unique
CA2711560A1 (fr) * 2008-01-10 2009-07-16 Pacific Biosciences Of California, Inc. Procedes et systemes d'analyse de reactions fluorescentes a excitation modulee
WO2009097368A2 (fr) 2008-01-28 2009-08-06 Complete Genomics, Inc. Procédés et compositions pour un appel de base efficace dans des réactions de séquençage
US8652781B2 (en) 2008-02-12 2014-02-18 Pacific Biosciences Of California, Inc. Cognate sampling kinetics
US9017973B2 (en) 2008-03-19 2015-04-28 Intelligent Biosystems, Inc. Methods and compositions for incorporating nucleotides
WO2009120374A2 (fr) * 2008-03-28 2009-10-01 Pacific Biosciences Of California, Inc. Procédés et compositions pour la préparation d’échantillon d’acide nucléique
US8628940B2 (en) * 2008-09-24 2014-01-14 Pacific Biosciences Of California, Inc. Intermittent detection during analytical reactions
WO2009145818A1 (fr) 2008-03-31 2009-12-03 Pacific Biosciences Of California, Inc Procédés et compositions de charge de molécule individuelle
ES2614078T3 (es) * 2008-03-31 2017-05-29 Pacific Biosciences Of California, Inc. Generación de polimerasas modificadas para precisión mejorada en secuenciación de una única molécula
US8999676B2 (en) 2008-03-31 2015-04-07 Pacific Biosciences Of California, Inc. Recombinant polymerases for improved single molecule sequencing
EP2274446B1 (fr) 2008-03-31 2015-09-09 Pacific Biosciences of California, Inc. Systèmes et procédés d'enzyme polymérase à deux étapes lentes
US8420366B2 (en) * 2008-03-31 2013-04-16 Pacific Biosciences Of California, Inc. Generation of modified polymerases for improved accuracy in single molecule sequencing
US9637380B2 (en) 2008-03-31 2017-05-02 Pacific Biosciences Of California, Inc. Nanoscale apertures having islands of functionality
US20090247426A1 (en) * 2008-03-31 2009-10-01 Pacific Biosciences Of California, Inc. Focused library generation
EP2274445A2 (fr) 2008-04-11 2011-01-19 University of Utah Research Foundation Procédés et compositions d'analyse de méthylation basée sur des séries quantitatives
GB2461026B (en) * 2008-06-16 2011-03-09 Plc Diagnostics Inc System and method for nucleic acids sequencing by phased synthesis
US8198023B2 (en) 2008-08-05 2012-06-12 Pacific Biosciences Of California, Inc. Prevention and alleviation of steric hindrance during single molecule nucleic acid synthesis by a polymerase
US20100036110A1 (en) * 2008-08-08 2010-02-11 Xiaoliang Sunney Xie Methods and compositions for continuous single-molecule nucleic acid sequencing by synthesis with fluorogenic nucleotides
US20100227327A1 (en) * 2008-08-08 2010-09-09 Xiaoliang Sunney Xie Methods and compositions for continuous single-molecule nucleic acid sequencing by synthesis with fluorogenic nucleotides
EP2342362B1 (fr) 2008-09-03 2017-03-01 Nabsys 2.0 LLC Utilisation d'électrodes nanométriques longitudinalement déplacées pour une détection de tension de biomolécules et autres analytes dans des canaux fluidiques
US9650668B2 (en) 2008-09-03 2017-05-16 Nabsys 2.0 Llc Use of longitudinally displaced nanoscale electrodes for voltage sensing of biomolecules and other analytes in fluidic channels
US8262879B2 (en) 2008-09-03 2012-09-11 Nabsys, Inc. Devices and methods for determining the length of biopolymers and distances between probes bound thereto
US8795961B2 (en) * 2008-09-05 2014-08-05 Pacific Biosciences Of California, Inc. Preparations, compositions, and methods for nucleic acid sequencing
CA2735979A1 (fr) * 2008-09-05 2010-03-11 Pacific Biosciences Of California, Inc. Polymerases genetiquement modifiees et conditions reactionnelles pour l'obtention de proprietes d'incorporation modifiees
US8383345B2 (en) 2008-09-12 2013-02-26 University Of Washington Sequence tag directed subassembly of short sequencing reads into long sequencing reads
DK3629011T3 (da) * 2008-09-16 2024-01-29 Pacific Biosciences California Inc Integreret optisk indretning
US8921046B2 (en) 2008-09-19 2014-12-30 Pacific Biosciences Of California, Inc. Nucleic acid sequence analysis
US8481264B2 (en) * 2008-09-19 2013-07-09 Pacific Biosciences Of California, Inc. Immobilized nucleic acid complexes for sequence analysis
US8383369B2 (en) * 2008-09-24 2013-02-26 Pacific Biosciences Of California, Inc. Intermittent detection during analytical reactions
WO2010036287A1 (fr) * 2008-09-24 2010-04-01 Pacific Biosciences Of California, Inc. Détection intermittente durant des réactions analytiques
CN102227659B (zh) 2008-09-30 2014-12-24 加利福尼亚太平洋生物科学股份有限公司 超高多路分析系统和方法
US8486865B2 (en) 2008-11-03 2013-07-16 The Regents Of The University Of California Methods for detecting modification resistant nucleic acids
US8486630B2 (en) 2008-11-07 2013-07-16 Industrial Technology Research Institute Methods for accurate sequence data and modified base position determination
US8993230B2 (en) * 2008-12-04 2015-03-31 Pacific Biosciences of Californ, Inc. Asynchronous sequencing of biological polymers
US9175338B2 (en) 2008-12-11 2015-11-03 Pacific Biosciences Of California, Inc. Methods for identifying nucleic acid modifications
US12527665B2 (en) 2008-12-11 2026-01-20 Pacific Biosciences Of California, Inc. Methods for identifying modified bases in nucleic acid templates
CN102317473A (zh) * 2008-12-11 2012-01-11 加利福尼亚太平洋生物科学股份有限公司 核酸模板的分类
WO2010075188A2 (fr) 2008-12-23 2010-07-01 Illumina Inc. Libération multibase pour lectures longues dans le séquençage par des protocoles de synthèse
EP3020830A1 (fr) * 2009-01-20 2016-05-18 The Board Of Trustees Of The Leland Stanford Junior University Expression du gène de cellule unique pour le diagnostic, pronostic et identification de cibles de médicaments
WO2010099220A2 (fr) * 2009-02-25 2010-09-02 California Institute Of Technology Procédés pour fabriquer des sondes à facteur de forme élevé et déformer des nanopiliers et des micropiliers à facteur de forme élevé
US9778188B2 (en) 2009-03-11 2017-10-03 Industrial Technology Research Institute Apparatus and method for detection and discrimination molecular object
US8455260B2 (en) * 2009-03-27 2013-06-04 Massachusetts Institute Of Technology Tagged-fragment map assembly
US20100243449A1 (en) * 2009-03-27 2010-09-30 Oliver John S Devices and methods for analyzing biomolecules and probes bound thereto
US20100255487A1 (en) 2009-03-27 2010-10-07 Life Technologies Corporation Methods and apparatus for single molecule sequencing using energy transfer detection
WO2010117420A2 (fr) 2009-03-30 2010-10-14 Pacific Biosciences Of California, Inc. Composés marqués par fret et leurs utilisations
WO2010117470A2 (fr) 2009-04-10 2010-10-14 Pacific Biosciences Of California, Inc. Dispositifs et procédés de séquençage de nanopore
CA2760155A1 (fr) 2009-04-27 2010-11-11 Pacific Biosciences Of California, Inc. Procedes et systemes de sequencage en temps reel
KR20120035912A (ko) * 2009-04-29 2012-04-16 피엘씨 다이아그노스틱스, 인크. 스캐닝 광원을 갖는 도파로 기반 검출 시스템
US20100279882A1 (en) * 2009-05-01 2010-11-04 Mostafa Ronaghi Sequencing methods
US20120064527A1 (en) * 2009-05-27 2012-03-15 Akira Maekawa Nucleic acid analysis device, nucleic acid analysis apparatus, and nucleic acid analysis method
US8246799B2 (en) * 2009-05-28 2012-08-21 Nabsys, Inc. Devices and methods for analyzing biomolecules and probes bound thereto
US8316738B2 (en) * 2009-06-10 2012-11-27 Magna Powertrain Of America, Inc. Compact transfer case with beveloid gearset
WO2010144150A2 (fr) 2009-06-12 2010-12-16 Pacific Biosciences Of California, Inc. Procédés et systèmes analytiques en temps réel
WO2010144151A2 (fr) * 2009-06-12 2010-12-16 Pacific Biosciences Of California, Inc. Analyse monomoléculaire en temps réel de la protéinogenèse
US8080468B2 (en) 2009-06-26 2011-12-20 California Institute Of Technology Methods for fabricating passivated silicon nanowires and devices thus obtained
US9671558B2 (en) * 2009-06-30 2017-06-06 Intel Corporation Chemically induced optical signals and DNA sequencing
US8501406B1 (en) 2009-07-14 2013-08-06 Pacific Biosciences Of California, Inc. Selectively functionalized arrays
US9416409B2 (en) 2009-07-31 2016-08-16 Ibis Biosciences, Inc. Capture primers and capture sequence linked solid supports for molecular diagnostic tests
ES2573277T3 (es) 2009-08-14 2016-06-07 Epicentre Technologies Corporation Métodos, composiciones y kits de generación de muestras empobrecidas en ARNr o de aislamiento del ARNr de las muestras
US8367159B2 (en) 2009-09-11 2013-02-05 Pacific Biosciences Of California, Inc. Methods for producing ZMWs with islands of functionality
US8906670B2 (en) * 2009-09-11 2014-12-09 Pacific Bioscience Of California, Inc. Zero-mode waveguides with non-reflecting walls
CA2808576A1 (fr) 2009-09-30 2011-04-07 Quantapore, Inc. Sequencage ultrarapide de polymeres biologiques au moyen de nanopores marques
WO2011050127A1 (fr) * 2009-10-22 2011-04-28 The University Of North Carolina At Chapel Hill Particules permettant la fabrication efficace de microréseaux et nanoréseaux et utilisation de ceux-ci
US8809093B2 (en) * 2009-11-19 2014-08-19 California Institute Of Technology Methods for fabricating self-aligning semicondutor heterostructures using silicon nanowires
US9018684B2 (en) 2009-11-23 2015-04-28 California Institute Of Technology Chemical sensing and/or measuring devices and methods
CA2781581C (fr) 2009-12-01 2018-03-06 Oxford Nanopore Technologies Limited Instrument d'analyse biochimique
ES2596655T3 (es) 2010-02-01 2017-01-11 Illumina Inc. Procedimientos de enfoque y sistemas y conjuntos ópticos que usan los mismos
WO2011103507A1 (fr) 2010-02-19 2011-08-25 Pacific Biosciences Of California, Inc. Systeme et procede de detection et de collecte d'optique
US8994946B2 (en) 2010-02-19 2015-03-31 Pacific Biosciences Of California, Inc. Integrated analytical system and method
WO2011112465A1 (fr) 2010-03-06 2011-09-15 Illumina, Inc. Systèmes, procédés et appareils permettant de détecter des signaux optiques provenant d'un échantillon
US20130095514A1 (en) * 2010-03-12 2013-04-18 Jpt Peptide Technologies Gmbh Method for Determining the Concentration of a Peptide
US9482615B2 (en) * 2010-03-15 2016-11-01 Industrial Technology Research Institute Single-molecule detection system and methods
SI2556171T1 (sl) 2010-04-05 2016-03-31 Prognosys Biosciences, Inc. Prostorsko kodirane biološke analize
US20190300945A1 (en) 2010-04-05 2019-10-03 Prognosys Biosciences, Inc. Spatially Encoded Biological Assays
US10787701B2 (en) 2010-04-05 2020-09-29 Prognosys Biosciences, Inc. Spatially encoded biological assays
US8652779B2 (en) 2010-04-09 2014-02-18 Pacific Biosciences Of California, Inc. Nanopore sequencing using charge blockade labels
US8470261B2 (en) 2010-05-06 2013-06-25 Ibis Biosciences, Inc. Integrated sample preparation systems and stabilized enzyme mixtures
CA2796822C (fr) 2010-05-07 2021-10-05 The Board Of Trustees Of The Leland Standford Junior University Mesure et comparaison de diversite immunitaire par sequencage a haut debit
US9670243B2 (en) 2010-06-02 2017-06-06 Industrial Technology Research Institute Compositions and methods for sequencing nucleic acids
US8865078B2 (en) 2010-06-11 2014-10-21 Industrial Technology Research Institute Apparatus for single-molecule detection
US8865077B2 (en) 2010-06-11 2014-10-21 Industrial Technology Research Institute Apparatus for single-molecule detection
WO2012009206A2 (fr) 2010-07-12 2012-01-19 Pacific Biosciences Of California, Inc. Réactions de séquençage avec des cations de métaux alcalins pour le contrôle de largeur d'impulsion
US8993737B2 (en) 2010-08-25 2015-03-31 Pacific Biosciences, Inc. Phospholinked dye analogs with an amino acid linker
EP2635553A4 (fr) 2010-09-16 2015-11-18 Ibis Biosciences Inc Stabilisation de colorants fluorescents labiles à l'ozone par une thiourée
EP3327140B1 (fr) * 2010-09-16 2025-08-06 Gen-Probe Incorporated Sondes de capture immobilisables par queue de nucléotide l
US8715933B2 (en) 2010-09-27 2014-05-06 Nabsys, Inc. Assay methods using nicking endonucleases
EP2633069B1 (fr) 2010-10-26 2015-07-01 Illumina, Inc. Procédés de séquençage
EP2635703B1 (fr) 2010-11-01 2018-03-21 Gen-Probe Incorporated Capture et amplification integrees d'acides nucleiques en vue de sequencage
EP2635679B1 (fr) 2010-11-05 2017-04-19 Illumina, Inc. Liaison entre des lectures de séquences à l'aide de codes marqueurs appariés
US9074251B2 (en) 2011-02-10 2015-07-07 Illumina, Inc. Linking sequence reads using paired code tags
WO2012067911A1 (fr) 2010-11-16 2012-05-24 Nabsys, Inc. Procédés de séquençage d'une biomolécule par détection de positions relatives de sondes hybridées
WO2012074855A2 (fr) 2010-11-22 2012-06-07 The Regents Of The University Of California Procédés d'identification d'un transcrit cellulaire naissant d'arn
WO2012092265A1 (fr) 2010-12-27 2012-07-05 Ibis Biosciences, Inc. Procédés et compositions pour préparer des échantillons d'acides nucléiques
US8951781B2 (en) 2011-01-10 2015-02-10 Illumina, Inc. Systems, methods, and apparatuses to image a sample for biological or chemical analysis
CN103403188B (zh) 2011-01-31 2016-03-30 伊鲁米那股份有限公司 用于降低核酸损伤的方法
CN103443338B (zh) 2011-02-02 2017-09-22 华盛顿大学商业化中心 大规模平行邻接作图
US11274341B2 (en) 2011-02-11 2022-03-15 NABsys, 2.0 LLC Assay methods using DNA binding proteins
EP2689028B1 (fr) 2011-03-23 2017-08-30 Pacific Biosciences Of California, Inc. Isolement de complexes polymérase-acide nucléique et charge sur des substrats
US9611510B2 (en) 2011-04-06 2017-04-04 The University Of Chicago Composition and methods related to modification of 5-methylcytosine (5-mC)
WO2012142301A2 (fr) 2011-04-12 2012-10-18 Quanterix Corporation Procédé de détermination d'un protocole de traitement et/ou d'un pronostic de rétablissement d'un patient à la suite d'un traumatisme cérébral
GB201106254D0 (en) 2011-04-13 2011-05-25 Frisen Jonas Method and product
US8778848B2 (en) 2011-06-09 2014-07-15 Illumina, Inc. Patterned flow-cells useful for nucleic acid analysis
US9670538B2 (en) 2011-08-05 2017-06-06 Ibis Biosciences, Inc. Nucleic acid sequencing by electrochemical detection
EP2753714B1 (fr) 2011-09-06 2017-04-12 Gen-Probe Incorporated Gabarits mis sous forme circulaire pour séquençage
EP2753712B1 (fr) 2011-09-06 2017-03-22 Gen-Probe Incorporated Structures fermées d'acide nucléique
US10152569B2 (en) 2011-09-26 2018-12-11 Gen-Probe Incorporated Algorithms for sequence determinations
WO2013056241A2 (fr) 2011-10-14 2013-04-18 Pacific Biosciences Of California, Inc. Séquençage rédox en temps réel
WO2013059746A1 (fr) 2011-10-19 2013-04-25 Nugen Technologies, Inc. Compositions et procédés pour l'amplification et le séquençage directionnels d'acide nucléique
WO2013063308A1 (fr) 2011-10-25 2013-05-02 University Of Massachusetts Procédé enzymatique pour l'enrichissement en arn coiffés, trousses pour la mise en œuvre de celui-ci et compositions issues de ce procédé
EP3305400A3 (fr) 2011-10-28 2018-06-06 Illumina, Inc. Système et procédé de fabrication de microréseau
WO2013070627A2 (fr) 2011-11-07 2013-05-16 Illumina, Inc. Appareils de séquençage intégré et procédés d'utilisation
EP2788499B1 (fr) 2011-12-09 2016-01-13 Illumina, Inc. Base de numération étendue pour étiquettes polymères
WO2013096819A2 (fr) 2011-12-22 2013-06-27 Ibis Biosciences, Inc. Positionnement d'une macromolécule par potentiel électrique
ES2645418T3 (es) 2011-12-22 2017-12-05 Ibis Biosciences, Inc. Amplificación de una secuencia de un ácido ribonucleico
EP3211100A1 (fr) 2011-12-22 2017-08-30 Ibis Biosciences, Inc. Amorces et procédés d'amplification
US9803231B2 (en) 2011-12-29 2017-10-31 Ibis Biosciences, Inc. Macromolecule delivery to nanowells
EP2872523B1 (fr) 2011-12-30 2018-01-17 Abbott Molecular Inc. Purification d'acide nucléique d'un micro-organisme à partir d'échantillons-hôtes
CN108611398A (zh) 2012-01-13 2018-10-02 Data生物有限公司 通过新一代测序进行基因分型
WO2013105025A1 (fr) * 2012-01-13 2013-07-18 Koninklijke Philips N.V. Séquençage d'adn avec recyclage du réactif sur la grille métallique
WO2013112923A1 (fr) 2012-01-26 2013-08-01 Nugen Technologies, Inc. Compositions et procédés pour l'enrichissement en séquence d'acide nucléique ciblée et la génération d'une banque à efficacité élevée
US9238836B2 (en) 2012-03-30 2016-01-19 Pacific Biosciences Of California, Inc. Methods and compositions for sequencing modified nucleic acids
US9528107B2 (en) 2012-01-31 2016-12-27 Pacific Biosciences Of California, Inc. Compositions and methods for selection of nucleic acids
EP3222627B1 (fr) 2012-02-15 2019-08-07 Pacific Biosciences of California, Inc. Substrats d'enzymes de polymérase avec blindage de protéine
NO2694769T3 (fr) 2012-03-06 2018-03-03
EP2825314A1 (fr) * 2012-03-16 2015-01-21 Life Technologies Corporation Systèmes et procédés d'analyse biologique
JP2015512508A (ja) * 2012-03-16 2015-04-27 ライフ テクノロジーズ コーポレーション 生物学的反応システムのための被覆されている基材
EP2828218B9 (fr) 2012-03-20 2021-04-07 University Of Washington Through Its Center For Commercialization Méthodes permettant de faire baisser le taux d'erreurs observées lors d'un séquençage massif d'adn eu parallèle en faisant appel à un séquençage par consensus duplex
US9803239B2 (en) 2012-03-29 2017-10-31 Complete Genomics, Inc. Flow cells for high density array chips
US20130261984A1 (en) 2012-03-30 2013-10-03 Illumina, Inc. Methods and systems for determining fetal chromosomal abnormalities
WO2013148400A1 (fr) 2012-03-30 2013-10-03 Pacific Biosciences Of California, Inc. Procédés et composition permettant le séquençage d'acides nucléiques modifiés
KR102118211B1 (ko) 2012-04-03 2020-06-02 일루미나, 인코포레이티드 핵산 서열분석에 유용한 통합 광전자 판독 헤드 및 유체 카트리지
US20130274148A1 (en) 2012-04-11 2013-10-17 Illumina, Inc. Portable genetic detection and analysis system and method
WO2013163207A1 (fr) 2012-04-24 2013-10-31 Pacific Biosciences Of California, Inc. Identification d'une modification de type 5-méthyl-c dans des matrices d'acides nucléiques
EP2844774B1 (fr) 2012-05-02 2018-07-18 Ibis Biosciences, Inc. Séquençage d'adn
US10584377B2 (en) 2012-05-02 2020-03-10 Ibis Biosciences, Inc. DNA sequencing
ES2683707T3 (es) 2012-05-02 2018-09-27 Ibis Biosciences, Inc. Secuenciación de ADN
WO2013173844A1 (fr) 2012-05-18 2013-11-21 Pacific Biosciences Of California, Inc. Colorants hétéroarylcyanines
US9315864B2 (en) 2012-05-18 2016-04-19 Pacific Biosciences Of California, Inc. Heteroarylcyanine dyes with sulfonic acid substituents
US9012022B2 (en) 2012-06-08 2015-04-21 Illumina, Inc. Polymer coatings
GB2559073A (en) 2012-06-08 2018-07-25 Pacific Biosciences California Inc Modified base detection with nanopore sequencing
US9267168B2 (en) 2012-06-12 2016-02-23 Pacific Biosciences Of California, Inc. Methods and compositions for isolating template nucleic acids
US8895249B2 (en) 2012-06-15 2014-11-25 Illumina, Inc. Kinetic exclusion amplification of nucleic acid libraries
US9372308B1 (en) 2012-06-17 2016-06-21 Pacific Biosciences Of California, Inc. Arrays of integrated analytical devices and methods for production
SG11201408478QA (en) 2012-06-18 2015-02-27 Nugen Technologies Inc Compositions and methods for negative selection of non-desired nucleic acid sequences
CA2878291A1 (fr) 2012-07-03 2014-01-09 Sloan Kettering Institute For Cancer Research Evaluation quantitative de la reconstitution du repertoire des cellules t chez l'homme apres une greffe allogenique de cellules souches hematopoietiques
US20150011396A1 (en) 2012-07-09 2015-01-08 Benjamin G. Schroeder Methods for creating directional bisulfite-converted nucleic acid libraries for next generation sequencing
NL2017959B1 (en) 2016-12-08 2018-06-19 Illumina Inc Cartridge assembly
WO2014031157A1 (fr) 2012-08-20 2014-02-27 Illumina, Inc. Procédé et système de séquençage reposant sur la durée de vie de fluorescence
US9637782B2 (en) 2012-09-28 2017-05-02 Pacific Biosciences Of California, Inc. Charged triplet-state quenchers for mitigation of photo-induced damage
US9399766B2 (en) 2012-10-01 2016-07-26 Pacific Biosciences Of California, Inc. Recombinant polymerases for incorporation of protein shield nucleotide analogs
JP6510978B2 (ja) 2012-10-16 2019-05-08 アボツト・モレキユラー・インコーポレイテツド 核酸を配列決定する方法および装置
EP3901280B1 (fr) 2012-10-17 2025-03-12 10x Genomics Sweden AB Procédés et produits pour optimiser la detection localisée ou spatiale de l'expression génique dans un échantillon de tissu
US9181583B2 (en) 2012-10-23 2015-11-10 Illumina, Inc. HLA typing using selective amplification and sequencing
US9651539B2 (en) 2012-10-28 2017-05-16 Quantapore, Inc. Reducing background fluorescence in MEMS materials by low energy ion beam treatment
US9223084B2 (en) 2012-12-18 2015-12-29 Pacific Biosciences Of California, Inc. Illumination of optical analytical devices
US9914966B1 (en) 2012-12-20 2018-03-13 Nabsys 2.0 Llc Apparatus and methods for analysis of biomolecules using high frequency alternating current excitation
US9683230B2 (en) 2013-01-09 2017-06-20 Illumina Cambridge Limited Sample preparation on a solid support
WO2014113557A1 (fr) 2013-01-18 2014-07-24 Nabsys, Inc. Liaison améliorée d'une sonde
WO2014116729A2 (fr) 2013-01-22 2014-07-31 The Board Of Trustees Of The Leland Stanford Junior University Haplotypage de loci hla par séquençage ultra-profond à l'aveugle
US9805407B2 (en) 2013-01-25 2017-10-31 Illumina, Inc. Methods and systems for using a cloud computing environment to configure and sell a biological sample preparation cartridge and share related data
US9624540B2 (en) 2013-02-22 2017-04-18 Pacific Biosciences Of California, Inc. Integrated illumination of optical analytical devices
US9512422B2 (en) * 2013-02-26 2016-12-06 Illumina, Inc. Gel patterned surfaces
EP2969479B1 (fr) 2013-03-13 2021-05-05 Illumina, Inc. Dispositifs fluidiques multicouches et leurs procédés de fabrication
EP3553175B1 (fr) 2013-03-13 2021-07-14 Illumina, Inc. Procédé de préparation d'une banque de séquençage d'acides nucléiques
EP2971070B2 (fr) 2013-03-14 2021-03-03 Illumina, Inc. Polymérases modifiées pour l'incorporation améliorée d'analogues nucléotidiques
WO2014139596A1 (fr) 2013-03-15 2014-09-18 Illumina Cambridge Limited Nucléosides ou nucléotides modifiés
US9193998B2 (en) 2013-03-15 2015-11-24 Illumina, Inc. Super resolution imaging
US20140274747A1 (en) 2013-03-15 2014-09-18 Illumina, Inc. Super resolution imaging
US10428379B2 (en) 2013-03-15 2019-10-01 Ibis Biosciences, Inc. Nucleotide analogs for sequencing
WO2014144092A1 (fr) 2013-03-15 2014-09-18 Nugen Technologies, Inc. Séquençage séquentiel
EP3597772A1 (fr) 2013-04-17 2020-01-22 Agency For Science, Technology And Research Procédé de génération lectures de séquence étendue
US9708656B2 (en) 2013-05-06 2017-07-18 Pacific Biosciences Of California, Inc. Real-time electronic sequencing
US9862997B2 (en) 2013-05-24 2018-01-09 Quantapore, Inc. Nanopore-based nucleic acid analysis with mixed FRET detection
US10544449B2 (en) 2013-06-14 2020-01-28 Pacific Biosciences Of California, Inc. Bis-biotinylation tags
WO2014201265A1 (fr) 2013-06-14 2014-12-18 Pacific Biosciences Of California, Inc. Étiquettes de bis-biotinylation
CN105849275B (zh) 2013-06-25 2020-03-17 普罗格诺西斯生物科学公司 检测样品中生物靶标的空间分布的方法和系统
HUE041318T2 (hu) 2013-07-01 2019-05-28 Illumina Inc Katalizátormentes felület-funkcionalizálás és polimer-ojtás
EP3241913B1 (fr) 2013-07-03 2019-02-20 Illumina, Inc. Système de séquençage par synthèse orthogonale
US9612245B2 (en) * 2013-07-03 2017-04-04 University Of North Texas Health Science Center At Fort Worth Multiple-pulse pumping for enhanced fluorescence detection and molecular imaging in cells and tissue
EP3030683B1 (fr) 2013-08-05 2022-03-02 Pacific Biosciences of California, Inc. Composés réactifs fluorescents protégés
CA3091557C (fr) 2013-08-08 2022-10-18 Illumina, Inc. Systeme fluidique pour l'apport de reactif a une cuve a circulation
WO2015026853A2 (fr) 2013-08-19 2015-02-26 Abbott Molecular Inc. Bibliothèques de séquençage de nouvelle génération
JP2016539343A (ja) 2013-08-30 2016-12-15 イルミナ インコーポレイテッド 親水性または斑状親水性表面上の液滴の操作
US9879318B2 (en) 2013-09-06 2018-01-30 Pacific Biosciences Of California, Inc. Methods and compositions for nucleic acid sample preparation
US9416414B2 (en) 2013-10-24 2016-08-16 Pacific Biosciences Of California, Inc. Delaying real-time sequencing
US10540783B2 (en) * 2013-11-01 2020-01-21 Illumina, Inc. Image analysis useful for patterned objects
CA2929596C (fr) 2013-11-13 2022-07-05 Nugen Technologies, Inc. Compositions et procedes pour l'identification d'une lecture de sequencage en double
CA2930832A1 (fr) 2013-11-17 2015-05-21 Quantum-Si Incorporated Systeme optique et puce d'analyse pour sonder, detecter et analyser des molecules
LT3077943T (lt) 2013-12-03 2020-10-12 Illumina, Inc. Vaizdo duomenų analizės būdai ir sistemos
EP3077545B1 (fr) 2013-12-05 2020-09-16 Centrillion Technology Holdings Corporation Procédés de séquençage d'acides nucléiques
WO2015085268A1 (fr) 2013-12-05 2015-06-11 Centrillion Technology Holdings Corporation Surfaces modifiées
CN106460032B (zh) 2013-12-05 2019-12-24 生捷科技控股公司 图案化阵列的制备
KR102271774B1 (ko) 2013-12-10 2021-06-30 일루미나, 인코포레이티드 생물학적 또는 화학적 분석을 위한 바이오센서들 및 이를 제조하기 위한 방법
WO2015095355A2 (fr) 2013-12-17 2015-06-25 The Brigham And Women's Hospital, Inc. Détection d'un anticorps dirigé contre un agent pathogène
US12183436B2 (en) 2013-12-18 2024-12-31 Pacific Biosciences Of California, Inc. String graph assembly for polyploid genomes
AU2014364926B2 (en) 2013-12-20 2017-12-14 Illumina, Inc. Preserving genomic connectivity information in fragmented genomic DNA samples
CN106062212A (zh) 2013-12-23 2016-10-26 伊鲁米那股份有限公司 用于改进光发射检测的结构化基底和关于其的方法
US9677132B2 (en) 2014-01-16 2017-06-13 Illumina, Inc. Polynucleotide modification on solid support
CA2936751C (fr) 2014-01-16 2022-03-29 Illumina, Inc. Preparation et sequencage d'amplicons sur supports solides
BR122022007092B8 (pt) 2014-02-18 2023-01-31 Illumina Inc Método para construir um perfil de dna, método para construir uma biblioteca de ácido nucléico, biblioteca de ácido nucléico, pluralidade de iniciadores e kit
US9745614B2 (en) 2014-02-28 2017-08-29 Nugen Technologies, Inc. Reduced representation bisulfite sequencing with diversity adaptors
US10018566B2 (en) 2014-02-28 2018-07-10 Ldip, Llc Partially encapsulated waveguide based sensing chips, systems and methods of use
EP3116651B1 (fr) 2014-03-11 2020-04-22 Illumina, Inc. Cartouche microfluidique intégrée jetable, et procédés pour la fabriquer
US11060139B2 (en) 2014-03-28 2021-07-13 Centrillion Technology Holdings Corporation Methods for sequencing nucleic acids
SG10201912516SA (en) 2014-04-29 2020-02-27 Illumina Inc Multiplexed single cell gene expression analysis using template switch and tagmentation
WO2015175832A1 (fr) 2014-05-16 2015-11-19 Illumina, Inc. Techniques de synthèse d'acide nucléique
BR112016027815B1 (pt) 2014-05-27 2022-07-12 Illumina, Inc. Sistemas e métodos para análise bioquímica incluindo um instrumento de base e cartucho removível
WO2015187670A2 (fr) 2014-06-03 2015-12-10 Illumina, Inc. Compositions, systèmes et procédés pour détecter des événements à l'aide d'attaches ancrées ou adjacentes à des nanopores
US20150353989A1 (en) 2014-06-09 2015-12-10 Illumina Cambridge Limited Sample preparation for nucleic acid amplification
EP3715468A1 (fr) 2014-06-13 2020-09-30 Illumina Cambridge Limited Procédés et compositions pour préparer des bibliothèques de séquençage
US10829814B2 (en) 2014-06-19 2020-11-10 Illumina, Inc. Methods and compositions for single cell genomics
WO2015200378A1 (fr) 2014-06-23 2015-12-30 The General Hospital Corporation Identification non biaisée, pangénomique, de dsb évaluée par séquençage (guide-seq)
EP3161157B1 (fr) 2014-06-24 2024-03-27 Bio-Rad Laboratories, Inc. "barcoding" par pcr numérique
US10017759B2 (en) 2014-06-26 2018-07-10 Illumina, Inc. Library preparation of tagged nucleic acid
WO2015200833A2 (fr) 2014-06-27 2015-12-30 Abbott Laboratories Compositions et procédés pour détecter le pegivirus humain 2 (hp-v-2)
JP6737710B2 (ja) 2014-06-27 2020-08-12 イルミナ インコーポレイテッド ヌクレオチドアナログの取り込みを改善するための修飾ポリメラーゼ
ES2713153T3 (es) 2014-06-30 2019-05-20 Illumina Inc Métodos y composiciones que utilizan la transposición unilateral
US10605766B2 (en) 2014-07-15 2020-03-31 Illumina, Inc. Biochemically activated electronic device
CN112941065A (zh) 2014-07-21 2021-06-11 亿明达股份有限公司 使用crispr-cas系统的多核苷酸富集
WO2016022833A1 (fr) 2014-08-06 2016-02-11 Nugen Technologies, Inc. Mesures numériques à partir de séquençage ciblé
GB201414098D0 (en) 2014-08-08 2014-09-24 Illumina Cambridge Ltd Modified nucleotide linkers
CN112903638B (zh) 2014-08-08 2025-05-16 宽腾矽公司 用于对分子进行探测、检测和分析的带外部光源的集成装置
CA2957540A1 (fr) 2014-08-08 2016-02-11 Quantum-Si Incorporated Dispositif integre de compartimentation temporelle de photons recus
CA2957543A1 (fr) 2014-08-08 2016-02-11 Quantum-Si Incorporated Systeme optique et puce d'analyse pour sonder, detecter, et analyser des molecules
US10435685B2 (en) 2014-08-19 2019-10-08 Pacific Biosciences Of California, Inc. Compositions and methods for enrichment of nucleic acids
WO2016028887A1 (fr) 2014-08-19 2016-02-25 Pacific Biosciences Of California, Inc. Compositions et méthodes pour l'enrichissement d'acides nucléiques
CN107076739B (zh) 2014-08-21 2018-12-25 伊卢米纳剑桥有限公司 可逆表面官能化
CA2959518A1 (fr) 2014-08-27 2016-03-03 Pacific Biosciences Of California, Inc. Reseaux de dispositifs analytiques integres
WO2016040602A1 (fr) 2014-09-11 2016-03-17 Epicentre Technologies Corporation Séquence au bisulfite à représentation réduite utilisant de l'uracile n-glycosylase (ung) et de l'endonucléase iv
WO2016040607A1 (fr) 2014-09-12 2016-03-17 Illumina, Inc. Compositions, systèmes et procédés de détection de la présence de sous-motifs polymères par chimioluminescence
CA2961743A1 (fr) 2014-09-17 2016-03-24 Ibis Biosciences, Inc. Sequencage par synthese a l'aide de dispositifs optiques de lecture a impulsions
KR102538753B1 (ko) 2014-09-18 2023-05-31 일루미나, 인코포레이티드 핵산 서열결정 데이터를 분석하기 위한 방법 및 시스템
DK3201355T3 (en) 2014-09-30 2019-10-14 Illumina Inc Modified polymerases for improved incorporation of nucleotide analogues
JP6668336B2 (ja) 2014-10-09 2020-03-18 イラミーナ インコーポレーテッド 非混和性液体を分離して少なくとも１つの液体を効果的に単離する方法及び装置
ES2789000T3 (es) 2014-10-10 2020-10-23 Quantapore Inc Análisis de polinucleótidos basado en nanoporos con marcadores fluorescentes que se inactivan mutuamente
US9897791B2 (en) 2014-10-16 2018-02-20 Illumina, Inc. Optical scanning systems for in situ genetic analysis
AU2015331739B2 (en) 2014-10-17 2021-12-02 Illumina Cambridge Limited Contiguity preserving transposition
CN107002126B (zh) 2014-10-24 2021-05-25 昆塔波尔公司 使用纳米结构阵列的聚合物的高效光学分析
CA2965578C (fr) 2014-10-31 2024-03-19 Illumina Cambridge Limited Polymeres et revetements copolymeres d'adn
ES2768762T3 (es) 2014-11-05 2020-06-23 Illumina Cambridge Ltd Reducción del daño al ADN durante la preparación de muestras y secuenciación usando agentes quelantes sideróforos
GB201419731D0 (en) 2014-11-05 2014-12-17 Illumina Cambridge Ltd Sequencing from multiple primers to increase data rate and density
PL3218511T3 (pl) 2014-11-11 2020-10-19 Illumina Cambridge Limited Sposoby i macierze do wytwarzania i sekwencjonowania monoklonalnych klastrów kwasu nukleinowego
HK1243464B (en) 2014-11-11 2019-08-16 Illumina, Inc. Polynucleotide amplification using CRISPR-CAS system
US10233490B2 (en) 2014-11-21 2019-03-19 Metabiotech Corporation Methods for assembling and reading nucleic acid sequences from mixed populations
US10616219B2 (en) * 2014-12-11 2020-04-07 FlowJo, LLC Single cell data management and analysis systems and methods
US10350570B2 (en) 2014-12-15 2019-07-16 Illumina, Inc. Compositions and methods for single molecular placement on a substrate
US10783984B2 (en) 2014-12-18 2020-09-22 Pacific Biosciences Of California, Inc. De novo diploid genome assembly and haplotype sequence reconstruction
US10302972B2 (en) 2015-01-23 2019-05-28 Pacific Biosciences Of California, Inc. Waveguide transmission
US10150872B2 (en) 2015-02-04 2018-12-11 Pacific Biosciences Of California, Inc. Multimeric protected fluorescent reagents
JP7001475B2 (ja) 2015-02-10 2022-01-19 イラミーナ インコーポレーテッド 細胞の構成成分を分析するための方法及び組成物
WO2016138427A1 (fr) 2015-02-27 2016-09-01 Indx Lifecare, Inc. Système de détection à guide d'ondes à source de lumière à balayage
US10487356B2 (en) 2015-03-16 2019-11-26 Pacific Biosciences Of California, Inc. Integrated devices and systems for free-space optical coupling
WO2016154038A1 (fr) 2015-03-20 2016-09-29 Illumina, Inc. Cartouche fluidique pour une utilisation dans la position verticale ou sensiblement verticale
CA3077811C (fr) 2015-03-24 2024-02-27 Illumina, Inc. Procedes, ensembles de support, et systemes pour l'imagerie d'echantillons pour une analyse biologique ou chimique
US10300452B2 (en) 2015-03-24 2019-05-28 Pacific Biosciences Of California, Inc. Methods and compositions for single molecule composition loading
US20180100192A1 (en) 2015-03-31 2018-04-12 lllumina Cambridge Limited Surface concatemerization of templates
CA2982146A1 (fr) 2015-04-10 2016-10-13 Spatial Transcriptomics Ab Analyse de plusieurs acides nucleiques spatialement differencies de specimens biologiques
CN112229834B (zh) 2015-04-14 2024-11-26 亿明达股份有限公司 用于改进对光发射的检测的结构化基底及涉及其的方法
US10844428B2 (en) 2015-04-28 2020-11-24 Illumina, Inc. Error suppression in sequenced DNA fragments using redundant reads with unique molecular indices (UMIS)
EP3292220B1 (fr) 2015-05-07 2022-07-13 Pacific Biosciences of California, Inc. Architecture pipeline de multiprocesseur
ES2826880T3 (es) 2015-05-11 2021-05-19 Illumina Inc Plataforma para el descubrimiento y análisis de agentes terapéuticos
KR102054571B1 (ko) 2015-05-29 2019-12-10 일루미나 케임브리지 리미티드 클러스터에서 표면 프라이머의 향상된 이용
WO2016196358A1 (fr) 2015-05-29 2016-12-08 Epicentre Technologies Corporation Méthodes d'analyse d'acides nucléiques
BR112017025587B1 (pt) 2015-05-29 2022-09-27 Illumina, Inc Portador de amostra e sistema de ensaio para conduzir reações designadas
RU2713396C2 (ru) 2015-06-03 2020-02-05 Иллюмина, Инк. Композиции, системы и способы секвенирования полинуклеотидов с применением соединительных структур, прикрепленных к полимеразам, расположенным вблизи нанопор
EP3527672B1 (fr) 2015-06-09 2022-10-05 Centrillion Technology Holdings Corporation Matrices aux oligonucleotides pour la séquençage d'acides nucléiques
US10365434B2 (en) 2015-06-12 2019-07-30 Pacific Biosciences Of California, Inc. Integrated target waveguide devices and systems for optical coupling
WO2017007757A1 (fr) 2015-07-06 2017-01-12 Illumina, Inc. Modulation à courant alternatif équilibré pour entraîner des électrodes d'opérations de gouttelettes
EP3320111B1 (fr) 2015-07-06 2021-05-05 Illumina Cambridge Limited Préparation d'échantillon pour l'amplification d'acide nucléique
CN107924121B (zh) 2015-07-07 2021-06-08 亿明达股份有限公司 经由纳米压印的选择性表面图案化
US20180207920A1 (en) 2015-07-17 2018-07-26 Illumina, Inc. Polymer sheets for sequencing applications
EP3329012B1 (fr) 2015-07-27 2021-07-21 Illumina, Inc. Cartographie spatiale d'informations de séquence d'acide nucléique
ES2864677T3 (es) 2015-07-30 2021-10-14 Illumina Inc Desbloqueo ortogonal de nucleótidos
CN108139345B (zh) 2015-08-06 2021-04-20 加利福尼亚太平洋生物科学股份有限公司 用于选择性寻址稀疏布置的电子测量装置的系统和方法
ES2868195T3 (es) 2015-08-14 2021-10-21 Illumina Inc Sistemas y métodos que utilizan sensores magnéticamente sensibles para determinar una característica genética
CN108474805A (zh) 2015-08-24 2018-08-31 亿明达股份有限公司 用于生物和化学测定的线路内蓄压器和流量控制系统
SG10201912283RA (en) 2015-08-28 2020-02-27 Illumina Inc Nucleic acid sequence analysis from single cells
HK1253910A1 (zh) 2015-09-02 2019-07-05 伊卢米纳剑桥有限公司 改善流控系统中的液滴操作的系统和方法
CA3000816A1 (fr) 2015-09-11 2017-03-16 The General Hospital Corporation Interrogation complete de dsb nucleasiques et sequencage (find-seq)
US10450598B2 (en) 2015-09-11 2019-10-22 Illumina, Inc. Systems and methods for obtaining a droplet having a designated concentration of a substance-of-interest
CA3000762A1 (fr) 2015-09-30 2017-04-06 The General Hospital Corporation Rapport in vitro complet d'evenements de clivage par sequencage (circle-seq)
EP3365108B1 (fr) 2015-10-22 2024-06-12 Illumina, Inc. Fluide de remplissage pour dispositifs fluidiques
US10814299B2 (en) 2015-11-18 2020-10-27 Pacific Biosciences Of California, Inc. Loading nucleic acids onto substrates
US10731211B2 (en) 2015-11-18 2020-08-04 Pacific Biosciences Of California, Inc. Methods and compositions for loading of polymerase complexes
CN108350487B (zh) 2015-11-19 2022-05-27 加利福尼亚太平洋生物科学股份有限公司 用于改善信号检测的化合物和系统
CN113321943A (zh) 2015-11-20 2021-08-31 加利福尼亚太平洋生物科学股份有限公司 标记的核苷酸类似物、反应混合物以及测序方法和系统
WO2017087974A1 (fr) 2015-11-20 2017-05-26 Pacific Biosciences Of California, Inc. Réactifs marqués par un colorant protégés
US10676788B2 (en) 2015-11-20 2020-06-09 Pacific Biosciences Of California, Inc. Modified nucleotide reagents
EP3384046B1 (fr) 2015-12-01 2021-04-28 Illumina, Inc. Système microfluidique numérique pour l'isolement de cellules uniques et la caractérisation d'analytes
CN109072300B (zh) 2015-12-17 2023-01-31 伊路敏纳公司 区分复杂生物样品中的甲基化水平
EP3400298B1 (fr) 2016-01-08 2024-03-06 Bio-Rad Laboratories, Inc. Résolution de multiples particules par gouttelette
TW201730563A (zh) 2016-01-11 2017-09-01 伊路米納有限公司 具有顯微螢光計、流體系統和流動單元閂鎖夾持模組的檢測裝置
WO2017165703A1 (fr) 2016-03-24 2017-09-28 Illumina, Inc. Dispositifs et compositions à base de super-réseau photonique destinés à être utilisés en imagerie par luminescence, et leurs procédés d'utilisation
US20170274374A1 (en) 2016-03-28 2017-09-28 Ilumina, Inc. Multi-plane microarrays
WO2017177017A1 (fr) 2016-04-07 2017-10-12 Omniome, Inc. Procédés de quantification d'acides nucléiques cibles et d'identification de variants de séquences
WO2017176896A1 (fr) 2016-04-07 2017-10-12 Illumina, Inc. Procédés et systèmes de construction de banques d'acides nucléiques normalisées
SG11201809171VA (en) 2016-04-22 2018-11-29 Illumina Inc Photonic stucture-based devices and compositions for use in luminescent imaging of multiple sites within a pixel, and methods of using the same
US10597643B2 (en) 2016-04-29 2020-03-24 Omniome, Inc. Polymerases engineered to reduce nucleotide-independent DNA binding
US10584379B2 (en) 2016-04-29 2020-03-10 Omniome, Inc. Method of nucleic acid sequence determination
CN109477137B (zh) 2016-05-11 2023-05-30 伊鲁米那股份有限公司 使用argonaute系统的多核苷酸富集和扩增
WO2017201198A1 (fr) 2016-05-18 2017-11-23 Illumina, Inc. Formation de motifs auto-assemblée utilisant des surfaces hydrophobes à motifs
US10544457B2 (en) 2016-06-14 2020-01-28 Pacific Biosciences Of California, Inc. Methods and compositions for enriching compositions for polymerase enzyme complexes
US10370701B2 (en) 2016-06-17 2019-08-06 Pacific Biosciences Of California, Inc. Methods and compositions for generating asymmetrically-tagged nucleic acid fragments
WO2018009346A1 (fr) 2016-07-05 2018-01-11 Quantapore, Inc. Séquencement de nanopores à base optique
WO2018013558A1 (fr) 2016-07-12 2018-01-18 Life Technologies Corporation Compositions et procédés pour détecter un acide nucléique
EP3485037B1 (fr) 2016-07-18 2022-02-09 F. Hoffmann-La Roche AG Modèles asymétriques et procédé asymétrique de séquençage d'acides nucléiques
CN109312391B (zh) 2016-07-18 2022-06-03 豪夫迈·罗氏有限公司 生成用于单分子测序的单链环状dna文库的方法
US10711300B2 (en) 2016-07-22 2020-07-14 Pacific Biosciences Of California, Inc. Methods and compositions for delivery of molecules and complexes to reaction sites
KR102709499B1 (ko) 2016-07-22 2024-09-24 오레곤 헬스 앤드 사이언스 유니버시티 단일 세포 전체 게놈 라이브러리 및 이의 제조를 위한 조합 인덱싱 방법
CN109844136A (zh) 2016-08-15 2019-06-04 欧姆尼奥姆股份有限公司 测序核酸的方法和系统
WO2018042251A1 (fr) 2016-08-29 2018-03-08 Oslo Universitetssykehus Hf Analyses chip-seq
WO2018064116A1 (fr) 2016-09-28 2018-04-05 Illumina, Inc. Procédés et systèmes de traitement de données
US10190155B2 (en) 2016-10-14 2019-01-29 Nugen Technologies, Inc. Molecular tag attachment and transfer
MY194951A (en) 2016-10-14 2022-12-28 Illumina Inc Cartridge assembly
PL3529380T3 (pl) 2016-10-19 2022-10-24 Illumina, Inc. Sposoby chemicznej ligacji kwasów nukleinowych
EP3529400B1 (fr) 2016-10-24 2021-02-17 Geneinfosec, Inc. Dissimulation d'informations présentes dans des acides nucléiques
JP7113838B2 (ja) 2016-11-16 2022-08-05 イルミナ インコーポレイテッド 配列バリアントコールのための有効化方法およびシステム
GB201619458D0 (en) 2016-11-17 2017-01-04 Spatial Transcriptomics Ab Method for spatial tagging and analysing nucleic acids in a biological specimen
WO2018100724A1 (fr) * 2016-12-01 2018-06-07 株式会社日立ハイテクノロジーズ Substrat de réseau de taches, procédé d'analyse d'acide nucléique, et dispositif d'analyse d'acide nucléique
CA3049961A1 (fr) 2016-12-09 2018-06-14 The Broad Institute, Inc. Diagnostics bases sur un systeme effecteur crispr
US12300357B2 (en) 2016-12-14 2025-05-13 FlowJo, LLC Applied computer technology for management, synthesis, visualization, and exploration of parameters in large multi-parameter data sets
CN118441026A (zh) 2016-12-19 2024-08-06 生物辐射实验室股份有限公司 液滴加标的相邻性保留的标签化dna
MX2019006616A (es) 2016-12-22 2019-10-24 Illumina Inc Arreglo que incluye cebador secuenciador y entidad no secuenciadora.
US11512339B2 (en) 2016-12-22 2022-11-29 Illumina, Inc. Arrays including a resin film and a patterned polymer layer
US20180195115A1 (en) 2016-12-22 2018-07-12 Illumina, Inc. Arrays with quality control tracers
US11248254B2 (en) 2016-12-30 2022-02-15 Omniome, Inc. Method and system employing distinguishable polymerases for detecting ternary complexes and identifying cognate nucleotides
GB201704754D0 (en) 2017-01-05 2017-05-10 Illumina Inc Kinetic exclusion amplification of nucleic acid libraries
ES2917403T3 (es) 2017-01-06 2022-07-08 Illumina Inc Corrección de ajuste de fase
WO2018132389A1 (fr) 2017-01-10 2018-07-19 Omniome, Inc. Polymérases modifiées pour réduire la liaison d'adn indépendante des nucléotides
KR102326612B1 (ko) 2017-01-17 2021-11-15 일루미나, 인코포레이티드 종양원성 스플라이스 변이체 결정
EP4534692A3 (fr) 2017-01-18 2025-06-18 Illumina, Inc. Procédés et systèmes de génération et de correction d'erreur d'ensembles d'indices moléculaires uniques ayant des longueurs moléculaires hétérogènes
US9932631B1 (en) 2017-09-11 2018-04-03 Omniome, Inc. Genotyping by polymerase binding
CN110177884B (zh) 2017-01-20 2021-08-06 欧姆尼欧美公司 通过聚合酶结合进行的基因分型
US10975427B2 (en) 2017-01-20 2021-04-13 Omniome, Inc. Process for cognate nucleotide detection in a nucleic acid sequencing workflow
AU2017394644B2 (en) 2017-01-20 2020-02-06 Pacific Biosciences Of California, Inc. Allele-specific capture of nucleic acids
EP3354746B1 (fr) 2017-01-30 2019-05-29 Gregor Mendel Institute of Molecular Plant Biology GmbH Nouveaux oligonucléotides insérés pour normaliser les données de séquence
GB201701686D0 (en) 2017-02-01 2017-03-15 Illunina Inc System & method with fiducials having offset layouts
GB201701689D0 (en) 2017-02-01 2017-03-15 Illumia Inc System and method with fiducials of non-closed shapes
EP3576868A4 (fr) 2017-02-01 2021-03-17 Illumina, Inc. Système et procédé avec repères répondant à de multiples fréquences d'excitation
GB201701688D0 (en) 2017-02-01 2017-03-15 Illumia Inc System and method with fiducials in non-recliner layouts
WO2018152162A1 (fr) 2017-02-15 2018-08-23 Omniome, Inc. Distinction des séquences par détection de dissociation de polymérase
IL303805B2 (en) 2017-02-21 2024-08-01 Illumina Inc Tegumentation using fixed transpososomes with linkers
US11104937B2 (en) 2017-03-15 2021-08-31 The Broad Institute, Inc. CRISPR effector system based diagnostics
US11021740B2 (en) 2017-03-15 2021-06-01 The Broad Institute, Inc. Devices for CRISPR effector system based diagnostics
KR20190140918A (ko) 2017-03-15 2019-12-20 더 브로드 인스티튜트, 인코퍼레이티드 바이러스 검출을 위한 crispr 이펙터 시스템 기반 진단
US11174515B2 (en) 2017-03-15 2021-11-16 The Broad Institute, Inc. CRISPR effector system based diagnostics
SG11201908680YA (en) 2017-03-20 2019-10-30 Illumina Inc Methods and compositions for preparing nucleic acid libraries
WO2018187013A1 (fr) 2017-04-04 2018-10-11 Omniome, Inc. Appareil fluidique et procédés utiles pour des réactions chimiques et biologiques
US12492430B2 (en) 2017-04-11 2025-12-09 Tecan Genomics, Inc. Library quantitation and qualification
SG11201909914RA (en) 2017-04-23 2019-11-28 Illumina Cambridge Ltd Compositions and methods for improving sample identification in indexed nucleic acid libraries
CN110770353B (zh) 2017-04-23 2024-11-26 伊鲁米那股份有限公司 用于改进编索引的核酸文库中的样品鉴定的组合物和方法
DK3872187T3 (da) 2017-04-23 2022-12-05 Illumina Cambridge Ltd Sammensætninger og fremgangsmåder til forbedring af prøveidentificering i indekserede nukleinsyrebiblioteker
US10161003B2 (en) 2017-04-25 2018-12-25 Omniome, Inc. Methods and apparatus that increase sequencing-by-binding efficiency
CN110799653B (zh) 2017-05-01 2025-01-03 伊鲁米那股份有限公司 用于多重大规模平行测序的最佳索引序列
CN110832087B (zh) 2017-05-08 2025-03-18 伊鲁米那股份有限公司 用于多核苷酸样品的索引的通用短衔接子
ES2980676T3 (es) 2017-05-25 2024-10-02 Flowjo Llc Visualización, análisis comparativo y detección de diferencias automatizada para grandes conjuntos de datos multiparamétricos
CN110997932B (zh) 2017-06-07 2024-06-04 俄勒冈健康科学大学 用于甲基化测序的单细胞全基因组文库
WO2018236631A1 (fr) 2017-06-20 2018-12-27 Illumina, Inc. Procédés et compositions pour résoudre les inefficacités dans des réactions d'amplification
US11186862B2 (en) 2017-06-20 2021-11-30 Bio-Rad Laboratories, Inc. MDA using bead oligonucleotide
JP7390285B2 (ja) 2017-06-26 2023-12-01 ウニヴェルズィテート・フューア・ボーデンクルトゥーア・ウィーン 老化細胞を検出するための新規バイオマーカー
WO2019018366A1 (fr) 2017-07-18 2019-01-24 Omniome, Inc. Procédé de modification chimique de surfaces plastiques
EP3662482A1 (fr) 2017-07-31 2020-06-10 Illumina Inc. Système de séquençage comportant une agrégation d'échantillons biologiques multiplexés
AU2018312560B2 (en) 2017-08-01 2022-03-10 Illumina, Inc. Hydrogel beads for nucleotide sequencing
ES2999650T3 (en) 2017-08-01 2025-02-26 Illumina Inc Spatial indexing of genetic material and library preparation using hydrogel beads and flow cells
EP3545106B1 (fr) 2017-08-01 2022-01-19 Helitec Limited Procédés d'enrichissement et de détermination de séquences nucléotidiques cibles
KR20200075814A (ko) 2017-08-15 2020-06-26 옴니옴 인코포레이티드 화학적 및 생물학적 분석물의 검출에 유용한 스캐닝 장치 및 방법
EP3682025A1 (fr) 2017-09-14 2020-07-22 H. Hoffnabb-La Roche Ag Nouveau procédé pour la génération de bibliothèques d'adn simple brin circulaire
US11447818B2 (en) 2017-09-15 2022-09-20 Illumina, Inc. Universal short adapters with variable length non-random unique molecular identifiers
WO2019053215A1 (fr) 2017-09-15 2019-03-21 F. Hoffmann-La Roche Ag Stratégie d'hybridation-extension-ligature pour générer des banques d'adn simple brin circulaires
CN118899030A (zh) 2017-09-20 2024-11-05 夸登特健康公司 用于区分体细胞变异和种系变异的方法和系统
EP3692166A1 (fr) 2017-10-06 2020-08-12 H. Hoffnabb-La Roche Ag Procédés de circularisation pour la préparation d'échantillons de séquençage de molécules uniques
US11397889B2 (en) 2017-10-16 2022-07-26 Illumina, Inc. Aberrant splicing detection using convolutional neural networks (CNNs)
EP3622521A1 (fr) 2017-10-16 2020-03-18 Illumina, Inc. Réseaux neuronaux à convolution profonde de classification de variants
US11099202B2 (en) 2017-10-20 2021-08-24 Tecan Genomics, Inc. Reagent delivery system
US11162138B2 (en) 2017-10-30 2021-11-02 Pacific Biosciences Of California, Inc. Multi-amplitude modular labeled compounds
EP4180534A1 (fr) 2017-11-02 2023-05-17 Bio-Rad Laboratories, Inc. Analyse génomique basée sur une transposase
WO2019086531A1 (fr) 2017-11-03 2019-05-09 F. Hoffmann-La Roche Ag Séquençage consensus linéaire
AU2018366213B2 (en) 2017-11-08 2025-05-15 Twinstrand Biosciences, Inc. Reagents and adapters for nucleic acid sequencing and methods for making such reagents and adapters
JP6857235B2 (ja) * 2017-11-21 2021-04-14 日本碍子株式会社 光導波路構造、蛍光体素子および光導波路構造の製造方法
US10655168B2 (en) 2017-12-22 2020-05-19 Pacific Biosciences Of California, Inc. Modified biotin-binding proteins for immobilization
BR112020013252A2 (pt) 2017-12-29 2020-12-01 Nautilus Biotechnology, Inc. abordagens de decodificação para a identificação de proteína
CA3279137A1 (en) 2018-01-08 2025-10-30 Illumina, Inc. Systems and devices for high-throughput sequencing with semiconductor-based detection
US11561196B2 (en) 2018-01-08 2023-01-24 Illumina, Inc. Systems and devices for high-throughput sequencing with semiconductor-based detection
CN110832510B (zh) 2018-01-15 2024-10-22 因美纳有限公司 基于深度学习的变体分类器
BR112020015093A2 (pt) 2018-01-29 2020-12-08 The Broad Institute, Inc. Diagnóstico baseado no sistema de efetor crispr
EP3746552B1 (fr) 2018-01-31 2023-12-27 Bio-Rad Laboratories, Inc. Procédés et compositions pour déconvolutionner des codes barres de séparation
JP7096893B2 (ja) 2018-02-05 2022-07-06 エフ．ホフマン－ラ ロシュ アーゲー 単一分子のための一本鎖環状ｄｎａ鋳型の作製
KR20200139671A (ko) 2018-02-06 2020-12-14 옴니옴 인코포레이티드 핵산 프라이머 연장을 위한 조성물 및 기술
JP7027459B2 (ja) 2018-02-13 2022-03-01 イルミナ インコーポレイテッド ヒドロゲルビーズを用いるｄｎａ配列決定
WO2019161253A1 (fr) 2018-02-16 2019-08-22 Ultima Genomics, Inc. Procédés de séquençage avec détection de fréquence unique
WO2019166530A1 (fr) 2018-03-02 2019-09-06 F. Hoffmann-La Roche Ag Génération de matrices d'adn circulaire monocaténaire pour séquençage de molécule unique
SG11201912279RA (en) 2018-03-29 2020-01-30 Illumina Inc Illumination for fluorescence imaging using objective lens
AU2019248635B2 (en) 2018-04-02 2022-01-27 Illumina, Inc. Compositions and methods for making controls for sequence-based genetic testing
NL2020861B1 (en) 2018-04-12 2019-10-22 Illumina Inc Variant classifier based on deep neural networks
WO2019200338A1 (fr) 2018-04-12 2019-10-17 Illumina, Inc. Classificateur de variantes basé sur des réseaux neuronaux profonds
US11512002B2 (en) 2018-04-18 2022-11-29 University Of Virginia Patent Foundation Silica materials and methods of making thereof
CA3097583A1 (fr) 2018-04-19 2019-10-24 Omniome, Inc. Amelioration de la precision d'appels de base dans des procedes de sequencage d'acide nucleique
JP7511344B2 (ja) 2018-04-20 2024-07-05 イルミナ インコーポレイテッド 単一細胞を封入する方法、封入された細胞およびその使用
CN112567047B (zh) 2018-04-26 2025-03-14 加利福尼亚太平洋生物科学股份有限公司 用于稳定核酸-核苷酸-聚合酶复合物的方法和组合物
AU2019271121B2 (en) 2018-05-15 2021-05-20 Illumina Cambridge Limited Compositions and methods for chemical cleavage and deprotection of surface-bound oligonucleotides
IL271454B2 (en) 2018-05-17 2025-04-01 Illumina Inc Rapid single-cell genetic sequencing with low Aggregation bias
WO2019226689A1 (fr) 2018-05-22 2019-11-28 Axbio Inc. Procédés, systèmes et compositions pour le séquençage d'acides nucléiques
WO2019227015A1 (fr) 2018-05-25 2019-11-28 Illumina, Inc. Signatures d'arn circulants spécifiques de la prééclampsie
AU2019276719B2 (en) 2018-05-31 2025-11-13 Pacific Biosciences Of California, Inc. Increased signal to noise in nucleic acid sequencing
US11180794B2 (en) 2018-05-31 2021-11-23 Omniome, Inc. Methods and compositions for capping nucleic acids
SG11202000905PA (en) 2018-06-04 2020-02-27 Illumina Inc High-throughput single-cell transcriptome libraries and methods of making and of using
JP2021526791A (ja) 2018-06-04 2021-10-11 ガーダント ヘルス， インコーポレイテッド セルフリー核酸の細胞起源を決定するための方法およびシステム
WO2020006421A1 (fr) 2018-06-29 2020-01-02 Pacific Biosciences Of California, Inc. Procédés et compositions pour l'administration de molécules et de complexes à des sites de réaction
US12073922B2 (en) 2018-07-11 2024-08-27 Illumina, Inc. Deep learning-based framework for identifying sequence patterns that cause sequence-specific errors (SSEs)
SG11202100141SA (en) 2018-07-12 2021-02-25 Twinstrand Biosciences Inc Methods and reagents for characterizing genomic editing, clonal expansion, and associated applications
WO2020023420A2 (fr) 2018-07-23 2020-01-30 Guardant Health, Inc. Procédés et systèmes pour ajuster la charge de mutation de tumorale par fraction et couverture tumorales
WO2020023362A1 (fr) 2018-07-24 2020-01-30 Omniome, Inc. Formation en série d'espèces de complexe ternaire
KR20210056329A (ko) 2018-08-07 2021-05-18 더 브로드 인스티튜트, 인코퍼레이티드 신규 cas12b 효소 및 시스템
KR102893591B1 (ko) 2018-08-15 2025-12-02 일루미나, 인코포레이티드 라이브러리 인리치먼트를 개선하기 위한 조성물 및 방법
EP4249651B1 (fr) 2018-08-20 2025-01-29 Bio-Rad Laboratories, Inc. Génération de séquences nucléotidiques par co-localisation de billes à code-barres dans des partitions
CN112970068B (zh) 2018-08-30 2025-03-18 夸登特健康公司 用于检测样品之间的污染的方法和系统
CA3109539A1 (fr) 2018-08-31 2020-03-05 Guardant Health, Inc. Detection d'instabilite des microsatellites dans un adn libre circulant
WO2020096691A2 (fr) 2018-09-04 2020-05-14 Guardant Health, Inc. Procédés et systèmes pour détecter un déséquilibre allélique dans des échantillons d'acides nucléiques acellulaires
WO2020060811A1 (fr) 2018-09-17 2020-03-26 Omniome, Inc. Polymérases modifiées pour un séquençage amélioré
WO2020058472A1 (fr) 2018-09-20 2020-03-26 Tamirna Gmbh Signatures de micro-arn pour la prédiction d'un dysfonctionnement hépatique
WO2020072816A1 (fr) 2018-10-03 2020-04-09 The Broad Institute, Inc. Diagnostics basés sur un système effecteur crispr pour la détection de fièvres hémorragiques
SG11201911777QA (en) 2018-10-15 2020-05-28 Illumina Inc Deep learning-based techniques for pre-training deep convolutional neural networks
AU2019364545B2 (en) 2018-10-26 2025-09-04 Illumina, Inc. Modulating polymer beads for DNA processing
IL299237B2 (en) 2018-10-31 2024-12-01 Illumina Inc Polymerases, compositions, and methods of use
ES2996898T3 (en) 2018-10-31 2025-02-13 Guardant Health Inc Method and system for calibrating epigenetic partitioning assays
NL2022043B1 (en) 2018-11-21 2020-06-03 Akershus Univ Hf Tagmentation-Associated Multiplex PCR Enrichment Sequencing
KR20210098491A (ko) 2018-11-30 2021-08-10 진인포섹, 아이엔씨. 무작위 올리고뉴클레오티드를 생성하고 그 서열을 결정하는 방법
AU2019390388B2 (en) 2018-11-30 2025-08-07 Illumina, Inc. Analysis of multiple analytes using a single assay
EP3891304A1 (fr) 2018-12-04 2021-10-13 Omniome, Inc. Fluides à phase mixte pour séquençage d'acide nucléique et autres essais analytiques
KR20210098844A (ko) 2018-12-05 2021-08-11 일루미나, 인코포레이티드 중합효소, 조성물, 및 사용 방법
SG11202012762WA (en) 2018-12-05 2021-01-28 Illumina Cambridge Ltd Methods and compositions for cluster generation by bridge amplification
US20220162667A1 (en) 2018-12-07 2022-05-26 Octant, Inc. Systems for protein-protein interaction screening
WO2020123309A1 (fr) 2018-12-10 2020-06-18 10X Genomics, Inc. Résolution de réseaux spatiaux par déconvolution basée sur la proximité
GB201820341D0 (en) 2018-12-13 2019-01-30 10X Genomics Inc Method for transposase-mediated spatial tagging and analysing genomic DNA in a biological specimen
GB201820300D0 (en) 2018-12-13 2019-01-30 10X Genomics Inc Method for spatial tagging and analysing genomic DNA in a biological specimen
EP3894598B1 (fr) 2018-12-14 2024-01-03 Illumina Cambridge Limited Réduction du phasage avec des nucléotides non marqués pendant le séquençage
WO2020126595A1 (fr) 2018-12-17 2020-06-25 Illumina Cambridge Limited Oligonucléotide d'amorce pour le séquençage
ES3032918T3 (en) 2018-12-17 2025-07-28 Illumina Cambridge Ltd Method of polynucleotide sequencing
CN112739830A (zh) 2018-12-18 2021-04-30 伊卢米纳剑桥有限公司 使用单一表面引物进行配对末端测序的方法和组合物
CN112654715B (zh) 2018-12-19 2024-12-31 亿明达股份有限公司 改进多核苷酸簇克隆性优先性的方法
SG11202104701XA (en) 2018-12-20 2021-06-29 Guardant Health Inc Methods, compositions, and systems for improving recovery of nucleic acid molecules
EP3899032A2 (fr) 2018-12-20 2021-10-27 Omniome, Inc. Régulation de température pour l'analyse d'acides nucléiques et d'autres analytes
US11293061B2 (en) 2018-12-26 2022-04-05 Illumina Cambridge Limited Sequencing methods using nucleotides with 3′ AOM blocking group
EP3674702A1 (fr) 2018-12-27 2020-07-01 Imec VZW Procédé de séquençage d'un polynucléotide à l'aide d'un biofet
US11649485B2 (en) 2019-01-06 2023-05-16 10X Genomics, Inc. Generating capture probes for spatial analysis
US11926867B2 (en) 2019-01-06 2024-03-12 10X Genomics, Inc. Generating capture probes for spatial analysis
CA3114732A1 (fr) 2019-01-11 2020-07-16 Illumina Cambridge Limited Complexes de transposomes lies a une surface complexe
ES3013495T3 (en) 2019-01-31 2025-04-14 Guardant Health Inc Method for isolating and sequencing cell-free dna
US11499189B2 (en) 2019-02-14 2022-11-15 Pacific Biosciences Of California, Inc. Mitigating adverse impacts of detection systems on nucleic acids and other biological analytes
CN113613787B (zh) 2019-02-20 2023-06-13 加利福尼亚太平洋生物科学股份有限公司 用于检测化学和生物分析物的扫描装置和方法
WO2020176659A1 (fr) 2019-02-27 2020-09-03 Guardant Health, Inc. Procédés et systèmes pour déterminer l'origine cellulaire d'acides nucléiques acellulaires
CA3131682A1 (fr) 2019-02-28 2020-09-03 Pacific Biosciences Of California, Inc. Alignement ameliore en utilisant des lectures de sequencage a effondrement d'homopolymeres
SG11202102530QA (en) 2019-03-01 2021-04-29 Illumina Inc High-throughput single-nuclei and single-cell libraries and methods of making and of using
US11783917B2 (en) 2019-03-21 2023-10-10 Illumina, Inc. Artificial intelligence-based base calling
NL2023310B1 (en) 2019-03-21 2020-09-28 Illumina Inc Training data generation for artificial intelligence-based sequencing
WO2020205296A1 (fr) 2019-03-21 2020-10-08 Illumina, Inc. Génération à base d'intelligence artificielle de métadonnées de séquençage
NL2023311B9 (en) 2019-03-21 2021-03-12 Illumina Inc Artificial intelligence-based generation of sequencing metadata
US11210554B2 (en) 2019-03-21 2021-12-28 Illumina, Inc. Artificial intelligence-based generation of sequencing metadata
NL2023314B1 (en) 2019-03-21 2020-09-28 Illumina Inc Artificial intelligence-based quality scoring
NL2023316B1 (en) 2019-03-21 2020-09-28 Illumina Inc Artificial intelligence-based sequencing
NL2023312B1 (en) 2019-03-21 2020-09-28 Illumina Inc Artificial intelligence-based base calling
CN109827918B (zh) * 2019-04-03 2021-08-13 广西壮族自治区冶金产品质量检验站 掺锡氧化铟粉中硅含量的测定方法
US11423306B2 (en) 2019-05-16 2022-08-23 Illumina, Inc. Systems and devices for characterization and performance analysis of pixel-based sequencing
US11593649B2 (en) 2019-05-16 2023-02-28 Illumina, Inc. Base calling using convolutions
WO2020243164A1 (fr) 2019-05-28 2020-12-03 Octant, Inc. Système de relais de transcription
NL2023516B1 (en) 2019-05-28 2020-12-08 Illumina Inc Manufacturing a flowcell with a planar waveguide
WO2020243722A1 (fr) 2019-05-31 2020-12-03 Guardant Health, Inc. Procédés et systèmes pour améliorer une surveillance de patient après une intervention chirurgicale
WO2020252186A1 (fr) 2019-06-11 2020-12-17 Omniome, Inc. Détection de mise au point étalonnée
EP3990185B1 (fr) 2019-06-28 2024-09-11 Illumina Cambridge Limited Cuves à circulation avec guides d'ondes linéaires
JP7603586B2 (ja) 2019-07-12 2024-12-20 イルミナ ケンブリッジ リミテッド 電気泳動を用いた核酸ライブラリの調製
SG11202105836XA (en) 2019-07-12 2021-07-29 Illumina Cambridge Ltd Compositions and methods for preparing nucleic acid sequencing libraries using crispr/cas9 immobilized on a solid support
WO2021011803A1 (fr) 2019-07-16 2021-01-21 Omniome, Inc. Acides nucléiques synthétiques ayant des structures non naturelles
US10656368B1 (en) 2019-07-24 2020-05-19 Omniome, Inc. Method and system for biological imaging using a wide field objective lens
EP3999651A4 (fr) 2019-08-26 2023-07-26 PACT Pharma, Inc. Méthodes de réalisation de séquençage guide sur des lymphocytes t humains primaires
TW202124406A (zh) 2019-09-10 2021-07-01 美商歐姆尼歐美公司 核苷酸之可逆修飾
US20220290245A1 (en) 2019-09-11 2022-09-15 The United States Of America, As Represented By The Secretary, Department Of Health And Human Servic Cancer detection and classification
KR20220097404A (ko) 2019-10-10 2022-07-07 1859, 인크. 미세유체 스크리닝을 위한 방법 및 시스템
EP4045683B1 (fr) 2019-10-18 2025-02-19 Pacific Biosciences of California, Inc. Procédés pour le coiffage d'acides nucléiques
US20210139867A1 (en) 2019-11-08 2021-05-13 Omniome, Inc. Engineered polymerases for improved sequencing by binding
CN113677807A (zh) 2019-11-22 2021-11-19 因美纳有限公司 先兆子痫特异性的循环rna标记
US20210214800A1 (en) 2019-11-26 2021-07-15 Guardant Health, Inc. Methods, compositions and systems for improving the binding of methylated polynucleotides
DE202019106694U1 (de) 2019-12-02 2020-03-19 Omniome, Inc. System zur Sequenzierung von Nukleinsäuren in Fluidschaum
DE202019106695U1 (de) 2019-12-02 2020-03-19 Omniome, Inc. System zur Sequenzierung von Nukleinsäuren in Fluidschaum
CA3131632A1 (fr) 2019-12-04 2021-06-10 Tong Liu Preparation de bibliotheques de sequencage d'adn pour la detection d'agents pathogenes d'adn dans le plasma
CA3164781A1 (fr) 2019-12-18 2021-06-24 F. Hoffmann-La Roche Ag Procedes de sequencage par synthese au moyen d'un schema de marquage consecutif
KR20220118295A (ko) 2019-12-19 2022-08-25 일루미나, 인코포레이티드 고 처리량 단일 세포 라이브러리, 및 이의 제조 방법 및 사용 방법
US11821035B1 (en) 2020-01-29 2023-11-21 10X Genomics, Inc. Compositions and methods of making gene expression libraries
WO2021152586A1 (fr) 2020-01-30 2021-08-05 Yeda Research And Development Co. Ltd. Procédés d'analyse du microbiome, du profil d'immunoglobuline et de l'état physiologique
US12076701B2 (en) 2020-01-31 2024-09-03 10X Genomics, Inc. Capturing oligonucleotides in spatial transcriptomics
US12059674B2 (en) 2020-02-03 2024-08-13 Tecan Genomics, Inc. Reagent storage system
US12110541B2 (en) 2020-02-03 2024-10-08 10X Genomics, Inc. Methods for preparing high-resolution spatial arrays
CN115243792A (zh) 2020-02-04 2022-10-25 加利福尼亚太平洋生物科学股份有限公司 流通池及其制造和使用方法
US12535420B2 (en) 2020-02-18 2026-01-27 Pacific Biosciences Of California, Inc. Highly multiplexed nucleic acid sequencing systems
US20210265016A1 (en) 2020-02-20 2021-08-26 Illumina, Inc. Data Compression for Artificial Intelligence-Based Base Calling
EP4107735B1 (fr) 2020-02-20 2025-11-26 Illumina, Inc. Appel de base de plusieurs à plusieurs basé sur l'intelligence artificielle
US12354008B2 (en) 2020-02-20 2025-07-08 Illumina, Inc. Knowledge distillation and gradient pruning-based compression of artificial intelligence-based base caller
US20210265015A1 (en) 2020-02-20 2021-08-26 Illumina, Inc. Hardware Execution and Acceleration of Artificial Intelligence-Based Base Caller
CN115516104A (zh) 2020-03-03 2022-12-23 加利福尼亚太平洋生物科学股份有限公司 用于对双链核酸进行测序的方法和组合物
US20230105642A1 (en) 2020-03-30 2023-04-06 Illumina, Inc. Method and compositions for preparing nucleic acid libraries
WO2021214766A1 (fr) 2020-04-21 2021-10-28 Yeda Research And Development Co. Ltd. Procédés de diagnostic d'infections virales et vaccins associés
JP2023524681A (ja) 2020-04-30 2023-06-13 ガーダント ヘルス， インコーポレイテッド 分配された核酸を使用した配列決定のための方法
US20230203592A1 (en) 2020-05-05 2023-06-29 Akershus Universitetssykehus Hf Compositions and methods for characterizing bowel cancer
US11188778B1 (en) 2020-05-05 2021-11-30 Illumina, Inc. Equalization-based image processing and spatial crosstalk attenuator
CN115836135A (zh) 2020-05-05 2023-03-21 加利福尼亚太平洋生物科学股份有限公司 用于修饰聚合酶-核酸复合物的组合物和方法
CA3177286A1 (fr) 2020-05-12 2021-11-18 Illumina Inc. Generation d'acides nucleiques avec des bases modifiees au moyen de desoxynucleotidyl transferase terminale recombinante
JP2023526252A (ja) 2020-05-14 2023-06-21 ガーダント ヘルス， インコーポレイテッド 相同組換え修復欠損の検出
US12031177B1 (en) 2020-06-04 2024-07-09 10X Genomics, Inc. Methods of enhancing spatial resolution of transcripts
US20230193356A1 (en) 2020-06-08 2023-06-22 The Broad Institute, Inc. Single cell combinatorial indexing from amplified nucleic acids
WO2021252617A1 (fr) 2020-06-09 2021-12-16 Illumina, Inc. Procédés pour augmenter le rendement de bibliothèques de séquençage
EP4165549A1 (fr) 2020-06-11 2023-04-19 Nautilus Biotechnology, Inc. Procédés et systèmes pour le décodage computationnel d'entités biologiques, chimiques et physiques
MX2022016492A (es) 2020-06-22 2023-03-06 Illumina Cambridge Ltd Nucleosidos y nucleotidos con grupo de bloqueo de acetal 3'.
CN111665617A (zh) * 2020-06-24 2020-09-15 武汉中纪生物科技有限公司 一种对焦方法及系统
BR112022026806A2 (pt) 2020-07-02 2023-04-25 Illumina Inc Método para calibrar a eficiência de semeadura de biblioteca de ácidos nucleicos em células de fluxo
JP2023532905A (ja) 2020-07-08 2023-08-01 イルミナ インコーポレイテッド トランスポソーム担体としてのビーズ
WO2023282916A1 (fr) 2021-07-09 2023-01-12 Guardant Health, Inc. Procédés de détection de réarrangements génomiques à l'aide d'acides nucléiques acellulaires
ES2987661T3 (es) 2020-07-10 2024-11-15 Guardant Health Inc Métodos de detección de reordenamientos genómicos utilizando ácidos nucleicos libres de células
WO2022026761A1 (fr) 2020-07-30 2022-02-03 Guardant Health, Inc. Compositions et méthodes pour isoler de l'adn acellulaire
BR112023002051A2 (pt) 2020-08-06 2023-04-18 Illumina Inc Preparação de bibliotecas de sequenciamento de rna e dna usando transpossomos ligados a microesferas
IL299783A (en) 2020-08-18 2023-03-01 Illumina Inc Sequence-Specific Targeted Transposition, Selection and Sorting of Nucleic Acids
WO2022046947A1 (fr) 2020-08-25 2022-03-03 Guardant Health, Inc. Méthodes et systèmes pour prédire l'origine d'un variant
US20220067489A1 (en) 2020-08-28 2022-03-03 Illumina, Inc. Detecting and Filtering Clusters Based on Artificial Intelligence-Predicted Base Calls
US12371743B2 (en) 2022-03-04 2025-07-29 Element Biosciences, Inc. Double-stranded splint adaptors and methods of use
US12359193B2 (en) 2022-03-04 2025-07-15 Element Biosciences, Inc. Single-stranded splint strands and methods of use
US12469162B2 (en) 2020-08-31 2025-11-11 Element Biosciences, Inc. Primary analysis in next generation sequencing
US11200446B1 (en) 2020-08-31 2021-12-14 Element Biosciences, Inc. Single-pass primary analysis
US12505571B2 (en) 2020-08-31 2025-12-23 Element Biosciences, Inc. Primary analysis in next generation sequencing
AU2021339945A1 (en) 2020-09-11 2023-03-02 Illumina Cambridge Limited Methods of enriching a target sequence from a sequencing library using hairpin adaptors
CA3193090A1 (fr) 2020-09-30 2022-04-07 Guardant Health, Inc. Procedes et systemes pour ameliorer le rapport signal sur bruit de dosages de partitionnement de methylation d'adn
AU2021360589A1 (en) 2020-10-15 2023-06-22 Board Of Regents, The University Of Texas System Detection and analysis of structural variations in genomes
MX2023004461A (es) 2020-10-21 2023-05-03 Illumina Inc Plantillas de secuenciacion que comprenden multiples insertos y composiciones y metodos para mejorar la productividad de secuenciacion.
EP4232599A1 (fr) 2020-10-23 2023-08-30 Guardant Health, Inc. Compositions et procédés d'analyse d'adn par division et conversion de base
US20220162680A1 (en) * 2020-11-20 2022-05-26 Illumina, Inc. Compositions and methods for sequencing using fluorophores and quenchers or donors
WO2022140629A1 (fr) 2020-12-23 2022-06-30 Guardant Health, Inc. Procédés et systèmes d'analyse des polynucléotides méthylés
CN112812954A (zh) * 2020-12-29 2021-05-18 中国科学院长春光学精密机械与物理研究所 一种基因测序芯片
CA3204784A1 (fr) 2021-01-13 2022-07-21 Alex Nemiroski Structuration de surface a l'aide d'un ensemble colloidal
AU2022209365A1 (en) 2021-01-21 2023-07-20 Nautilus Subsidiary, Inc. Systems and methods for biomolecule preparation
CN117043352A (zh) 2021-01-29 2023-11-10 伊鲁米纳公司 使用多核苷酸改善流通池的接种效率的方法、组合物和试剂盒
DK4288562T3 (da) 2021-02-04 2025-02-10 Illumina Inc Indekseret long-read-generering på transposom-bundne beads
WO2022174054A1 (fr) 2021-02-13 2022-08-18 The General Hospital Corporation Procédés et compositions pour la détection de macromolécules in situ et utilisations correspondantes
KR20230156364A (ko) 2021-03-05 2023-11-14 가던트 헬쓰, 인크. 분자 반응을 분석하기 위한 방법 및 관련 측면
WO2022192889A1 (fr) 2021-03-09 2022-09-15 Guardant Health, Inc. Détection de la présence d'une tumeur sur la base de données de séquençage de polynucléotide hors cible
IL305336B2 (en) 2021-03-11 2025-09-01 Nautilus Subsidiary Inc Systems and methods for preserving biomolecules
US20220301657A1 (en) 2021-03-16 2022-09-22 Illumina, Inc. Tile location and/or cycle based weight set selection for base calling
WO2022197752A1 (fr) 2021-03-16 2022-09-22 Illumina, Inc. Sélection d'ensembles de poids basée sur l'emplacement d'un élément et/ou le cycle pour appel de base
AU2022245985A1 (en) 2021-03-22 2023-09-21 Illumina Cambridge Limited Methods for improving nucleic acid cluster clonality
CA3214278A1 (fr) 2021-03-29 2022-10-06 Illumina, Inc Procedes ameliores de preparation de banques
MX2023011219A (es) 2021-03-29 2023-10-02 Illumina Inc Composiciones y métodos para evaluar el daño del adn en una genoteca y normalizar el sesgo del tamaño del amplicón.
BR112023019959A2 (pt) 2021-03-30 2024-01-30 Illumina Inc Métodos aprimorados de preparação isotérmica de biblioteca e dna complementar
WO2022208171A1 (fr) 2021-03-31 2022-10-06 UCL Business Ltd. Procédés de détection d'analytes
JP2024511760A (ja) 2021-03-31 2024-03-15 イルミナ インコーポレイテッド エラー補正のための固有分子識別子を有するトランスポゾンベースの技術を使用した指向性タグメンテーション配列決定ライブラリーの調製方法
CA3183578A1 (fr) 2021-03-31 2022-10-06 Illumina Inc. Appelant de base a base d'intelligence artificielle avec reconnaissance contextuelle
EP4315342A1 (fr) 2021-04-02 2024-02-07 Illumina, Inc. Modèle d'apprentissage automatique pour la détection d'une bulle dans une lame d'échantillon de nucléotide pour séquençage
US12217829B2 (en) 2021-04-15 2025-02-04 Illumina, Inc. Artificial intelligence-based analysis of protein three-dimensional (3D) structures
US20220336057A1 (en) 2021-04-15 2022-10-20 Illumina, Inc. Efficient voxelization for deep learning
US12070744B2 (en) 2021-04-22 2024-08-27 Illumina, Inc. Valve assemblies and related systems
CN117642514A (zh) 2021-05-04 2024-03-01 加利福尼亚太平洋生物科学股份有限公司 具有缩小尺度单元阱的集成分析器件的阵列
EP4337786A1 (fr) 2021-05-10 2024-03-20 Pacific Biosciences of California, Inc. Ensemencement et amplification monomoléculaire sur une surface
US20220356515A1 (en) 2021-05-10 2022-11-10 Pacific Biosciences Of California, Inc. Dna amplification buffer replenishment during rolling circle amplification
CA3216735A1 (fr) 2021-05-20 2022-11-24 Patrizia IAVICOLI Compositions et procedes de sequencage par synthese
WO2022265994A1 (fr) 2021-06-15 2022-12-22 Illumina, Inc. Fonctionnalisation de surface exempte d'hydrogel pour séquençage
EP4359555A1 (fr) 2021-06-23 2024-05-01 Illumina, Inc. Compositions, procédés, kits, cartouches et systèmes de séquençage de réactifs
WO2022271954A1 (fr) 2021-06-24 2022-12-29 Illumina, Inc. Procédés et compositions pour l'indexation combinatoire d'acides nucléiques à base de billes
WO2023278184A1 (fr) 2021-06-29 2023-01-05 Illumina, Inc. Procédés et systèmes pour corriger une diaphonie dans un éclairage émis par des sites de réaction
AU2022300970A1 (en) 2021-06-29 2024-01-18 Illumina, Inc. Self-learned base caller, trained using oligo sequences
CA3224402A1 (fr) 2021-06-29 2023-01-05 Eric Jon Ojard Metrique de rapport signal-sur-bruit pour determiner des identifications de bases nucleotidiques et qualite d'identification de bases
AU2022301321A1 (en) 2021-06-29 2024-01-18 Illumina, Inc. Machine-learning model for generating confidence classifications for genomic coordinates
US12530882B2 (en) 2021-07-01 2026-01-20 Illumina, Inc. Efficient artificial intelligence-based base calling of index sequences
WO2023287617A1 (fr) 2021-07-13 2023-01-19 Illumina, Inc. Procédés et systèmes pour l'extraction en temps réel de diaphonie dans un éclairage émis à partir de sites de réaction
KR20240031968A (ko) 2021-07-19 2024-03-08 일루미나, 인코포레이티드 염기 호출에 대한 보간 및 적응을 갖는 강도 추출
US11455487B1 (en) 2021-10-26 2022-09-27 Illumina Software, Inc. Intensity extraction and crosstalk attenuation using interpolation and adaptation for base calling
US20230021577A1 (en) 2021-07-23 2023-01-26 Illumina Software, Inc. Machine-learning model for recalibrating nucleotide-base calls
CN117813391A (zh) 2021-07-23 2024-04-02 因美纳有限公司 制备用于dna测序的基底表面的方法
EP4377960A1 (fr) 2021-07-28 2024-06-05 Illumina, Inc. Étalonnage de score de qualité de systèmes d'appel de bases
JP2024529843A (ja) 2021-08-03 2024-08-14 イルミナ インコーポレイテッド 複数のベースコーラモデルを使用するベースコール
US12077789B2 (en) 2021-08-14 2024-09-03 Illumina, Inc. Polymerases, compositions, and methods of use
AU2022331421A1 (en) 2021-08-17 2024-01-04 Illumina, Inc. Methods and compositions for identifying methylated cytosines
WO2023034076A1 (fr) 2021-08-31 2023-03-09 Illumina, Inc. Cellule d'écoulement à résolution d'imagerie de puits améliorée
US12529049B2 (en) 2021-09-09 2026-01-20 Nautilus Subsidiary, Inc. Characterization and localization of protein modifications
US20230093253A1 (en) 2021-09-17 2023-03-23 Illumina, Inc. Automatically identifying failure sources in nucleotide sequencing from base-call-error patterns
EP4405954A1 (fr) 2021-09-21 2024-07-31 Illumina, Inc. Génome de référence de graphe et stratégie d'appel de bases utilisant des haplotypes attribués
WO2023049073A1 (fr) 2021-09-22 2023-03-30 Nautilus Biotechnology, Inc. Procédés et systèmes pour déterminer des interactions polypeptidiques
WO2023049215A1 (fr) 2021-09-22 2023-03-30 Illumina, Inc. Appel de base basé sur l'état compressé
CN118318049A (zh) 2021-09-28 2024-07-09 安序源有限公司 用于处理核酸样品的方法以及核酸样品的组合物
WO2023056328A2 (fr) 2021-09-30 2023-04-06 Illumina, Inc. Supports solides et procédés d'appauvrissement et/ou d'enrichissement de fragments de bibliothèque préparés à partir de bioéchantillons
US12480157B2 (en) 2021-09-30 2025-11-25 Illumina, Inc. Polynucleotide sequencing
AU2022367166A1 (en) 2021-10-11 2024-04-04 Nautilus Subsidiary, Inc. Highly multiplexable analysis of proteins and proteomes
US12257600B2 (en) * 2021-10-19 2025-03-25 Femtika, UAB Aluminum surface treatment method to increase adhesion with polyurethane coating
US20250236865A1 (en) 2021-10-20 2025-07-24 Illumina, Inc. Methods for capturing library dna for sequencing
US20230149883A1 (en) 2021-11-03 2023-05-18 Nautilus Biotechnology, Inc. Systems and methods for surface structuring
WO2023081485A1 (fr) 2021-11-08 2023-05-11 Pacific Biosciences Of California, Inc. Séquençage par étapes d'un polynucléotide à l'aide d'un mélange réactionnel homogène
EP4441743A1 (fr) 2021-12-02 2024-10-09 Illumina, Inc. Génération de corrections de signal spécifique à un groupe pour déterminer des appels de base nucléotidique
WO2023122363A1 (fr) 2021-12-23 2023-06-29 Illumina Software, Inc. Résumés dynamiques d'état graphique pour séquençage de nucléotides
US20230215515A1 (en) 2021-12-23 2023-07-06 Illumina Software, Inc. Facilitating secure execution of external workflows for genomic sequencing diagnostics
US12403467B2 (en) 2021-12-23 2025-09-02 Illumina, Inc. Systems and related temperature calibration methods
US20230207050A1 (en) 2021-12-28 2023-06-29 Illumina Software, Inc. Machine learning model for recalibrating nucleotide base calls corresponding to target variants
KR20240131386A (ko) 2021-12-29 2024-08-30 일루미나, 인코포레이티드 유전체 분석 애플리케이션을 위한 변이 분석 모델 버전 자동 스위칭
US20240294967A1 (en) 2022-01-20 2024-09-05 Illumina, Inc. Methods of detecting methylcytosine and hydroxymethylcytosine by sequencing
US20230313271A1 (en) 2022-02-25 2023-10-05 Illumina, Inc. Machine-learning models for detecting and adjusting values for nucleotide methylation levels
KR20240152324A (ko) 2022-02-25 2024-10-21 일루미나, 인코포레이티드 뉴클레오티드 서열분석을 위한 교정 서열
EP4500536A1 (fr) 2022-03-25 2025-02-05 Illumina, Inc. Appel de bases séquence par séquence
CA3245990A1 (fr) 2022-03-29 2023-10-05 Nautilus Subsidiary, Inc. Réseaux intégrés pour processus à analyte unique
AU2023248405A1 (en) 2022-04-07 2024-01-04 Illumina, Inc. Altered cytidine deaminases and methods of use
WO2023212490A1 (fr) 2022-04-25 2023-11-02 Nautilus Subsidiary, Inc. Systèmes et procédés d'évaluation et d'amélioration de la qualité de dosages moléculaires multiplex
US20230340571A1 (en) 2022-04-26 2023-10-26 Illumina, Inc. Machine-learning models for selecting oligonucleotide probes for array technologies
US20230348967A1 (en) 2022-04-29 2023-11-02 Illumina Cambridge Limited Methods and systems for encapsulating lyophilised microspheres
US20230360725A1 (en) 2022-05-09 2023-11-09 Guardant Health, Inc. Detecting degradation based on strand bias
WO2023220627A1 (fr) 2022-05-10 2023-11-16 Illumina Software, Inc. Réseau neuronal adaptatif pour séquençage de nucléotides
US20230392207A1 (en) 2022-06-03 2023-12-07 Illumina, Inc. Circulating rna biomarkers for preeclampsia
WO2023240093A1 (fr) 2022-06-06 2023-12-14 Element Biosciences, Inc. Procédés d'assemblage et de lecture de séquences d'acides nucléiques à partir de populations mixtes
WO2023250364A1 (fr) 2022-06-21 2023-12-28 Nautilus Subsidiary, Inc. Procédé pour la détection d'analytes sur des sites dont la distance n'est pas optiquement résolvable
WO2023250504A1 (fr) 2022-06-24 2023-12-28 Illumina Software, Inc. Amélioration d'alignement de lecture de division en identifiant et en évaluant de manière intelligente des groupes de divisions candidats
US20230420082A1 (en) 2022-06-27 2023-12-28 Illumina Software, Inc. Generating and implementing a structural variation graph genome
WO2024006779A1 (fr) 2022-06-27 2024-01-04 Illumina, Inc. Accélérateurs pour un modèle d'imputation de génotypes
WO2024006705A1 (fr) 2022-06-27 2024-01-04 Illumina Software, Inc. Génotypage amélioré d'antigène leucocytaire humain (hla)
IL317477A (en) 2022-07-05 2025-02-01 Element Biosciences Inc PCR-free library preparation using double-stranded probe adapters and methods of use
WO2024015962A1 (fr) 2022-07-15 2024-01-18 Pacific Biosciences Of California, Inc. Adaptateurs en épingle à cheveux asymétriques bloqués
US20240038327A1 (en) 2022-07-26 2024-02-01 Illumina Software, Inc. Rapid single-cell multiomics processing using an executable file
AU2023325064A1 (en) 2022-08-15 2025-03-06 Element Biosciences, Inc. Spatially resolved surface capture of nucleic acids
WO2024039516A1 (fr) 2022-08-19 2024-02-22 Illumina, Inc. Détection de la troisième paire de bases de l'adn spécifique de site
KR20250086817A (ko) 2022-09-12 2025-06-13 엘리먼트 바이오사이언스, 인크. 범용 긴 스플린트 가닥을 갖는 이중가닥 스플린트 어댑터 및 사용 방법
WO2024059655A1 (fr) 2022-09-15 2024-03-21 Nautilus Subsidiary, Inc. Caractérisation de l'accessibilité de structures macromoléculaires
CN120019440A (zh) 2022-09-22 2025-05-16 加利福尼亚太平洋生物科学股份有限公司 用于串联重复序列映射的系统和方法
WO2024073599A1 (fr) 2022-09-29 2024-04-04 Nautilus Subsidiary, Inc. Préparation de surfaces de réseaux pour des procédés à analyte unique
CN118974830A (zh) 2022-09-29 2024-11-15 因美纳有限公司 用于推算靶变体的靶变体参考组
US20240141427A1 (en) 2022-09-30 2024-05-02 Illumina, Inc. Polymerases, compositions, and methods of use
EP4594482A1 (fr) 2022-09-30 2025-08-06 Illumina, Inc. Cytidine désaminases et procédés d'utilisation dans la cartographie de nucléotides cytosine modifiés
WO2024069581A1 (fr) 2022-09-30 2024-04-04 Illumina Singapore Pte. Ltd. Complexes hélicase-cytidine désaminase et procédés d'utilisation
US20250388894A1 (en) 2022-09-30 2025-12-25 Illumina, Inc. Methods of using cpg binding proteins in mapping modified cytosine nucleotides
CN118974831A (zh) 2022-09-30 2024-11-15 因美纳有限公司 用于细化结构变体检出的机器学习模型
US20240127905A1 (en) 2022-10-05 2024-04-18 Illumina, Inc. Integrating variant calls from multiple sequencing pipelines utilizing a machine learning architecture
WO2024077202A2 (fr) 2022-10-06 2024-04-11 Illumina, Inc. Sondes pour améliorer la surveillance d'échantillons environnementaux
EP4482978A1 (fr) 2022-10-06 2025-01-01 Illumina, Inc. Sondes servant à appauvrir un petit arn non codant abondant
EP4482987A2 (fr) 2022-10-06 2025-01-01 Illumina, Inc. Sondes pour améliorer la surveillance d'échantillons de coronavirus
WO2024081649A1 (fr) 2022-10-11 2024-04-18 Illumina, Inc. Détection et correction de valeurs de méthylation à partir de dosages de séquençage de méthylation
CN120188220A (zh) 2022-11-15 2025-06-20 夸登特健康公司 用来自基线循环肿瘤DNA(ctDNA)水平和ctDNA水平随时间纵向变化的信号从ctDNA预测非小细胞肺癌(NSCLC)患者药物响应或直至死亡或癌症进展的时间的方法
EP4627583A1 (fr) 2022-11-30 2025-10-08 Illumina, Inc. Prédiction précise de variants à partir de données de séquençage de méthylation
WO2024118903A1 (fr) 2022-11-30 2024-06-06 Illumina, Inc. Correction chimio-enzymatique de transformations d'uracile faux positives
KR20250143080A (ko) 2022-12-07 2025-09-30 엘리먼트 바이오사이언스, 인크. 수용성 시아닌 유도체 및 핵산 시퀀싱 방법에서의 이의 용도
EP4630816A1 (fr) 2022-12-09 2025-10-15 Nautilus Subsidiary, Inc. Procédé comprenant la mise en oeuvre sur un réseau d'analytes uniques d'au moins 50 cycles d'un processus
WO2024129672A1 (fr) 2022-12-12 2024-06-20 The Broad Institute, Inc. Arn en circulation pour évaluer la connectivité cellule-cellule et la neuroanatomie
EP4634400A1 (fr) 2022-12-14 2025-10-22 Illumina, Inc. Systèmes et procédés de capture et d'enrichissement de billes en grappe sur des substrats de cuve à circulation
EP4634285A1 (fr) 2022-12-15 2025-10-22 Nautilus Subsidiary, Inc. Inhibition de phénomènes photoniques sur des réseaux de molécules uniques
US20250354196A1 (en) 2022-12-16 2025-11-20 Illumina, Inc. Boranes on solid supports
WO2024137682A1 (fr) 2022-12-21 2024-06-27 Guardant Health, Inc. Détection de déficiences en recombinaison homologue sur la base de l'état de méthylation de molécules d'acide nucléique acellulaire
US20240229131A1 (en) 2022-12-22 2024-07-11 Illumina, Inc. Transition-metal catalyst compositions and methods for sequencing by synthesis
CN119095982A (zh) 2022-12-22 2024-12-06 伊路米纳有限公司 用于边合成边测序的钯催化剂组合物和方法
CA3246559A1 (fr) 2022-12-27 2024-07-04 Illumina, Inc. Procédés de séquençage utilisant des nucléotides bloqués par allyle 3'
CA3246669A1 (fr) 2023-01-06 2024-07-11 Illumina, Inc. Réduction des uraciles par polymérase
WO2024151373A1 (fr) 2023-01-12 2024-07-18 Nautilus Subsidiary, Inc. Caractérisation de glycanes et de glycoconjugués
EP4662663A1 (fr) 2023-02-06 2025-12-17 Illumina, Inc. Détermination et élimination d'interférence de lumière inter-groupes
US20250361562A1 (en) 2023-02-17 2025-11-27 Illumina, Inc. Cell-free dna signals as biomarkers of preeclampsia
WO2024206122A1 (fr) 2023-03-24 2024-10-03 Nautilus Subsidiary, Inc. Amélioration du transfert des nanoparticules sur les surfaces des réseaux
WO2024206394A1 (fr) 2023-03-30 2024-10-03 Illumina, Inc. Compositions et procédés de séquençage d'acides nucléiques
US20250384952A1 (en) 2023-03-30 2025-12-18 Illumina, Inc. Tandem repeat genotyping
WO2024229396A1 (fr) 2023-05-03 2024-11-07 Illumina, Inc. Modèle d'apprentissage automatique pour réétalonner des appels de génotype à partir de fichiers de données de séquençage existants
WO2024233502A1 (fr) 2023-05-05 2024-11-14 Guardant Health, Inc. Test à base de sang d'adn acellulaire pour le criblage du cancer
WO2024243237A1 (fr) 2023-05-24 2024-11-28 Nautilus Subsidiary, Inc. Systèmes et procédés de vision artificielle
WO2024249200A1 (fr) 2023-05-26 2024-12-05 Illumina, Inc. Procédés de conservation de l'état de méthylation pendant le regroupement
US20240404624A1 (en) 2023-05-31 2024-12-05 Illumina, Inc. Structural variant alignment and variant calling by utilizing a structural-variant reference genome
WO2024249973A2 (fr) 2023-06-02 2024-12-05 Illumina, Inc. Liaison de gènes humains à des phénotypes cliniques à l'aide de réseaux neuronaux graphiques
WO2024254003A1 (fr) 2023-06-05 2024-12-12 Illumina, Inc. Identification et cartographie de sites de méthylation
US20240420800A1 (en) 2023-06-15 2024-12-19 Guardant Health, Inc. METHOD FOR HRD DETECTION IN TARGETED cfDNA SAMPLES USING DE NOVO MUTATIONAL SIGNATURES
WO2025006432A1 (fr) 2023-06-26 2025-01-02 Pacific Biosciences Of California, Inc. Compositions et procédés d'extension d'acides nucléiques
CN121359206A (zh) 2023-06-27 2026-01-16 因美纳有限公司 使用甲基化水平估计的变体检出
WO2025006487A1 (fr) 2023-06-30 2025-01-02 Illumina, Inc. Utilisation des coordonnées spatiales des cuves à circulation pour relier les lectures afin d'améliorer l'analyse du génome
WO2025006874A1 (fr) 2023-06-30 2025-01-02 Illumina, Inc. Modèle d'apprentissage automatique pour réétalonner des appels de génotype correspondant à des variants de lignée germinale et variants de mosaïque somatique
WO2025010160A1 (fr) 2023-07-06 2025-01-09 Pacific Biosciences Of California, Inc. Procédés et compositions pour stabiliser des concatémères
AU2024298639A1 (en) 2023-07-24 2026-01-29 Element Biosciences, Inc. On-support circularization and amplification for generating immobilized nucleic acid concatemer molecules
WO2025024672A1 (fr) 2023-07-26 2025-01-30 Element Biosciences, Inc. Systèmes de séquençage d'adn et leur utilisation
WO2025034461A1 (fr) 2023-08-04 2025-02-13 Pacific Biosciences Of California, Inc. Détection de 5-méthylcytosine
GB202312147D0 (en) 2023-08-08 2023-09-20 Syndex Bio Ltd Methylation method
US20250066841A1 (en) 2023-08-24 2025-02-27 Nautilus Subsidiary, Inc. Compositions and methods for detecting binding interactions under equilibrium or non-equilibrium conditions
WO2025054389A1 (fr) 2023-09-07 2025-03-13 Illumina, Inc. Identification de la cytosine méthylée par l'utilisation de points de repère
WO2025059533A1 (fr) 2023-09-13 2025-03-20 The Broad Institute, Inc. Enzymes crispr et systèmes
WO2025062341A1 (fr) 2023-09-20 2025-03-27 Element Biosciences, Inc. Cibles de test optique à l'état solide et configurables et dispositifs de cuve à circulation
WO2025072783A1 (fr) 2023-09-28 2025-04-03 Illumina, Inc. Cytidine désaminases modifiées et méthodes d'utilisation
WO2025072368A1 (fr) 2023-09-28 2025-04-03 Illumina, Inc. Capture et libération sélective de matériau biologique
WO2025072467A1 (fr) 2023-09-29 2025-04-03 Guardant Health, Inc. Génotypage cyp2d6
US20250111899A1 (en) 2023-09-29 2025-04-03 Illumina, Inc. Predicting insert lengths using primary analysis metrics
WO2025072870A1 (fr) 2023-09-29 2025-04-03 Illumina, Inc. Suivi et modification d'emplacement de cluster sur des lames d'échantillon de nucléotides en temps réel
WO2025081064A2 (fr) 2023-10-11 2025-04-17 Illumina, Inc. Désaminase thermophile et procédés d'identification de cytosine modifiée
WO2025078657A1 (fr) 2023-10-13 2025-04-17 F. Hoffmann-La Roche Ag Flux de travail d'enrichissement cible sans amplification pour détection directe de modifications d'acide nucléique
WO2025090883A1 (fr) 2023-10-27 2025-05-01 Illumina, Inc. Détection de variants dans des séquences nucléotidiques sur la base d'une diversité d'haplotype
WO2025090646A1 (fr) 2023-10-27 2025-05-01 Guardant Health, Inc. Surveillance de réponse moléculaire par déséquilibre allélique
WO2025106431A1 (fr) 2023-11-17 2025-05-22 Illumina, Inc. Détermination de variantes structurelles
WO2025106629A1 (fr) 2023-11-17 2025-05-22 Illumina, Inc. Détection de variante structurale à l'aide de lectures spatialement liées
WO2025117738A1 (fr) 2023-11-28 2025-06-05 Illumina, Inc. Procédés d'amélioration d'efficacité de ligature d'indice moléculaire unique
EP4567128A1 (fr) 2023-12-07 2025-06-11 Max-Delbrück-Centrum für Molekulare Medizin in der Helmholtz-Gemeinschaft Procede et moyens ameliores de detection spatiale d'acides nucleiques in situ
US20250189519A1 (en) 2023-12-11 2025-06-12 Nautilus Subsidiary, Inc. Methods and compositions of particle-based arrays
WO2025129074A2 (fr) 2023-12-14 2025-06-19 Illumina, Inc. Techniques d'indexation pour bibliothèques d'adn tagmentés
WO2025129133A1 (fr) 2023-12-15 2025-06-19 Illumina, Inc. Modèles de maladie résiduelle minime (mrd) pour déterminer des probabilités qu'un sujet comprenne un cancer
US20250201346A1 (en) 2023-12-18 2025-06-19 Illumina, Inc. Using machine learning models for detecting minimum residual disease (mrd) in a subject
WO2025137222A1 (fr) 2023-12-19 2025-06-26 Illumina, Inc. Dosage de détection de méthylation
WO2025137341A1 (fr) 2023-12-20 2025-06-26 Illumina, Inc. Détermination directe de mesures de rapport signal sur bruit pour une convergence accélérée lors de la détermination d'appels de base nucléotidique et d'une qualité d'appel de base
WO2025137268A1 (fr) 2023-12-20 2025-06-26 Pacific Biosciences Of California, Inc. Procédés et compositions pour pour réduire la dépendance à la cg
WO2025137166A1 (fr) 2023-12-20 2025-06-26 Nautilus Subsidiary, Inc. Dispositifs fluidiques pour dosages d'analyte unique
CN121359207A (zh) 2023-12-21 2026-01-16 因美纳有限公司 利用具有等位基因-变体差异的改进单倍型数据结构增强核苷酸读段的映射和比对
WO2025144716A1 (fr) 2023-12-28 2025-07-03 Illumina, Inc. Nucléotides ayant des groupes de blocage 3'-o-glucoside clivables par voie enzymatique pour séquençage
WO2025160089A1 (fr) 2024-01-26 2025-07-31 Illumina, Inc. Construction de référence multigénome personnalisée pour une analyse de séquençage améliorée d'échantillons génomiques
WO2025174774A1 (fr) 2024-02-12 2025-08-21 Illumina, Inc. Détermination de corrections hors ligne pour des erreurs spécifiques de séquence provoquées par des séquences nucléotidiques de faible complexité
WO2025174708A1 (fr) 2024-02-13 2025-08-21 Illumina, Inc. Conception et procédé de compatibilité inter-plateformes de séquençage de cuves à circulation
WO2025184226A1 (fr) 2024-02-28 2025-09-04 Illumina, Inc. Nucléotides à coiffage terminal de phosphate
CN121359209A (zh) 2024-02-28 2026-01-16 因美纳有限公司 用于改进核苷酸读段的映射和比对以及改进基因型检出的个性化单倍型数据库
WO2025188906A1 (fr) 2024-03-08 2025-09-12 Illumina, Inc. Nucléotides d'adénosine modifiés
WO2025189105A1 (fr) 2024-03-08 2025-09-12 Illumina, Inc. Seuillage par taille de fragments d'adn
WO2025193747A1 (fr) 2024-03-12 2025-09-18 Illumina, Inc. Modèles d'apprentissage automatique pour ordonner et accélérer les tâches de séquençage ou les lames d'échantillons de nucléotides correspondantes
WO2025191535A1 (fr) 2024-03-15 2025-09-18 Element Biosciences, Inc. Adaptateurs attelle partiellement double brin et procédés d'utilisation
WO2025196650A1 (fr) 2024-03-19 2025-09-25 Element Biosciences, Inc. Dispositifs de cuve à circulation et leur utilisation
WO2025196727A1 (fr) 2024-03-22 2025-09-25 Element Biosciences, Inc. Conjugués multivalents macromoléculaires et leurs utilisations
WO2025196731A1 (fr) 2024-03-22 2025-09-25 Element Biosciences, Inc. Conjugués multivalents polymères et utilisations associées
WO2025199236A1 (fr) 2024-03-22 2025-09-25 Nautilus Subsidiary, Inc. Systèmes de particules plasmoniques pour dosages à analyte unique
WO2025207886A1 (fr) 2024-03-28 2025-10-02 Illumina, Inc. Kits et procédés de préparation de bibliothèque de cuves à circulation et de détection de méthylation
WO2025212338A1 (fr) 2024-04-01 2025-10-09 Nautilus Subsidiary, Inc. Nanostructures pour la modulation de conformation d'analytes
WO2025230914A1 (fr) 2024-04-29 2025-11-06 Illumina, Inc. Nucléotides avec lieurs auto-immolables déclenchés par des enzymes pour le séquençage par synthèse
WO2025235016A1 (fr) 2024-05-07 2025-11-13 Element Biosciences, Inc. Procédé de séquençage à flexibilité spatiale
CN121359208A (zh) 2024-05-13 2026-01-16 因美纳有限公司 在测序运行期间修改测序循环以满足针对目标基因组区域的定制化覆盖度估计
WO2025240909A1 (fr) 2024-05-17 2025-11-20 Element Biosciences, Inc. Capture et enrichissement de séquences d'acides nucléiques cibles par hybridation
WO2025240924A1 (fr) 2024-05-17 2025-11-20 Illumina, Inc. Systèmes d'égalisation aveugle pour applications d'appel de base
WO2025250996A2 (fr) 2024-05-31 2025-12-04 Illumina, Inc. Modèles de génération et de réétalonnage d'appel pour mettre en œuvre des haplotypes de référence diploïdes personnalisés dans un appel de génotype
WO2025264831A1 (fr) 2024-06-18 2025-12-26 Illumina, Inc. Procédés pour augmenter la qualité de séquençage de régions riches en gc
WO2025264836A1 (fr) 2024-06-18 2025-12-26 Illumina, Inc. Procédés pour augmenter la qualité de séquençage de régions riches en gc
WO2026006774A1 (fr) 2024-06-28 2026-01-02 Illumina, Inc. Cytidine désaminases modifiées et procédés d'utilisation
WO2026006771A1 (fr) 2024-06-28 2026-01-02 Illumina, Inc. Scores de filtrage de grappe
WO2026006314A1 (fr) 2024-06-28 2026-01-02 Illumina, Inc. Marquage de régions cibles avant le séquençage de nucléotides
WO2026019720A1 (fr) 2024-07-15 2026-01-22 Pacific Biosciences Of California, Inc. Chargement amélioré de molécules et de complexes dans des sites de réaction

Citations (79)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4994373A (en) * 1983-01-27 1991-02-19 Enzo Biochem, Inc. Method and structures employing chemically-labelled polynucleotide probes
US5001050A (en) * 1989-03-24 1991-03-19 Consejo Superior Investigaciones Cientificas PHφ29 DNA polymerase
US5198543A (en) * 1989-03-24 1993-03-30 Consejo Superior Investigaciones Cientificas PHI29 DNA polymerase
US5200313A (en) * 1983-08-05 1993-04-06 Miles Inc. Nucleic acid hybridization assay employing detectable anti-hybrid antibodies
US5243618A (en) * 1991-11-22 1993-09-07 Hughes Aircraft Company Cavity resonator incorporating waveguide filter
US5302509A (en) * 1989-08-14 1994-04-12 Beckman Instruments, Inc. Method for sequencing polynucleotides
US5403708A (en) * 1992-07-06 1995-04-04 Brennan; Thomas M. Methods and compositions for determining the sequence of nucleic acids
US5405747A (en) * 1991-09-25 1995-04-11 The Regents Of The University Of California Office Of Technology Transfer Method for rapid base sequencing in DNA and RNA with two base labeling
US5455166A (en) * 1991-01-31 1995-10-03 Becton, Dickinson And Company Strand displacement amplification
US5465151A (en) * 1993-01-21 1995-11-07 State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of The University Of Oregon Sensors employing interference of electromagnetic waves passing through waveguides having functionalized surfaces
US5470710A (en) * 1993-10-22 1995-11-28 University Of Utah Automated hybridization/imaging device for fluorescent multiplex DNA sequencing
US5547835A (en) * 1993-01-07 1996-08-20 Sequenom, Inc. DNA sequencing by mass spectrometry
US5601982A (en) * 1995-02-07 1997-02-11 Sargent; Jeannine P. Method and apparatus for determining the sequence of polynucleotides
US5620854A (en) * 1993-08-25 1997-04-15 Regents Of The University Of California Method for identifying biochemical and chemical reactions and micromechanical processes using nanomechanical and electronic signal identification
US5631134A (en) * 1992-11-06 1997-05-20 The Trustees Of Boston University Methods of preparing probe array by hybridation
US5646264A (en) * 1990-03-14 1997-07-08 The Regents Of The University Of California DNA complexes with dyes designed for energy transfer as fluorescent markers
US5661028A (en) * 1995-09-29 1997-08-26 Lockheed Martin Energy Systems, Inc. Large scale DNA microsequencing device
US5677769A (en) * 1995-05-30 1997-10-14 Imra America Optical sensor utilizing rare-earth-doped integrated-optic lasers
US5677196A (en) * 1993-05-18 1997-10-14 University Of Utah Research Foundation Apparatus and methods for multi-analyte homogeneous fluoro-immunoassays
US5688648A (en) * 1994-02-01 1997-11-18 The Regents Of The University Of California Probes labelled with energy transfer coupled dyes
US5695934A (en) * 1994-10-13 1997-12-09 Lynx Therapeutics, Inc. Massively parallel sequencing of sorted polynucleotides
US5703222A (en) * 1992-04-03 1997-12-30 The Perkin-Elmer Corporation Probe composition containing a binding domain and polymer chain and methods of use
US5846727A (en) * 1996-06-06 1998-12-08 Board Of Supervisors Of Louisiana State University And Agricultural & Mechanical College Microsystem for rapid DNA sequencing
US5854033A (en) * 1995-11-21 1998-12-29 Yale University Rolling circle replication reporter systems
US5858671A (en) * 1996-11-01 1999-01-12 The University Of Iowa Research Foundation Iterative and regenerative DNA sequencing method
US5922591A (en) * 1995-06-29 1999-07-13 Affymetrix, Inc. Integrated nucleic acid diagnostic device
US5961923A (en) * 1995-04-25 1999-10-05 Irori Matrices with memories and uses thereof
US6004744A (en) * 1991-03-05 1999-12-21 Molecular Tool, Inc. Method for determining nucleotide identity through extension of immobilized primer
US6027890A (en) * 1996-01-23 2000-02-22 Rapigene, Inc. Methods and compositions for enhancing sensitivity in the analysis of biological-based assays
US6048690A (en) * 1991-11-07 2000-04-11 Nanogen, Inc. Methods for electronic fluorescent perturbation for analysis and electronic perturbation catalysis for synthesis
US6210896B1 (en) * 1998-08-13 2001-04-03 Us Genomics Molecular motors
US6221592B1 (en) * 1998-10-20 2001-04-24 Wisconsin Alumi Research Foundation Computer-based methods and systems for sequencing of individual nucleic acid molecules
US6232075B1 (en) * 1998-12-14 2001-05-15 Li-Cor, Inc. Heterogeneous assay for pyrophosphate detection
US6263286B1 (en) * 1998-08-13 2001-07-17 U.S. Genomics, Inc. Methods of analyzing polymers using a spatial network of fluorophores and fluorescence resonance energy transfer
US6280939B1 (en) * 1998-09-01 2001-08-28 Veeco Instruments, Inc. Method and apparatus for DNA sequencing using a local sensitive force detector
US6287824B1 (en) * 1998-09-15 2001-09-11 Yale University Molecular cloning using rolling circle amplification
US20010029049A1 (en) * 1997-10-06 2001-10-11 David R. Walt "self - encoding sensor with microspheres "
US6323009B1 (en) * 2000-06-28 2001-11-27 Molecular Staging, Inc. Multiply-primed amplification of nucleic acid sequences
US6325553B1 (en) * 1998-02-24 2001-12-04 Gemfire Corporation Connection system for optical redundancy
US20020014850A1 (en) * 1998-05-26 2002-02-07 Zhongyi Xia Focusing electrode for field emission displays and method
US20020025529A1 (en) * 1999-06-28 2002-02-28 Stephen Quake Methods and apparatus for analyzing polynucleotide sequences
US6355420B1 (en) * 1997-02-12 2002-03-12 Us Genomics Methods and products for analyzing polymers
US6399335B1 (en) * 1999-11-16 2002-06-04 Advanced Research And Technology Institute, Inc. γ-phosphoester nucleoside triphosphates
US6403311B1 (en) * 1997-02-12 2002-06-11 Us Genomics Methods of analyzing polymers using ordered label strategies
US20020110939A1 (en) * 2001-02-13 2002-08-15 Mitsubishi Denki Kabushiki Kaisha Semiconductor device and method of inspecting the same
US20020164629A1 (en) * 2001-03-12 2002-11-07 California Institute Of Technology Methods and apparatus for analyzing polynucleotide sequences by asynchronous base extension
US6485944B1 (en) * 1997-10-10 2002-11-26 President And Fellows Of Harvard College Replica amplification of nucleic acid arrays
US20020180570A1 (en) * 2000-10-26 2002-12-05 The Trustees Of Princeton University Method and apparatus for dielectric spectroscopy or biological solustions
US6510263B1 (en) * 2000-01-27 2003-01-21 Unaxis Balzers Aktiengesellschaft Waveguide plate and process for its production and microtitre plate
US6515751B1 (en) * 1999-03-11 2003-02-04 Cornell Research Foundation Inc. Mechanically resonant nanostructures
US6524829B1 (en) * 1998-09-30 2003-02-25 Molecular Machines & Industries Gmbh Method for DNA- or RNA-sequencing
US6528780B1 (en) * 1998-02-05 2003-03-04 Seiko Instruments Inc. Optical probe for proximity field
US20030044781A1 (en) * 1999-05-19 2003-03-06 Jonas Korlach Method for sequencing nucleic acid molecules
US20030064366A1 (en) * 2000-07-07 2003-04-03 Susan Hardin Real-time sequence determination
US20030092034A1 (en) * 2000-01-14 2003-05-15 Jonathan Cooper Analytical chip
US6573089B1 (en) * 1999-01-08 2003-06-03 Applera Corporation Method for using and making a fiber array
US20030123827A1 (en) * 2001-12-28 2003-07-03 Xtalight, Inc. Systems and methods of manufacturing integrated photonic circuit devices
US20030137313A1 (en) * 2001-01-20 2003-07-24 Bert Jannsen Resonant microwave sensor
US20030143556A1 (en) * 2001-04-03 2003-07-31 Gary Blackburn Nucleic acid reactions using labels with different redox potentials
US20030148542A1 (en) * 2000-06-02 2003-08-07 Michael Pawlak Kit and method for determining a plurality of analytes
US6618537B2 (en) * 2002-01-14 2003-09-09 Applied Wdm, Inc. Optical waveguide structures and methods of fabrication
US6617137B2 (en) * 2001-10-15 2003-09-09 Molecular Staging Inc. Method of amplifying whole genomes without subjecting the genome to denaturing conditions
US20030175780A1 (en) * 1996-11-01 2003-09-18 University Of Iowa Research Foundation Iterative and regenerative DNA sequencing method
US20030174992A1 (en) * 2001-09-27 2003-09-18 Levene Michael J. Zero-mode metal clad waveguides for performing spectroscopy with confined effective observation volumes
US20030186255A1 (en) * 2001-06-06 2003-10-02 Li-Cor, Inc. Single molecule detection systems and methods
US6642034B2 (en) * 1997-10-08 2003-11-04 Yale University Multiple displacement amplification
US6670126B2 (en) * 2000-05-12 2003-12-30 Molecular Staging, Inc. Poly-primed amplification of nucleic acid sequences
US6713672B1 (en) * 2001-12-07 2004-03-30 Laird Technologies, Inc. Compliant shaped EMI shield
US6740865B1 (en) * 1998-07-21 2004-05-25 Packard Instrument Company, Inc. Imaging system for luminescence assays
US20040110180A1 (en) * 2001-04-06 2004-06-10 Shirley Recipon Kinases and phosphatases
US6753200B2 (en) * 1999-01-13 2004-06-22 Cornell Research Foundation Monolithic nanofluid sieving structures for DNA manipulation
US20040132155A1 (en) * 2000-05-30 2004-07-08 Sugen Incorporated Mammalian protein phosphatases
US20040157306A1 (en) * 2000-11-13 2004-08-12 Sugen Incorporated Mammalian protein phosphatases
US20040203097A1 (en) * 2001-05-24 2004-10-14 Henry Yue Kinases and phosphatases
US6961125B2 (en) * 2001-01-03 2005-11-01 Packard Instrument Company, Inc. Luminescence imager
US20060017917A1 (en) * 2004-07-22 2006-01-26 Cullum Brian M Surface enhanced Raman spectroscopic nano-imaging probe and uses therefor
US20060063264A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Apparatus and method for performing nucleic acid analysis
US7170050B2 (en) * 2004-09-17 2007-01-30 Pacific Biosciences Of California, Inc. Apparatus and methods for optical analysis of molecules
US20070206187A1 (en) * 2006-02-13 2007-09-06 Pacific Biosciences Of California, Inc. Methods and systems for simultaneous real-time monitoring of optical signals from multiple sources

Family Cites Families (52)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1338457C (fr) 1986-08-22 1996-07-16 Henry A. Erlich Enzyme thermostable purifiee
GB8910880D0 (en) 1989-05-11 1989-06-28 Amersham Int Plc Sequencing method
CA2044616A1 (fr) * 1989-10-26 1991-04-27 Roger Y. Tsien Sequencage de l'adn
IL97222A (en) 1990-02-16 1995-08-31 Orion Yhtymae Oy Method and reagent for determining specific nucleotide variations
WO1992010588A1 (fr) 1990-12-06 1992-06-25 Affymax Technologies N.V. Mise en sequence par hybridation d'un acide nucleique cible en une matrice d'oligonucleotides determines
US5255083A (en) * 1991-06-05 1993-10-19 Sony Corporation Of America Digital color correction system and method
US5221592A (en) * 1992-03-06 1993-06-22 Hoechst Celanese Corporation Diazo ester of a benzolactone ring compound and positive photoresist composition and element utilizing the diazo ester
GB9208733D0 (en) 1992-04-22 1992-06-10 Medical Res Council Dna sequencing method
US5874239A (en) 1993-07-30 1999-02-23 Affymax Technologies N.V. Biotinylation of proteins
WO1996027025A1 (fr) 1995-02-27 1996-09-06 Ely Michael Rabani Dispositif, composes, algorithmes, et procedes de caracterisation et manipulation avec parallelisme moleculaire
EP0745686A1 (fr) 1995-06-01 1996-12-04 Roche Diagnostics GmbH L'utilisation de l'activité de supression 3'-intrinsèque de l'ADN polymérase
WO1998033939A1 (fr) * 1997-01-31 1998-08-06 Hitachi, Ltd. Procede pour determiner une sequence de base d'acide nucleique et appareil correspondant
US5773308A (en) * 1997-02-10 1998-06-30 The United States Of America As Represented By The Secretary Of The Navy Photoactivatable o-nitrobenzyl polyethylene glycol-silane for the production of patterned biomolecular arrays
DE69824716D1 (de) 1997-04-01 2004-07-29 Manteia S A Methode zur sequenzierung von nukleinsäuren
US5866366A (en) 1997-07-01 1999-02-02 Smithkline Beecham Corporation gidB
CN1152140C (zh) 1997-07-28 2004-06-02 医疗生物系统有限公司 核酸序列分析
WO1999019341A1 (fr) 1997-10-10 1999-04-22 President & Fellows Of Harvard College Amplification par replique de reseaux d'acides nucleiques
DE69928265T3 (de) 1998-07-30 2013-11-28 Illumina Cambridge Ltd. Matrizen von biomolekülen und ihre verwendung in sequenzierung
WO2002061126A2 (fr) 2001-01-30 2002-08-08 Solexa Ltd. Preparation d'un reseau de polypeptides
GB0002310D0 (en) 2000-02-01 2000-03-22 Solexa Ltd Polynucleotide sequencing
JP2002522780A (ja) 1998-08-13 2002-07-23 ユー．エス．ゲノミクス ポリマーの光学的な特性決定
GB9828785D0 (en) 1998-12-30 1999-02-17 Amersham Pharm Biotech Ab Sequencing systems
CA2375769A1 (fr) * 1999-01-11 2000-07-20 President And Fellows Of Harvard College Amplification isotherme d'adn
US7270951B1 (en) 1999-03-10 2007-09-18 Asm Scientific, Inc. Method for direct nucleic acid sequencing
AU3567900A (en) 1999-03-30 2000-10-16 Solexa Ltd. Polynucleotide sequencing
GB9907813D0 (en) 1999-04-06 1999-06-02 Medical Biosystems Ltd Synthesis
GB9907812D0 (en) 1999-04-06 1999-06-02 Medical Biosystems Ltd Sequencing
WO2001013088A1 (fr) 1999-08-13 2001-02-22 U.S. Genomics, Inc. Procede et appareil d'etirage de polymeres
AU7086800A (en) 1999-08-30 2001-03-26 Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services, The High speed parallel molecular nucleic acid sequencing
US7244559B2 (en) * 1999-09-16 2007-07-17 454 Life Sciences Corporation Method of sequencing a nucleic acid
US6274320B1 (en) * 1999-09-16 2001-08-14 Curagen Corporation Method of sequencing a nucleic acid
EP1218543A2 (fr) 1999-09-29 2002-07-03 Solexa Ltd. Sequen age de polynucleotides
GB9923644D0 (en) 1999-10-06 1999-12-08 Medical Biosystems Ltd DNA sequencing
AU1471001A (en) 1999-11-04 2001-05-14 California Institute Of Technology Methods and apparatus for analyzing polynucleotide sequences
GB0002389D0 (en) 2000-02-02 2000-03-22 Solexa Ltd Molecular arrays
DE10017824B4 (de) * 2000-04-10 2004-03-18 Till I.D. Gmbh Vorrichtung zur parallelen photometrischen Fluoreszenz- oder Lumineszenzanalyse mehrerer voneinander getrennter Probenbereiche auf einem Objekt
CA2412567A1 (fr) 2000-06-07 2001-12-13 Li-Cor, Inc. Nucleotides a commutation de charges
GB0016472D0 (en) 2000-07-05 2000-08-23 Amersham Pharm Biotech Uk Ltd Sequencing method and apparatus
GB0016473D0 (en) 2000-07-05 2000-08-23 Amersham Pharm Biotech Uk Ltd Sequencing method
WO2002029106A2 (fr) 2000-10-03 2002-04-11 California Institute Of Technology Dispositifs microfluidiques et procedes d'utilisation
GB0112238D0 (en) 2001-05-18 2001-07-11 Medical Biosystems Ltd Sequencing method
US20020187508A1 (en) 2001-06-08 2002-12-12 Wong Gordon G. Methods and products for analyzing nucleic acids using nick translation
AU2002337653A1 (en) 2001-07-25 2003-02-17 The Trustees Of Princeton University Nanochannel arrays and their preparation and use for high throughput macromolecular analysis
GB0119719D0 (en) 2001-08-13 2001-10-03 Solexa Ltd DNA sequence analysis
WO2003020734A2 (fr) 2001-08-29 2003-03-13 Amersham Biosciences Corp Polyphosphates de nucleoside marques
JP2003185569A (ja) * 2001-12-14 2003-07-03 Mitsubishi Chemicals Corp 表面プラズモン共鳴を利用した試料の分析装置及び表面プラズモン共鳴分析用センサチップ
JP3897703B2 (ja) * 2002-01-11 2007-03-28 キヤノン株式会社 センサ装置およびそれを用いた検査方法
DE10217568A1 (de) 2002-04-19 2003-11-13 Infineon Technologies Ag Wellenleiter in porösen Substraten
JP2003344433A (ja) * 2002-05-22 2003-12-03 Okutekku:Kk マイクロアレイ、マイクロアレイシステム及び被検物質の測定方法
US6977153B2 (en) * 2002-12-31 2005-12-20 Qiagen Gmbh Rolling circle amplification of RNA
US20050186317A1 (en) * 2003-06-23 2005-08-25 Richard Dempster Determination of dough development using near infrared radiation
CA2557177A1 (fr) * 2004-02-19 2005-09-01 Stephen Quake Procedes et kits pour analyser des sequences de polynucleotides

Patent Citations (91)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4994373A (en) * 1983-01-27 1991-02-19 Enzo Biochem, Inc. Method and structures employing chemically-labelled polynucleotide probes
US5200313A (en) * 1983-08-05 1993-04-06 Miles Inc. Nucleic acid hybridization assay employing detectable anti-hybrid antibodies
US5001050A (en) * 1989-03-24 1991-03-19 Consejo Superior Investigaciones Cientificas PHφ29 DNA polymerase
US5198543A (en) * 1989-03-24 1993-03-30 Consejo Superior Investigaciones Cientificas PHI29 DNA polymerase
US5302509A (en) * 1989-08-14 1994-04-12 Beckman Instruments, Inc. Method for sequencing polynucleotides
US5646264A (en) * 1990-03-14 1997-07-08 The Regents Of The University Of California DNA complexes with dyes designed for energy transfer as fluorescent markers
US5455166A (en) * 1991-01-31 1995-10-03 Becton, Dickinson And Company Strand displacement amplification
US6004744A (en) * 1991-03-05 1999-12-21 Molecular Tool, Inc. Method for determining nucleotide identity through extension of immobilized primer
US5405747A (en) * 1991-09-25 1995-04-11 The Regents Of The University Of California Office Of Technology Transfer Method for rapid base sequencing in DNA and RNA with two base labeling
US6048690A (en) * 1991-11-07 2000-04-11 Nanogen, Inc. Methods for electronic fluorescent perturbation for analysis and electronic perturbation catalysis for synthesis
US5243618A (en) * 1991-11-22 1993-09-07 Hughes Aircraft Company Cavity resonator incorporating waveguide filter
US5703222A (en) * 1992-04-03 1997-12-30 The Perkin-Elmer Corporation Probe composition containing a binding domain and polymer chain and methods of use
US5403708A (en) * 1992-07-06 1995-04-04 Brennan; Thomas M. Methods and compositions for determining the sequence of nucleic acids
US5631134A (en) * 1992-11-06 1997-05-20 The Trustees Of Boston University Methods of preparing probe array by hybridation
US5547835A (en) * 1993-01-07 1996-08-20 Sequenom, Inc. DNA sequencing by mass spectrometry
US5465151A (en) * 1993-01-21 1995-11-07 State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of The University Of Oregon Sensors employing interference of electromagnetic waves passing through waveguides having functionalized surfaces
US5677196A (en) * 1993-05-18 1997-10-14 University Of Utah Research Foundation Apparatus and methods for multi-analyte homogeneous fluoro-immunoassays
US5620854A (en) * 1993-08-25 1997-04-15 Regents Of The University Of California Method for identifying biochemical and chemical reactions and micromechanical processes using nanomechanical and electronic signal identification
US5470710A (en) * 1993-10-22 1995-11-28 University Of Utah Automated hybridization/imaging device for fluorescent multiplex DNA sequencing
US5688648A (en) * 1994-02-01 1997-11-18 The Regents Of The University Of California Probes labelled with energy transfer coupled dyes
US5695934A (en) * 1994-10-13 1997-12-09 Lynx Therapeutics, Inc. Massively parallel sequencing of sorted polynucleotides
US5601982A (en) * 1995-02-07 1997-02-11 Sargent; Jeannine P. Method and apparatus for determining the sequence of polynucleotides
US5961923A (en) * 1995-04-25 1999-10-05 Irori Matrices with memories and uses thereof
US5677769A (en) * 1995-05-30 1997-10-14 Imra America Optical sensor utilizing rare-earth-doped integrated-optic lasers
US5922591A (en) * 1995-06-29 1999-07-13 Affymetrix, Inc. Integrated nucleic acid diagnostic device
US5661028A (en) * 1995-09-29 1997-08-26 Lockheed Martin Energy Systems, Inc. Large scale DNA microsequencing device
US5854033A (en) * 1995-11-21 1998-12-29 Yale University Rolling circle replication reporter systems
US6027890A (en) * 1996-01-23 2000-02-22 Rapigene, Inc. Methods and compositions for enhancing sensitivity in the analysis of biological-based assays
US5846727A (en) * 1996-06-06 1998-12-08 Board Of Supervisors Of Louisiana State University And Agricultural & Mechanical College Microsystem for rapid DNA sequencing
US5858671A (en) * 1996-11-01 1999-01-12 The University Of Iowa Research Foundation Iterative and regenerative DNA sequencing method
US20030175780A1 (en) * 1996-11-01 2003-09-18 University Of Iowa Research Foundation Iterative and regenerative DNA sequencing method
US6403311B1 (en) * 1997-02-12 2002-06-11 Us Genomics Methods of analyzing polymers using ordered label strategies
US6355420B1 (en) * 1997-02-12 2002-03-12 Us Genomics Methods and products for analyzing polymers
US20050196317A1 (en) * 1997-10-06 2005-09-08 Trustees Of Tufts College Self-encoding sensor with microspheres
US20010029049A1 (en) * 1997-10-06 2001-10-11 David R. Walt "self - encoding sensor with microspheres "
US6642034B2 (en) * 1997-10-08 2003-11-04 Yale University Multiple displacement amplification
US6485944B1 (en) * 1997-10-10 2002-11-26 President And Fellows Of Harvard College Replica amplification of nucleic acid arrays
US6528780B1 (en) * 1998-02-05 2003-03-04 Seiko Instruments Inc. Optical probe for proximity field
US6325553B1 (en) * 1998-02-24 2001-12-04 Gemfire Corporation Connection system for optical redundancy
US20020014850A1 (en) * 1998-05-26 2002-02-07 Zhongyi Xia Focusing electrode for field emission displays and method
US6740865B1 (en) * 1998-07-21 2004-05-25 Packard Instrument Company, Inc. Imaging system for luminescence assays
US6210896B1 (en) * 1998-08-13 2001-04-03 Us Genomics Molecular motors
US6263286B1 (en) * 1998-08-13 2001-07-17 U.S. Genomics, Inc. Methods of analyzing polymers using a spatial network of fluorophores and fluorescence resonance energy transfer
US6280939B1 (en) * 1998-09-01 2001-08-28 Veeco Instruments, Inc. Method and apparatus for DNA sequencing using a local sensitive force detector
US6287824B1 (en) * 1998-09-15 2001-09-11 Yale University Molecular cloning using rolling circle amplification
US6524829B1 (en) * 1998-09-30 2003-02-25 Molecular Machines & Industries Gmbh Method for DNA- or RNA-sequencing
US6221592B1 (en) * 1998-10-20 2001-04-24 Wisconsin Alumi Research Foundation Computer-based methods and systems for sequencing of individual nucleic acid molecules
US6232075B1 (en) * 1998-12-14 2001-05-15 Li-Cor, Inc. Heterogeneous assay for pyrophosphate detection
US6306607B2 (en) * 1998-12-14 2001-10-23 Li-Cor, Inc. Heterogeneous assay for pyrophosphate
US6255083B1 (en) * 1998-12-14 2001-07-03 Li Cor Inc System and methods for nucleic acid sequencing of single molecules by polymerase synthesis
US20030194740A1 (en) * 1998-12-14 2003-10-16 Li-Cor, Inc. System and method for nucleic acid sequencing by polymerase synthesis
US6573089B1 (en) * 1999-01-08 2003-06-03 Applera Corporation Method for using and making a fiber array
US6753200B2 (en) * 1999-01-13 2004-06-22 Cornell Research Foundation Monolithic nanofluid sieving structures for DNA manipulation
US6515751B1 (en) * 1999-03-11 2003-02-04 Cornell Research Foundation Inc. Mechanically resonant nanostructures
US20030044781A1 (en) * 1999-05-19 2003-03-06 Jonas Korlach Method for sequencing nucleic acid molecules
US20030092005A1 (en) * 1999-05-19 2003-05-15 Levene Michael J. Optical field enhancement
US20020025529A1 (en) * 1999-06-28 2002-02-28 Stephen Quake Methods and apparatus for analyzing polynucleotide sequences
US6399335B1 (en) * 1999-11-16 2002-06-04 Advanced Research And Technology Institute, Inc. γ-phosphoester nucleoside triphosphates
US20030092034A1 (en) * 2000-01-14 2003-05-15 Jonathan Cooper Analytical chip
US6510263B1 (en) * 2000-01-27 2003-01-21 Unaxis Balzers Aktiengesellschaft Waveguide plate and process for its production and microtitre plate
US6670126B2 (en) * 2000-05-12 2003-12-30 Molecular Staging, Inc. Poly-primed amplification of nucleic acid sequences
US20040132155A1 (en) * 2000-05-30 2004-07-08 Sugen Incorporated Mammalian protein phosphatases
US20030148542A1 (en) * 2000-06-02 2003-08-07 Michael Pawlak Kit and method for determining a plurality of analytes
US6323009B1 (en) * 2000-06-28 2001-11-27 Molecular Staging, Inc. Multiply-primed amplification of nucleic acid sequences
US20030064366A1 (en) * 2000-07-07 2003-04-03 Susan Hardin Real-time sequence determination
US20020180570A1 (en) * 2000-10-26 2002-12-05 The Trustees Of Princeton University Method and apparatus for dielectric spectroscopy or biological solustions
US20040157306A1 (en) * 2000-11-13 2004-08-12 Sugen Incorporated Mammalian protein phosphatases
US6961125B2 (en) * 2001-01-03 2005-11-01 Packard Instrument Company, Inc. Luminescence imager
US20030137313A1 (en) * 2001-01-20 2003-07-24 Bert Jannsen Resonant microwave sensor
US20020110939A1 (en) * 2001-02-13 2002-08-15 Mitsubishi Denki Kabushiki Kaisha Semiconductor device and method of inspecting the same
US20020164629A1 (en) * 2001-03-12 2002-11-07 California Institute Of Technology Methods and apparatus for analyzing polynucleotide sequences by asynchronous base extension
US20030143556A1 (en) * 2001-04-03 2003-07-31 Gary Blackburn Nucleic acid reactions using labels with different redox potentials
US20040110180A1 (en) * 2001-04-06 2004-06-10 Shirley Recipon Kinases and phosphatases
US20040203097A1 (en) * 2001-05-24 2004-10-14 Henry Yue Kinases and phosphatases
US20030186255A1 (en) * 2001-06-06 2003-10-02 Li-Cor, Inc. Single molecule detection systems and methods
US20030174992A1 (en) * 2001-09-27 2003-09-18 Levene Michael J. Zero-mode metal clad waveguides for performing spectroscopy with confined effective observation volumes
US6917726B2 (en) * 2001-09-27 2005-07-12 Cornell Research Foundation, Inc. Zero-mode clad waveguides for performing spectroscopy with confined effective observation volumes
US6617137B2 (en) * 2001-10-15 2003-09-09 Molecular Staging Inc. Method of amplifying whole genomes without subjecting the genome to denaturing conditions
US6713672B1 (en) * 2001-12-07 2004-03-30 Laird Technologies, Inc. Compliant shaped EMI shield
US20030123827A1 (en) * 2001-12-28 2003-07-03 Xtalight, Inc. Systems and methods of manufacturing integrated photonic circuit devices
US6618537B2 (en) * 2002-01-14 2003-09-09 Applied Wdm, Inc. Optical waveguide structures and methods of fabrication
US20060017917A1 (en) * 2004-07-22 2006-01-26 Cullum Brian M Surface enhanced Raman spectroscopic nano-imaging probe and uses therefor
US20060063264A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Apparatus and method for performing nucleic acid analysis
US20060061754A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Arrays of optical confinements and uses thereof
US20060061755A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Apparatus and method for analysis of molecules
US7170050B2 (en) * 2004-09-17 2007-01-30 Pacific Biosciences Of California, Inc. Apparatus and methods for optical analysis of molecules
US20070206189A1 (en) * 2004-09-17 2007-09-06 Stephen Turner Optical analysis of molecules
US7302146B2 (en) * 2004-09-17 2007-11-27 Pacific Biosciences Of California, Inc. Apparatus and method for analysis of molecules
US7313308B2 (en) * 2004-09-17 2007-12-25 Pacific Biosciences Of California, Inc. Optical analysis of molecules
US7315019B2 (en) * 2004-09-17 2008-01-01 Pacific Biosciences Of California, Inc. Arrays of optical confinements and uses thereof
US20070206187A1 (en) * 2006-02-13 2007-09-06 Pacific Biosciences Of California, Inc. Methods and systems for simultaneous real-time monitoring of optical signals from multiple sources

Cited By (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070184475A1 (en) * 2000-07-07 2007-08-09 Susan Hardin Sequence determination by direct detection
US20030064366A1 (en) * 2000-07-07 2003-04-03 Susan Hardin Real-time sequence determination
US20100255463A1 (en) * 2000-07-07 2010-10-07 Susan Harsin Compositions and methods for sequence determination
US20070292867A1 (en) * 2000-07-07 2007-12-20 Susan Hardin Sequence determination using multiply tagged polymerizing agents
US20070172858A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Methods for sequence determination
US20070172868A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Compositions for sequence determination using tagged polymerizing agents and tagged monomers
US20070172861A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Mutant polymerases
US20070172866A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Methods for sequence determination using depolymerizing agent
US20070275395A1 (en) * 2000-07-07 2007-11-29 Susan Hardin Tagged monomers for use in sequence determination
US20070172863A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Compositions and methods for sequence determination
US20110184163A1 (en) * 2000-12-01 2011-07-28 Life Technologies Corporation Enzymatic Nucleic Acid Synthesis: Compositions and Methods for Inhibiting Pyrophosphorolysis
US8648179B2 (en) 2000-12-01 2014-02-11 Life Technologies Corporation Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis
US8314216B2 (en) 2000-12-01 2012-11-20 Life Technologies Corporation Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis
US20070172819A1 (en) * 2000-12-01 2007-07-26 Hardin Susan H Enzymatic nucleic acid synthesis: compositions including pyrophosphorolysis inhibitors
US9243284B2 (en) 2000-12-01 2016-01-26 Life Technologies Corporation Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis
US20100216122A1 (en) * 2000-12-01 2010-08-26 Life Technologies Corporation Enzymatic nucleic acid synthesis: methods for direct detection of tagged monomers
US20100235105A1 (en) * 2001-07-09 2010-09-16 Life Technologies Corporation Method for analyzing dynamic detectable events at the single molecule level
US10329555B2 (en) 2002-08-12 2019-06-25 Adimab, Llc High throughput generation and affinity maturation of humanized antibody
US9464286B2 (en) 2002-08-12 2016-10-11 Adimab, Llc High throughput generation and affinity maturation of humanized antibody
US20090186343A1 (en) * 2003-01-28 2009-07-23 Visigen Biotechnologies, Inc. Methods for preparing modified biomolecules, modified biomolecules and methods for using same
US7476503B2 (en) 2004-09-17 2009-01-13 Pacific Biosciences Of California, Inc. Apparatus and method for performing nucleic acid analysis
US8709725B2 (en) 2004-09-17 2014-04-29 Pacific Biosciences Of California, Inc. Arrays of optical confinements and uses thereof
US20060063264A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Apparatus and method for performing nucleic acid analysis
US20080156974A1 (en) * 2004-09-17 2008-07-03 Stephen Turner Arrays of optical confinements and uses thereof
US9709503B2 (en) 2004-09-17 2017-07-18 Pacific Biosciences Of California, Inc. Apparatus and method for performing nucleic acid analysis
US9588051B2 (en) 2004-09-17 2017-03-07 Pacific Biosciences Of California, Inc. Apparatus and method for performing nucleic acid analysis
US7315019B2 (en) 2004-09-17 2008-01-01 Pacific Biosciences Of California, Inc. Arrays of optical confinements and uses thereof
US7313308B2 (en) 2004-09-17 2007-12-25 Pacific Biosciences Of California, Inc. Optical analysis of molecules
US7906284B2 (en) 2004-09-17 2011-03-15 Pacific Biosciences Of California, Inc. Arrays of optical confinements and uses thereof
US7302146B2 (en) * 2004-09-17 2007-11-27 Pacific Biosciences Of California, Inc. Apparatus and method for analysis of molecules
US20060061755A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Apparatus and method for analysis of molecules
US20060061754A1 (en) * 2004-09-17 2006-03-23 Stephen Turner Arrays of optical confinements and uses thereof
US20070206189A1 (en) * 2004-09-17 2007-09-06 Stephen Turner Optical analysis of molecules
US20070250274A1 (en) * 2006-02-06 2007-10-25 Visigen Biotechnologies, Inc. Method for analyzing dynamic detectable events at the single molecule level
US7668697B2 (en) 2006-02-06 2010-02-23 Andrei Volkov Method for analyzing dynamic detectable events at the single molecule level
US20080241951A1 (en) * 2006-07-20 2008-10-02 Visigen Biotechnologies, Inc. Method and apparatus for moving stage detection of single molecular events
US20080091005A1 (en) * 2006-07-20 2008-04-17 Visigen Biotechnologies, Inc. Modified nucleotides, methods for making and using same
US20080241938A1 (en) * 2006-07-20 2008-10-02 Visigen Biotechnologies, Inc. Automated synthesis or sequencing apparatus and method for making and using same
US10189894B2 (en) 2007-09-14 2019-01-29 Adimab, Llc Rationally designed, synthetic antibody libraries and uses therefor
US10196635B2 (en) 2007-09-14 2019-02-05 Adimab, Llc Rationally designed, synthetic antibody libraries and uses therefor
US11008568B2 (en) 2007-09-14 2021-05-18 Adimab, Llc Rationally designed, synthetic antibody libraries and uses therefor
US11008383B2 (en) 2007-09-14 2021-05-18 Adimab, Llc Rationally designed, synthetic antibody libraries and uses therefor
US12529164B2 (en) 2007-09-14 2026-01-20 Adimab, Llc Rationally designed, synthetic antibody libraries and uses therefor
US11705217B2 (en) 2008-03-28 2023-07-18 Pacific Biosciences Of California, Inc. Sequencing using concatemers of copies of sense and antisense strands
US20160070034A1 (en) * 2008-06-02 2016-03-10 University Of Utah Research Foundation Localization of Near-Field Resonances in Bowtie Antennae: Influence of Adhesion Layers
US20120183970A1 (en) * 2009-08-06 2012-07-19 Ibis Biosciences, Inc. Non-mass determined base compositions for nucleic acid detection
US9080209B2 (en) * 2009-08-06 2015-07-14 Ibis Biosciences, Inc. Non-mass determined base compositions for nucleic acid detection

Also Published As

Publication number Publication date
EP3415641B1 (fr) 2023-11-01
CN101914620A (zh) 2010-12-15
CA2579150C (fr) 2014-11-25
EP3415641A1 (fr) 2018-12-19
JP6294064B2 (ja) 2018-03-14
AU2005296200A1 (en) 2006-04-27
CA2579150A1 (fr) 2006-04-27
US7315019B2 (en) 2008-01-01
GB2423819A (en) 2006-09-06
US7476503B2 (en) 2009-01-13
GB0608338D0 (en) 2006-06-07
US20060061755A1 (en) 2006-03-23
WO2006044078A3 (fr) 2008-02-21
GB2423819B (en) 2008-02-06
JP2008513782A (ja) 2008-05-01
US20060061754A1 (en) 2006-03-23
EP1790202A2 (fr) 2007-05-30
JP2016154560A (ja) 2016-09-01
US20060063264A1 (en) 2006-03-23
CN101914620B (zh) 2014-02-12
AU2005296200B2 (en) 2011-07-14
EP1790202A4 (fr) 2013-02-20
JP2014057601A (ja) 2014-04-03
US7302146B2 (en) 2007-11-27
WO2006044078A2 (fr) 2006-04-27

Similar Documents

Publication Publication Date Title
US9709503B2 (en) Apparatus and method for performing nucleic acid analysis
US7302146B2 (en) Apparatus and method for analysis of molecules
US7476504B2 (en) Use of reversible extension terminator in nucleic acid sequencing
US8993230B2 (en) Asynchronous sequencing of biological polymers
US20110217698A1 (en) Method to improve single molecule analyses
AU2011224119B2 (en) Apparatus and method for analysis of molecules

Legal Events

Date Code Title Description
AS Assignment

Owner name: PACIFIC BIOSCIENCES OF CALIFORNIA, INC., CALIFORNI

Free format text: CHANGE OF NAME;ASSIGNORS:TURNER, STEPHEN;KORLACH, JONAS;REEL/FRAME:017213/0840

Effective date: 20050811

AS Assignment

Owner name: PACIFIC BIOSCIENCES OF CALIFORNIA, INC., CALIFORNI

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:TURNER, STEPHEN;KORLACH, JONAS;REEL/FRAME:017282/0769

Effective date: 20051017

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION