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  • 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
  • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
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  • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
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  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
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  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
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  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/1429—Signal processing
  • G01N15/1433—Signal processing using image recognition
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  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/1484—Optical investigation techniques, e.g. flow cytometry microstructural devices
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  • 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
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
  • G01N21/251—Colorimeters; Construction thereof
  • G01N21/253—Colorimeters; Construction thereof for batch operation, i.e. multisample apparatus
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  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00277—Apparatus
  • B01J2219/00279—Features relating to reactor vessels
  • B01J2219/00306—Reactor vessels in a multiple arrangement
  • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
  • B01J2219/00315—Microtiter plates
  • B01J2219/00317—Microwell devices, i.e. having large numbers of wells
• • B—PERFORMING OPERATIONS; TRANSPORTING
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  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00583—Features relative to the processes being carried out
  • B01J2219/00603—Making arrays on substantially continuous surfaces
  • B01J2219/00646—Making arrays on substantially continuous surfaces the compounds being bound to beads immobilised on the solid supports
  • B01J2219/00648—Making arrays on substantially continuous surfaces the compounds being bound to beads immobilised on the solid supports by the use of solid beads
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00583—Features relative to the processes being carried out
  • B01J2219/00603—Making arrays on substantially continuous surfaces
  • B01J2219/00659—Two-dimensional arrays
• • 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
• • 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
  • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• • 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
  • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
  • B01L2300/0877—Flow chambers
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/06—Valves, specific forms thereof
  • B01L2400/0622—Valves, specific forms thereof distribution valves, valves having multiple inlets and/or outlets, e.g. metering valves, multi-way valves
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/06—Valves, specific forms thereof
  • B01L2400/0633—Valves, specific forms thereof with moving parts
  • B01L2400/0644—Valves, specific forms thereof with moving parts rotary valves
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
  • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/149—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N2015/0007—Investigating dispersion of gas
  • G01N2015/0015—Investigating dispersion of gas in solids

Definitions

• the invention relates generally to systems and apparatus for carrying out large scale parallel reactions on solid phase supports, and more particularly, to systems and apparatus for monitoring and carrying out reactions on arrays of microparticles.
• microparticles for synthesizing analytes or for capturing analytes for subsequent analysis, e.g. Lam et al (cited above); Benkovic et al, International patent application PCT US95/03355; Gavin et al, International patent application PCT/EP97/02039; Brenner et al, International patent application PCT/US96/09513, and the like.
• Lam et al cited above
• Benkovic et al International patent application PCT US95/03355
• Gavin et al International patent application PCT/EP97/02039
• Brenner et al International patent application PCT/US96/09513, and the like.
• microparticles generally facilitate the construction and manipulation of large repertoires of analytes with minimal reagent and/or sample consumption.
• handling and manipulating large numbers of microparticles e.g.
• objects of our invention include, but are not limited to, providing a system and apparatus for sequentially delivering reagents to a population of analytes anchored to separate microparticles; providing an apparatus for simultaneously monitoring the interactions of processing reagents and analytes on the surfaces of microparticles disposed in a planar array; providing an apparatus for detecting optical signals generated by, or as the result of, interactions of processing reagents and analytes on the surfaces of microparticles disposed in a planar array; providing an apparatus for detecting pluralities of optical signals, each such plurality being generated at the surface of the same microparticle as a result of interactions between processing reagents and an analyte anchored to the surface of such microparticle; providing an apparatus for simultaneously tracking the positions of individual microparticles in a population of microparticles disposed in a flow chamber as a closely packed planar array; and providing a system and apparatus for simultaneously analyzing the nucleotide sequences of a population of polynucleo
• our invention achieves these and other objects with an apparatus comprising a flow chamber for disposing a population of microparticles in a planar array; fluidic means for sequentially delivering processing reagents from one or more reagent reservoirs to the flow chamber; and detection means for detecting a sequence of optical signals from each of the microparticles of the population.
• the sequences of optical signals are generated as a result of a multi-step analytical process, such as nucleic acid sequence analysis.
• the invention provides a system for simultaneously monitoring a population of analytes which includes the apparatus of the invention, microparticles carrying the analytes, and software means for processing images of, and/or optical signals generated by, the microparticles when disposed in a planar array.
• the flow chamber includes constraining means for restricting the movement of microparticles during cycles of reagent delivery.
• the invention includes a system for simultaneously analyzing the nucleotide sequences of a population of polynucleotides. Copies of each kind of polynucleotide in the population are sorted onto and anchored to one or more microparticles so that a population of loaded microparticles is formed. Loaded microparticles are disposed in a planar array in a flow chamber through which processing reagents are sequentially delivered to the loaded microparticles from one or more reagent reservoirs by a fluidic means.
• Optical signals generated by, or produced as a result of, the interaction of processing reagents and polynucleotides on the microparticles are imaged by a detection means.
• analysis includes determining the nucleotide sequence of a portion of each polynucleotide on the different microparticles, massively parallel signature sequencing (MPSS) analysis is employed, e.g. as described in Albrecht et al, International patent application PCT/US97/09472.
• MPSS massively parallel signature sequencing
• Figure 1 a is a schematic representation of a flow chamber and fluidics and detection systems for observing a planar array of microparticles loaded with analyte molecules, such as cDNA molecules for sequencing.
• Figure 1 b is a schematic of a preferred holder for a flow chamber.
• Figure 2a is bilateral cut away view of a flow chamber.
• Figure 2b is a top view of a flow chamber.
• Figure 2c is an illustration of microparticles being loaded into a flow chamber.
• Figures 3a through 3d schematically illustrate microparticle constraining means for a flow chamber.
• Figure 4 is a schematic representation of a device for loading microparticles into a flow chamber.
• Figure 5 is a schematic representation of a fluidics system for use with the invention.
• Figures 6a and 6b schematically illustrate top-lighting and back-lighting approaches for determining microparticle centers in an array.
• Figure 7 schematically illustrates the assignment of pixels to microparticles for data processing.
• Figure 8 is a flow chart summarizing operation of the system of the invention.
• oligonucleotide tags refers to an oligonucleotide to which a oligonucleotide tag specifically hybridizes to form a perfectly matched duplex or triplex.
• the oligonucleotide tag may be selected to be either double stranded or single stranded.
• the term "complement” is meant to encompass either a double stranded complement of a single stranded oligonucleotide tag or a single stranded complement of a double stranded oligonucleotide tag.
• oligonucleotide includes linear oligomers of natural or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, anomeric forms thereof, peptide nucleic acids (PNAs), and the like, capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like.
• monomers are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g.
• oligonucleotide 3-4, to several tens of monomeric units, e.g. 40-60.
• ATGCCTG a sequence of letters, such as "ATGCCTG”
• A denotes deoxyadenosine
• C denotes deoxycytidine
• G denotes deoxyguanosine
• T denotes thymidine, unless otherwise noted.
• oligonucleotides of the invention comprise the four natural nucleotides; however, they may also comprise non- natural nucleotide analogs.
• oligonucleotides having natural or non-natural nucleotides may be employed, e.g. where processing by enzymes is called for, usually oligonucleotides consisting of natural nucleotides are required.
• Perfectly matched in reference to a duplex means that the poly- or oligonucleotide strands making up the duplex form a double stranded structure with one other such that every nucleotide in each strand undergoes Watson-Crick basepairing with a nucleotide in the other strand.
• the term also comprehends the pairing of nucleoside analogs, such as deoxyinosine, nucleosides with 2-aminopurine bases, and the like, that may be employed.
• the term means that the triplex consists of a perfectly matched duplex and a third strand in which every nucleotide undergoes Hoogsteen or reverse Hoogsteen association with a basepair of the perfectly matched duplex.
• a "mismatch" in a duplex between a tag and an oligonucleotide means that a pair or triplet of nucleotides in the duplex or triplex fails to undergo Watson-Crick and/or Hoogsteen and/or reverse Hoogsteen bonding.
• nucleoside includes the natural nucleosides, including 2'-deoxy and 2'- hydroxyl forms, e.g. as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992).
• "Analogs" in reference to nucleosides includes synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g. described by Scheit, Nucleotide Analogs (John Wiley, New York, 1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990), or the like, with the only proviso that they are capable of specific hybridization.
• Such analogs include synthetic nucleosides designed to enhance binding properties, reduce complexity, increase specificity, and the like.
• sequence determination or "determining a nucleotide sequence” in reference to polynucleotides includes determination of partial as well as full sequence information of the polynucleotide. That is, the term includes sequence comparisons, fingerprinting, and like levels of information about a target polynucleotide, as well as the express identification and ordering of nucleosides, usually each nucleoside, in a target polynucleotide. The term also includes the determination of the identification, ordering, and locations of one, two, or three of the four types of nucleotides within a target polynucleotide.
• sequence determination may be effected by identifying the ordering and locations of a single type of nucleotide, e.g. cytosines, within the target polynucleotide "CATCGC " so that its sequence is represented as a binary code, e.g. "100101 ... " for "C-(not C)-(not C)-C-(not C)-C ... " and the like.
• a single type of nucleotide e.g. cytosines
• the term "complexity" in reference to a population of polynucleotides means the number of different species of molecule present in the population. DETAILED DESCRIPTION OF THE INVENTION
• the system and apparatus of the invention is particularly applicable to the analysis of molecules that can be anchored in populations of duplicate copies to particulate solid phase supports. That is, in accordance with the invention, each analyte of a population is present on at least one microparticle in a quantity sufficient for the type of analysis being performed.
• Analyte populations particularly relevant for use with the present apparatus include combinatorial libraries synthesized on microparticle supports, e.g as disclosed in Lam et al, Chem. Rev., 97: 411-448 (1997); or Dower et al, U.S. patent 5,708,153, and polynucleotide libraries sorted onto microparticle supports, e.g. as disclosed in Brenner (cited above).
• FIG. 1 is a schematic representation of an embodiment of the invention for detecting fluorescent signals.
• Flow chamber (100) having inlet (102), outlet (104) and planar cavity (106) holds microparticles in a planar array from which optical signals (108) generated by analytes and/or reactants on microparticles can be collected and imaged.
• Flow chamber (100) is operationally associated with fluidic system (112) and detection system (114), so that delivery of fluids and collection of signals is under control of computer (116).
• optical signals are collected by microscope (118) and are imaged onto a solid state imaging device, such as charge- coupled device (CCD) (120) which is capable of generating a digital image of the physical image of the microparticle array with sufficient resolution for individual microparticles to be distinguished.
• CCD charge- coupled device
• detection system (114) usually includes appropriate bandpass filter (122) for optical signal (108), bandpass filter (124) for excitation beam (128) generated by light source (126), and other standard components.
• bandpass filter (122) for optical signal (108)
• bandpass filter (124) for excitation beam (128) generated by light source (126)
• other standard components such as a conventional fluorescence microscope is preferred which is configured for epiillumination.
• a key feature of the invention is flow chamber (100).
• Body (130) of flow chamber (100) preferably comprised inlet ( 102), outlet ( 104) and planar cavity ( 106) which are formed by standard micromachining techniques, e.g. Ekstrom et al, International patent application PCT/SE91/00327; Brown, U.S. patent 4,911,782; Harrison et al, Anal. Chem. 64: 1926-1932 (1992); and the like.
• Transparent plate (132) is sealingly attached to body (130) to form an operational flow chamber (100).
• Body (130) may be constructed from any of several different materials including glass, silicon, polyethylene, polyester, teflon, other plastics, and the like.
• transparent plate (132) is glass or quartz; and, when body (130) and transparent plate (132) are glass or silicon, transparent plate (132) is preferably attached to body (130) by anodic bonding, e.g. Pomerantz, U.S. patent 3,397,279.
• Key functions of the flow chamber include i) holding a population of microparticles in a substantially immobilized planar array, or monolayer, during a sequence of processing steps, ii) ensuring that processing reagents can access each microparticle during each step of a process, and iii) minimizing processing reagent usage.
• the degree of immobilization required may vary among different embodiments.
• microparticle movement within a planar array increases the computational and measurement burden of tracking positions of microparticles by image processing software.
• Design trade-offs therefore exist between the use of image processing software and the use of physical and/or chemical means for constraining microparticle movement.
• physical and/or chemical means are employed to constrain microparticle movement within the planar array of microparticles in flow chamber (100). Such means are referred to herein as "movement constraining means.”
• movement constraining means Most preferably, physical, or mechanical, movement constraining means are employed.
• microparticles are disposed in flow chamber (100) in a closely packed planar array.
• "closely packed” in reference to a planar array means either that the number of microparticles per unit area of a planar array is at least eighty percent of the number of microparticles in a hexagonal array of equal area, or that the average distance between centers of adjacent microparticles is less than two microparticle diameters.
• a "hexagonal" array of microparticles means a planar array of microparticles in which every microparticle in the array contacts at least six other adjacent microparticles, as shown in Figure 3 a. Additions features of flow chamber ( 100) of a preferred embodiment are illustrated in
• Figures 2a through 2c Figure 2a is a cross sectional view along a longitudinal plane that bisects flow chamber (100). The same view, in a more abstracted rendition, is shown in Figure 2c.
• inlet (102) fluidly communicates with planar cavity (106) and outlet (104).
• Microparticles (200) carrying analytes enter inlet (102) and are carried by a suspending buffer to planar cavity (106) where they become packed against dam (202) which prevents the microparticles from exiting the flow chamber through outlet (104).
• dam (202) may be formed by a sudden reduction of the vertical dimension of planar cavity (106).
• vertical dimension (204) of planar cavity (106) is selected so that microparticles (200) are constrained to a plane, i.e. a monolayer, when they pack against dam (202). More preferably, vertical dimension (204) is selected to be between about 120 to 150 percent of the diameter of the microparticles employed. For example, when microparticles are employed that have diameters of 5 ⁇ m, vertical dimension (204) may be 7 ⁇ m. Magnetic microparticles may be constrained to a plane and constrained from movement by applying a magnetic field so that the microparticles are attracted to the ceiling or to the floor of planar cavity (106).
• Width (206) of planar cavity (106) is not a critical dimension; however, for convenience and efficiency, width (206) may be selected to correspond to the dimensions of the signal collection region of detection system (114).
• Such regions labeled 1 through k in Figure 2b are referred to herein as "tiles.” That is, the region of planar cavity (106) occupied by microparticles may be divided into non-overlapping areas, referred to as "tiles," that cover the entire occupied region.
• Figure 2b which is a top view of the flow chamber of Figure 2a, also shows inlet (102), planar cavity (106), dam (202), and outlet (104) that lie in sequence along axis (217) of flow chamber (100).
• Many movement constraining means may be selected for use with the flow chamber, either alone or in combination.
• Such means include loading microparticles with trace amounts of a chemically reactive species which may be activated and cross-linked; providing physical, or mechanical structures, such as ridges, within the flow chamber; providing magnetically responsive microparticles which may be immobilized by an external magnetic field; providing a second population of microparticles that are loaded into a flow chamber after the analyte-containing population, which forces the analyte-containing population against dam (202); and the like.
• Exemplary chemically reactive species for use with nucleic acid analytes are disclosed in Summerton et al, U.S. patent 4,123,610; Gamper et al, J. Mol.
• microparticle movement is constrained by providing a flow chamber with planar cavity (106) containing a plurality of ridges running parallel to axis (217) of the flow chamber, i.e. parallel to the direction of reagent flow, so that microparticles are arranged into rows, which may be single-file, or several microparticles wide, as shown in Figures 3a and 3b.
• Figures 3a and 3b illustrate two possible distances between parallel ridges. In Figure 3a, the distance is selected to permit maximal packing of microparticles into a hexagonal array, and in Figure 3b, the distance is selected for less efficient packing, but for increased reagent access to microparticle surfaces.
• Figures 3c and 3d are axial views of the flow chamber showing the microparticle arrangements of Figures 3a and 3b, respectively.
• the inner surfaces of flow chamber (100) may be passivated, that is, treated to render such surfaces inert and/or non- adsorbing with respect to enzymes.
• the type of treatment depends on the sensitivity of the enzymes used in the process, and their affinity for the surfaces.
• Surface treatments include silanization, e.g. with commercially available reagents (Pierce, Rockford, IL); and/or adsorption of various blocking polymers, such as poly-a-alanine, polyglycine, polyadenylic acid, polymaleimide, polyvinylpyrrolidone, or the like, e.g. Shoffner et al, Nucleic Acids Research, 24:
• glass inner surfaces of flow chamber (100) are covalently coated with a neutral coating, such as allyl methacrylate, using the technique disclosed in Sandoval et al, U.S. patent 5,326,738, which is incorporated by reference.
• Figure lb illustrates flow chamber (100) mounted between holders (140) and (142) which sealingly connect inlet ( 102) to inlet tubing ( 144) and outlet ( 104) to outlet tubing ( 146), respectively.
• holder (140) contains a rotary valve (not shown) operated by actuator (148) that shunts fluid flowing through inlet tubing (144) to inlet (102) or to waste line (150).
• actuator (148) operated by actuator (148) that shunts fluid flowing through inlet tubing (144) to inlet (102) or to waste line (150).
• Such a valve minimizes the amount of process reagent from a previous step that must be passed through flow chamber (100) prior to the initiation of the next process step. That is, such a rotary valve permits reagent in inlet tubing (144) to be shunted to waste and replaced by processing reagent required for the next step in the process being executed.
• peltier block (152) is employed to control temperature in flow chamber (100) and the entire assembly including flow chamber (100) and peltier block (152) is mounted on xyz-stage (154) which is under control of computer (116).
• microparticles are loaded into flow chamber ( 100) prior to attachment of holders (140) and (142) and the initiation of processing steps.
• Figure 4 illustrates a microparticle loader for loading microparticles into flow chamber (100).
• Flow chamber (100) is mounted between holders (400), (402), (404), and (406).
• Holders (400) and (402) sealingly clamp onto the inlet end (101) of flow chamber (100) and holders (404) and (406) sealingly clamp onto the outlet end (103) of flow chamber (100) so that inlet tubing (408) is in fluid communication with outlet tubing (410) when the microparticle loader is assembled.
• Inlet tubing (408) is connected to syringe (416) which is used to drive fluid through flow chamber (100).
• Holder (400) is constructed to have conical passage (412) which narrows to match the diameter of inlet (102) of flow chamber (100). After assembly of holders (400), (402), (404), and (406) a suspension of microparticles is placed in the conical passage after which fitting (414) is sealingly connected to holder (400).
• Fluid pressure and flow generated by syringe (416) then drives the microparticles into planar cavity (106) and against dam (202).
• approximately 500 thousand microparticles are loaded into flow chamber (100) by placing 5 ⁇ L of a 100 thousand microparticle/ ⁇ L solution (TE buffer, pH 8.0, Sambrook et al, Molecular Cloning, Second Edition (Cold Spring Harbor
• process reagents are delivered to flow chamber (100) by the fluidic system illustrated in Figure 5 which has the capacity to handle many different reagents for complex analytical processes.
• the fluidics system may accommodate up to 38 reagents, including wash buffers, rinses, enzymes, hybridization probes, adaptors, and the like.
• the function of the fluidics system is the sequential metering of selected processing reagents to flow chamber (100).
• Inlet (102) of flow chamber (100) is sealingly connected to holder (140) which contains rotary valve (actuator shown as 148) (not shown in Figure 5).
• rotary valve actuator shown as 148) (not shown in Figure 5).
• a variety of means may be employed for moving processing reagents from reservoirs, through tubing, and into flow chamber (100), including gravity feed, pressure feed, and pumps, e.g. peristaltic, syringe, and the like.
• common syringe pump (500) is employed for removing predetermined amounts processing reagents from reservoirs and for forcing such reagents through flow chamber (100) at a predetermined flow rate.
• pump (500) in operational association with valve block (502) and rotary valve (504) removes a predetermined amount of processing reagent from a selected reservoir by siphoning reagent out of the reservoir on the out-stroke of plunger (501) of pump (500).
• rotary valve (504) directs processing reagent from tubing (503) to reservoir (505) of pump (500).
• state of rotary valve (504) is changed to direct processing reagent from reservoir (505) to inlet tubing (144).
• Tubing (503) connects rotary valve (504) with manifold (508) which, in turn, is connected to a plurality (five shown) of banks of zero dead volume valves (506).
• Zero dead volume valves (506) connect individual reservoirs holding processing reagents to a common passageway (not shown in Figure 5) that runs through each of the banks of valves connecting to manifold (508).
• a preferred zero dead volume valve is described in U.S. patents 4,558, 845 and 4,703,913, which are incorporated by reference.
• Process reagents from reservoirs (514) are distributed to the banks of dead volume valves by way of manifold (510).
• Alternative valve blocks for controlling delivery of process reagents to flow chamber (100) include the valve matrix disclosed in U.S.
• detection means (114) of the invention is the ability to keep track of individual microparticles through multiple process steps and/or cycles. In connection with such tracking, detection means (114) periodically records optical characteristics of individual microparticles that provide a close approximation microparticle centers.
• the optical characteristic is the focused back light from the microparticles. That is, in reference to Figure 6a, back light (600) passes vertically through flow chamber (100) where it is focused by microparticles (602) onto focal plane (604).
• the image of focal plane (604) in this configuration appears as a field of bright points, where each point is located at the approximate center of its corresponding microparticle.
• top light (610)
• microparticles 602 where it scatters from the top surface of the microparticles.
• the optical characteristic is the scatter center of a microparticle.
• an image is collected from the plane containing scatter centers (612) resulting from such top lighting.
• the image of the scatter centers provides a convenient way to readily determine the approximate centers of the microparticles.
• microparticle centers (700) are determined, pixels (702) are assigned for determining characteristics, e.g. intensity, of an optical signal generated at each microparticle (602).
• the size of microparticle (602) and pixel area determine how many pixels are assigned to each microparticle.
• important factors include the degree to which the calculated center of a microparticle (as described above) is likely to deviate from the geometric center, the extent to which optical signal collected from the edge of an image contains spurious information (e.g. signal from an overlapping or adjacent microparticle), the uniformity of microparticle diameter and shape, and the like.
• the pixel dimensions of the CCD detector are about .9 ⁇ m x .9 ⁇ m.
• nine pixels fit easily within the interior of a microparticle image with a margin of at least about 1 ⁇ m between any pixel and the edge of the microparticle image.
• an initial pixel is assigned which encloses the computed center of a microparticle, e.g. pixel "5" in Figure 7. Thereafter, additional pixels are assigned, usually the immediately adjacent pixels.
• the value of the optical signal generated by a process at the surface of a microparticle is the average value of the optical signals collected by pixels assigned to that microparticle.
• microparticles with anchored analytes have been loaded into flow chamber (100) which has been operationally mounted in holders 140 and 142.
• the initial operation is the calibration of the microparticle focal plane (802). That is, the vertical, or "z", position of the xyz-stage is determined which optimizes the focus of either the scatter centers of the microparticles, i.e. the microparticle tops for top-lighting, or the focus points of the microparticles for back-lighting.
• the optimization is carried out by a conventional autofocusing algorithm which provides an image contrast function constructed from a predetermined sample of regions within a collected image.
• the contrast function may be evaluated iteratively for sequence of z-positions so that the differences of successive values of the contrast function can be determined. These are tested until a difference is found below a predetermined threshold, which is taken as the maximum of the contrast function. Focal plane location is taken as the z position which maximizes the image contrast function.
• a predetermined threshold which is taken as the maximum of the contrast function.
• Focal plane location is taken as the z position which maximizes the image contrast function.
• process steps are initiated (804) by way of a fluidics controller operationally associated with computer (116).
• stage settings are adjusted to place the first tile into focus using the autofocus algorithm (806), which places the focal plane of the microscope objective approximately at the tops of the microparticles.
• Stage settings are then adjusted (808) to bring the focal plane of the microscope objective to the approximate centers of the microparticles, as illustrated (606) in Figures 6a and 6b. The amount of stage movement in this re- focusing depends on the diameter of the microparticles being used.
• filters (124) and (122) After appropriate selection of filters (124) and (122), a fluorescent image of the first tile is collected (810) and transferred to data server (812).
• Fluorescent images are collected on the plane of the microparticle centers because of imperfections in the planar array. That is, microparticles in planar cavity (106) do not lie in a perfect planar array for a variety of reasons. For example, some microparticles are elevated above others as a result of packing into the flow chamber; there is some variability in the size and shape of the microparticles; and, the floor of planar cavity (106) may be uneven.
• the focal plane of the microscope objective is returned (814) to the microparticle focal plane, where another image is collected (816) for the purpose of computing microparticle centers as described above.
• the image of microparticle centers is transferred to data server (812) where data processor (818) assigns pixels of the fluorescent image to each microparticle center, as described above.
• data server data processor (818) assigns pixels of the fluorescent image to each microparticle center, as described above.
• the stage is moved so that an image of the next tile can be collected (822). If there are no further tiles of microparticles (820), then the next steps and/or cycles of the process are executed (826). If there are no further process steps (824), then the process is complete and the apparatus is placed in a holding mode.
• Optical signals collected in the course of analysis may be generated by a variety of mechanisms, including absorption and fluorescence, chemiluminescence, electrochemiluminescence, or bioluminescence emission.
• optical signaling means e.g. Kessler, editor, Nonradioactive Labeling and Detection of Biomolecules (Springer-Nerlag, Berlin); Keller and Manak, D ⁇ A Probes, Second Edition (Stockton Press, New York, 1993); and the like.
• optical signals generated in processing steps are fluorescence emissions.
• microparticles An important feature of the system of the invention is the use of microparticles for carrying analytes.
• a variety of microparticles may be employed depending on particular applications.
• microparticles must consist of a material compatible with the reagents and chemistry of the process steps being carried out and microparticle must be substantially mechanically rigid so that they retain their shape and size during process steps.
• substantially mechanically rigid means that microparticles neither swell nor contract by more than ten percent (as measure by diameter) in any process solvent or reagent.
• microparticles are microspheres of uniform size, i.e. microparticles are monodisperse.
• the diameters of spherical microparticles have a coefficient of variation less than five percent, and most preferably, less than two percent.
• Microparticle diameters are in the range of from 0J ⁇ m to 100 ⁇ m.
• microparticle diameters range from 1 ⁇ m to 20 ⁇ m.
• microparticle diameters are in the range of 1 to 5 ⁇ m.
• Suitable microparticle materials include inorganic support materials such as glass, e.g. controlled-pore glass, Balltoni beads; silica, zirconia, and the like, e.g.
• Minimally cross-hybridizing sets of oligonucleotide tags and tag complements may be synthesized either combinatorially or individually depending on the size of the set desired and the degree to which cross-hybridization is sought to be minimized (or stated another way, the degree to which specificity is sought to be enhanced).
• a minimally cross- hybridizing set may consist of a set of individually synthesized 10-mer sequences that differ from each other by at least 4 nucleotides, such set having a maximum size of 332 (when composed of 3 kinds of nucleotides and counted using a computer program such as disclosed in Appendix Ic of International patent application PCT/US96/09513).
• a minimally cross-hybridizing set of oligonucleotide tags may also be assembled combinatorially from subunits which themselves are selected from a minimally cross-hybridizing set.
• a set of minimally cross-hybridizing 12-mers differing from one another by at least three nucleotides may be synthesized by assembling 3 subunits selected from a set of minimally cross- hybridizing 4-mers that each differ from one another by three nucleotides.
• an oligonucleotide tag of the invention preferably consists of a plurality of subunits, each subunit consisting of an oligonucleotide of 3 to 9 nucleotides in length wherein each subunit is selected from the same minimally cross- hybridizing set.
• the number of oligonucleotide tags available depends on the number of subunits per tag and on the length of the subunits.
• 9 8 containing primers has a complexity of 8 , or about 1.34 x 10 .
• mRNA for library construction can be extracted from as few as 10-100 mammalian cells. Since a single mammalian cell contains about 5 x 10
• the cDNA library construction protocol results in a population containing no more than 37% of the total number of different tags. That is, without any overt sampling step at all, the protocol inherently generates a sample that comprises 37%, or less, of the tag repertoire. The probability of obtaining a double under these conditions is about 5%, which is within the preferred range.
• the fraction of the tag repertoire sampled is reduced to only 3.7%, even assuming that all the processing steps take place at 100% efficiency.
• the efficiencies of the processing steps for constructing cDNA libraries are very low, a "rule of thumb" being that good library should contain about 10 cDNA clones from mRNA extracted from 10 mammalian cells.
• a tag-polynucleotide conjugate mixture potentially contains every possible pairing of tags and types of mRNA or polynucleotide.
• overt sampling may be implemented by removing a sample volume after a serial dilution of the starting mixture of tag- polynucleotide conjugates. The amount of dilution required depends on the amount of starting material and the efficiencies of the processing steps, which are readily estimated.
• Such a sample is readily obtained as follows: Assume that the 5 x 10 mRNAs are perfectly converted into 5 x 10 vectors with tag-cDNA conjugates as inserts and that the 5 x 10 vectors are in a reaction solution having a volume of 100 ⁇ l. Four 10-fold serial dilutions may be carried out by transferring 10 ⁇ l from the original solution into a vessel containing 90 ⁇ l of an appropriate buffer, such as TE. This process may be repeated for three additional dilutions to obtain a 100 ⁇ l solution containing 5 x 10 vector molecules per ⁇ l. A 2 ⁇ l aliquot from this solution yields 10 vectors containing tag-cDNA conjugates as inserts.
• This sample is then amplified by straight forward transformation of a competent host cell followed by culturing.
• a competent host cell followed by culturing.
• no step in the above process proceeds with perfect efficiency.
• the step of transforming a host is very inefficient.
• no more than 1% of the vectors are taken up by the host and replicated.
• even fewer dilutions would be required to obtain a sample of 10 conjugates.
• a general method for exposing the single stranded tag after amplification involves digesting a target polynucleotide-containing conjugate with the 5'- 3' exonuclease activity of T4 DNA polymerase, or a like enzyme, e.g. as described in Kuijper et al, Gene, 112: 147-155 (1992).
• T4 DNA polymerase or a like enzyme, e.g. as described in Kuijper et al, Gene, 112: 147-155 (1992).
• the polynucleotides are mixed with microparticles containing the complementary sequences of the tags under conditions that favor the formation of perfectly matched duplexes between the tags and their complements.
• the hybridization conditions are sufficiently stringent so that only perfectly matched sequences form stable duplexes.
• GMA glycidalmethacrylate
• Polynucleotides loaded onto microparticles may be simultaneously sequenced in the instant apparatus using a "base-by-base” DNA sequencing methodology.
• Such sequencing methodology permits the stepwise identification of a sequence of nucleotides in a target polynucleotide, usually one base at a time, through successive cycles of treatment and detection.
• Base-by-base approaches are disclosed in the following references: Cheeseman, U.S. patent 5,302.509; Tsien et al, International application WO 91/06678; Rosenthal et al, International application WO 93/21340; Canard et al, Gene, 148: 1-6 (1994); Metzker et al, Nucleic Acids Research, 22: 4259-4267 (1994); and the like.
• Encoded adaptors may be used in an adaptor-based method of DNA sequencing that includes repeated cycles of ligation, identification, and cleavage, such as the method described in Brenner (cited above).
• a cDNA library is constructed in which an oligonucleotide tag consisting of 8 four-nucleotide "words" is attached to each cDNA.
• the repertoire of oligonucleotide tags of this size is sufficiently large (about 10 8 ) so that if the cDNAs are synthesized from a population of about 10 6 mRNAs, then there is a high probability that each cDNA will have a unique tag for sorting.
• the Bsm BI-Dpn II fragment containing the tag-cDNA conjugate is then inserted into a plasmid and amplified.
• tag-cDNA conjugates are amplified out of the plasmids by PCR in the presence of 5-Me-dCTP, using biotinylated and fluorescently labeled primers containing pre- defined restriction endonuclease sites.
• affinity purification with streptavidin coated magnetic beads the tag-cDNA conjugates are cleaved from the beads, treated with T4 DNA polymerase in the presence of dGTP to render the tags single stranded, and then combined with a repertoire of GMA beads having tag complements attached.
• poly(A + ) mRNA is extracted from DBY746 yeast cells using conventional protocols.
• First and second strand cDNA synthesis is carried out by combining 100- 150 pmoles of the following primer (SEQ ID NO: 1):
• V is G, C, or A
• [W,W,W,G] is a four-nucleotide word selected from Table II of Brenner, International patent application PCT/US96/09513, the single underlined portion is a Bsm BI recognition site, and the double underlined portion is a Pac I recognition site.
• tags of the conjugates are rendered single stranded by treating them with T4 DNA polymerase in the presence of dGTP. After the reaction is quenched, the tag-cDNA conjugate is purified by phenol-chloroform extraction and combined with 5.5 mm GMA beads carrying tag complements, each tag complement having a 5' phosphate. Hybridization is conducted under stringent conditions in the presence of a thermal stable ligase so that only tags forming perfectly matched duplexes with their complements are ligated.
• the GMA beads are washed and the loaded beads are concentrated by FACS sorting, using the fluorescently labeled cDNAs to identify loaded GMA beads.
• the tag-cDNA conjugates attached to the GMA beads are digested with Dpn II to remove the fluorescent label and treated with alkaline phosphatase to prepare the cDNAs for sequencing. That is, phasphatase is used to remove the 5' phosphate from the ends of the cDNAs to prevent unwanted cDNA-cDNA ligations by way of the palindromic Dpn II site.
• cleavage adaptor SEQ ID NO: 3
• SEQ ID NO: 3 The following cleavage adaptor is ligated to the Dpn II-digested and phosphatase treated cDNAs:
• the 3' phosphate is removed by alkaline phosphatase, the 5' strand of the cDNA is treated with T4 DNA kinase, and the nick between the cleavage adaptor and cDNA is ligated.
• the nick between the cleavage adaptor and cDNA is ligated.
• encoded adaptors are ligated to the ends of the cDNAs and the beads are ready for loading into the flow chamber.
• the polynucleotide-bead mixture is then washed, treated with a mixture of T4 polynucleotide kinase and T4 DNA ligase (New England Biolabs, Beverly, MA) to add a 5' phosphate at the gap between the target polynucleotide and the adaptor, and to complete the ligation of the adaptors to the target polynucleotide.
• T4 polynucleotide kinase and T4 DNA ligase New England Biolabs, Beverly, MA
• the bead-polynucleotide mixture is then washed in TE, diluted to a concentration of approximately 100 thousand beads per ⁇ L, and 5 ⁇ L of the resulting solution is loaded into a flow chamber with the help of the holders of Figure 4.
• top strands of the following 16 sets of 64 encoded adaptors (SEQ ID NO: 4 through SEQ ID NO: 19) are each separately synthesized on an automated DNA synthesizer (model 392 Applied Biosystems, Foster City) using standard methods.
• the bottom strand which is the same for all adaptors, is synthesized separately then hybridized to the respective top strands:
• Each of the 16 tag complements are separately synthesized as amino-derivatized oligonucleotides and are each labeled with a fluorescein molecule (using an NHS-ester of fluorescein, available from Molecular Probes, Eugene, OR) which is attached to the 5' end of the tag complement through a polyethylene glycol linker (Clonetech Laboratories, Palo Alto, CA).
• the sequences of the tag complements are simply the 12-mer complements of the tags listed above.
• a flow chamber of the design shown in Figures 2a and 2b is employed in association with an Olympus Optical Co., Ltd.
• Height (204) of flow chamber (201) is selected to be 7 ⁇ m, or approximately 140% of the diameter of the GMA beads.
• Width (210) of flow chamber (201 ) is selected so as to ensure that a 3x3 array of 9 image pixels will cover approximately 40-60% of a bead's image after lOx magnification (as illustrated in Figure 7). Thus, in order to capture images of tiles of about 100 thousand 5 ⁇ m GMA beads, width (210) is selected to have a value of 1.7 mm.
• Length (212) is selected so that the flow chamber can hold from 1 to 10 tiles of about one hundred thousand 5 ⁇ m diameter beads each.
• the cross section (220) of inlet passage (214) matches that of the inlet tubing and gradually enlarges to match that of flow chamber (201) in the region of the planar cavity, i.e. the region holding the GMA beads on which analysis is performed. It is desirable to have a constant cross section through the planar cavity of flow chamber (201) to minimize the creation of non-uniform flow patterns, as might occur with sudden constrictions and/or expansions in cross section.
• Both body (218) and cover (216) of flow chamber (201) are glass, and the planar cavity and channels of body (218) are formed by standard chemical etching techniques.
• Cross section (222) of outlet passage (224) is selected to match the cross section of flow chamber (201) at dam (202).
• the fluidics system of Figure 5a which includes all valves, syringe pump (500), and Peltier block (152), is controlled by code written in LabNIEW 5.0 (National Instruments, Austin, TX) and run on a Compact Deskpro Pentium-based microprocessor, which is connected to the various components of the fluidics system by standard I/O circuit boards. Detection system (114) and overall control of the instrument is effected through a Sun Microsystems (Mountain View, CA) Sparcstation 5. Three cycles of ligation, identification, and cleavage are carried out in flow chamber (201 ) to give the sequences of 12 nucleotides at the termini of each of appoximately 500,000 cDNAs. That is, five tiles of GMA beads are analyzed in the following series of process steps:
• the salt wash solution is 150 mM NaCl and 10 mM Tris- HC1 (pH 8.5), and the ethanol wash solution is 3:1 (v/v) solution of the salt wash solution and ethanol.
• the ligation and wash steps 13 and 14 are repeated once, after which the adaptors and the cDNAs are prepared for second strand ligation by passing T4 DNA kinase (New England Bioscience. Beverly, MA) at 7 units per ⁇ L through the flow chamber at 37°C with a flow rate of 1-2 ⁇ L per minute for 15-20 minutes.

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