WO2009105213A1 - Non-emulsion methods and masked biomolecules - Google Patents
Non-emulsion methods and masked biomolecules Download PDFInfo
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- WO2009105213A1 WO2009105213A1 PCT/US2009/001034 US2009001034W WO2009105213A1 WO 2009105213 A1 WO2009105213 A1 WO 2009105213A1 US 2009001034 W US2009001034 W US 2009001034W WO 2009105213 A1 WO2009105213 A1 WO 2009105213A1
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- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Definitions
- the present invention relates to methods and devices for amplifying nucleic acid, and, in particular, amplifying so as to generate products on a surface without the use of emulsions, hi a preferred embodiment, a plurality of groups of amplified product are generated on the surface, each group positioned in different locations on said surface so as to create an array.
- the present invention relates to methods and devices for amplifying nucleic acid, and, in particular, amplifying so as to generate products on a surface without the use of emulsions.
- a plurality of groups of amplified product are generated on the surface, each group positioned in different (typically predetermined) locations on said surface so as to create an array.
- each group is homogeneous, hi one embodiment, each group consists of amplified product of a single nucleic acid template.
- the method comprises performing limiting dilution PCR within closed compartments (e.g. sealed regions) created by two surfaces coming together.
- the present invention contemplates a method for making an array, comprising: a) providing: a first element comprising a first surface; a second element comprising a second surface; a plurality of indentations, wherein said indentations are on either said first surface or said second surface (or on both surfaces); and a solution comprising molecules selected from the group consisting of biomolecules and anchoring molecules; b) contacting said first surface with said solution under conditions such that at least a portion of said molecules attach to at least a portion of said first surface so as to create a modified surface comprising attached molecules; and c) positioning said second surface on top of said modified surface, so as to create a plurality of first regions defined by said indentations, said first regions comprising unmasked attached molecules, and second regions comprising masked attached molecules, thereby making an array.
- the present invention contemplates this embodiment of an array as a device.
- step b) causes the anchoring molecules to contact the indentations, the flat surface, or both.
- the biomolecules may contact the indentations, the flat surface, or both.
- said positioning of step c) causes at least a portion of said solution on said first surface to move off of said first surface.
- bringing the surfaces into contact can cause liquid to be put under pressure so that some portion of the solution volume moves off (e.g. drains off and that portion of the volume is removed or lost).
- the present invention be limited to how the solution is brought into contact with the surface in step b).
- the surface is dipped or immersed in the solution.
- said indentations have attached PCR primer(s) and said solution comprises nucleic acid template.
- said template has been diluted to a concentration such that less than one hundred molecules on average (more preferably, less than 10, still more preferably, less than 1 molecule) of template are in contact with any one indentation.
- said template has been diluted to a concentration such that less than three on average - but more than 1 molecule - of template are in contact with any one indentation (or are within any one region).
- said solution also contains reagents for PCR (e.g. polymerase, dNTPs, buffer, etc.).
- the template amplified in any one sealed region is homogeneous (i.e. only one type of template was amplified).
- different (diluted) template is introduced into at least two different sealed regions such that each of said different template is simultaneously amplified and each of said amplified product is homogeneous.
- said first surface is substantially flat (e.g. flat over 90% of the surface or comprising less than 10% deviation from flat). It can, but need not be, completely flat. It can be curved or only slightly curved. In one embodiment, said contacting of step b) results in substantially the entire first surface being contacted with said solution.
- said first surface comprises glass.
- said first surface comprises a surface of a microscope slide.
- said first surface comprises a surface of a microchip (e.g. a silicon surface), hi a preferred embodiment, one or both surfaces comprise a polymer. The use of an elastomer is believed to enhance the sealing of one surface against the other.
- indentations refers to a space, cavity, dent, crater, well, depression, hollow, recess or impression that is formed in the surface. In a preferred embodiment, indentations do not extend through the entire thickness of a surface. Useful dimensions for indentations are between .001 and 10,000 microns in diameter, with depths between 0.1 X and 1OX (i.e. 10 times) the diameter, and spacing (separation) of between IX and 3X (i.e. three times) the diameter.
- indentations are between 50 nanometers and 50 microns in diameter, with depths approximately 0.5X the diameter, and spacing approximately 1.2X and 2X (i.e. two times) the diameter.
- the present invention contemplates indentations of 50 nanometers in diameter, that are 25 nanometers deep, and that are spaced 60 nanometers apart.
- the present invention contemplates indentations of 5 microns in diameter, which are 2.5 microns deep, and spaced 7 microns apart.
- the latter size indentations can, in one embodiment, be loaded with beads of 5 microns in diameter.
- each of said indentations has a depth that extends up to the midpoint of said first or second element (i.e. the depth of the indentation is equal to or less than one-half the thickness of the surface).
- said second surface is crenellated and the gaps comprise said indentations.
- the indentations have raised edges. The term "raised edge” means that the edge of the indentation rises above the plane of the surface.
- there are particles in the indentations e.g. beads). It is not intended that the present invention be limited by the manner in which the indentations are manufactured.
- the indentations are introduced into the surface by treating the surface (e.g. etching a surface of glass, silicon or otherwise etchable surface).
- the indentations are introduced by casting or molding.
- the indentations are integrally molded using a polymeric surface (e.g. plastic).
- integral molding refers to the method of casting such that features are of unitary construction.
- unitary construction refers to an association of elements (e.g. the surface and the indentations) such that they are formed from the same piece of raw material without the need for further integration.
- the first surface comprises plastic and has indentations. In one embodiment, said first surface is elastomeric.
- said indentations are evenly spaced.
- said attached molecules after step b) are attached over substantially the entire first surface at a substantially even density, hi another embodiment, the molecules are attached randomly over the surface or a portion of the surface. It is not intended that the present invention be limited by the nature of the molecules.
- said molecules are biomolecules.
- said biomolecules are nucleic acid molecules (e.g. oligonucleotides, polynucleotides, etc.).
- said nucleic acid molecules comprise PCR primers.
- said nucleic acid molecules comprise probes for RCA.
- said nucleic acid molecules comprise hairpins (e.g. the hairpin allows the nucleic acid to self-prime in an extension reaction with a polymerase).
- the present invention contemplates a variety of orientations for the two surfaces.
- the two surfaces can be placed on edge (e.g. two microscope slides, or a microscope slide and a cover, can be brought together so that each contacts the other on the broad flat surface, but then can be placed on edge) for purposes of detection in a device.
- edge e.g. two microscope slides, or a microscope slide and a cover
- said first surface faces up
- said second surface faces down.
- the present invention contemplates a method of amplifying nucleic acid template, comprising: a) providing: i) a first element comprising a first surface; ii) a second element comprising a second surface; iii) a plurality of indentations, wherein said indentations are on said first surface, second surface or both; iv) a first solution comprising a plurality of first PCR primers; v) a second solution comprising a plurality of second PCR primers; and vi) a third solution comprising template, polymerase, dNTPs and primers, wherein said primers are first PCR primers, second PCR primers or both; b) contacting said first surface with said first solution under conditions such that at least a portion of said first PCR primers attach to at least a portion of said surface so as to create a first modified surface comprising attached molecules; c) contacting said second surface with said second solution under conditions such that at least a portion of said second solution under conditions such
- said positioning of step d) causes at least a portion of said solution on said surface of said first element to move off of said surface.
- each of said indentations has a depth that extends up to the midpoint between said bottom and top surfaces.
- said second surface is crenellated and the gaps comprise said indentations.
- said indentations are wells.
- said first surface is substantially fiat (10% or less deviation from flat).
- said second surface comprises indentations.
- said indentations comprise particles (e.g. beads).
- said contacting of step b) results in substantially the entire surface being contacted with said solution.
- said indentations of said second surface element are evenly spaced.
- said attached molecules after step b) are attached over substantially the entire surface at a substantially even density.
- said first element comprises glass.
- said first surface comprises plastic.
- said first surface comprises indentations.
- said indentations are formed at the time said plastic surface is molded, hi one embodiment, said first surface is elastomeric.
- said first element is a microscope slide.
- said first element is a microchip.
- said first PCR primers consist of forward PCR primers.
- said first PCR primers consist of reverse PCR primers
- hi one embodiment, said second PCR primers consist of reverse PCR primers.
- said second PCR primers consist of forward PCR primers.
- said template of said third solution has been diluted to a concentration such that less than one hundred molecules on average (more preferably, less than 10, still more preferably, less than 1 molecule) of template are in contact with any one indentation.
- the template amplified in any one sealed region is homogeneous (i.e. only one type of template was amplified), hi a preferred embodiment, different (diluted) template is introduced into at least two different sealed regions such that each of said different template is simultaneously amplified and each of said amplified product is homogeneous.
- a concentration such that less than one hundred molecules on average (more preferably, less than 10, still more preferably, less than 1 molecule) of template are in contact with any one indentation.
- the template amplified in any one sealed region is homogeneous (i.e. only one type of template was amplified)
- different (diluted) template is introduced into at least two different sealed regions such that each of said different template is simultaneously amplified and each of said amplified product is homogeneous.
- said template has been diluted to a concentration such that less than three on average - but more than 1 molecule - of template are in contact with any one indentation (or are within any one region), hi such a case, the amplified product may not always be homogeneous (e.g. it may be the product of two different templates).
- the present invention contemplates a method of amplifying nucleic acid template, comprising: a) providing: i) a first element comprising a first surface; ii) a second element comprising a second surfaces; iii) a plurality of indentations, wherein said indentations are on said first surface, second surface or both; iv) a first solution comprising a plurality of first and second PCR primers; v) a second solution comprising template, polymerase, dNTPs and primers, wherein said primers are first PCR primers, second PCR primers or both; b) contacting said first surface with said first solution under conditions such that at least a portion of said PCR primers attach to at least a portion of said first surface so as to create a modified surface comprising attached molecules; and c) positioning said second surface on top of said modified surface, so as to create a plurality of first regions defined by said indentations, said first regions comprising unmasked attached
- the method further comprises, prior to step c), contacting said modified surface with said second solution. In one embodiment, the method further comprises, after step c), contacting said modified surface with said second solution.
- said template of said second solution has been diluted to a concentration such that less than one hundred molecules on average (more preferably, less than 10, still more preferably, less than 1 molecule) of template are in contact with any one indentation. In one embodiment of the above-described method, said template of said second solution has been diluted to a concentration such that less than one hundred molecules (more preferably, less than 10, still more preferably, less than 1 molecule) of template are in within any one region.
- the template amplified in any one sealed region is homogeneous (i.e. only one type of template was amplified).
- different (diluted) template is introduced into at least two different sealed regions such that each of said different template is simultaneously amplified and each of said amplified product is homogeneous.
- said template has been diluted to a concentration such that less than three on average - but more than 1 molecule - of template are in contact with any one indentation (or are within any one region).
- the amplified product may not be homogeneous in every region where there is product.
- said positioning of step d) causes at least a portion of said second solution on said modified surface of said first element to move off of said surface.
- said indentations are wells.
- said indentations comprise particles (e.g. beads free or attached to wells).
- said first surface is substantially flat.
- said contacting of step b) results in substantially the entire first surface being contacted with said first solution, hi one embodiment, said first surface comprises indentations, hi one embodiment, said indentations are evenly spaced, hi one embodiment, said attached molecules after step b) are attached over substantially the entire surface at a substantially even density.
- said first element comprises glass.
- said first element is a microscope slide, hi one embodiment, said first surface comprises plastic, hi one embodiment, said first surface comprises indentations, hi one embodiment, said indentations are formed at the time said plastic surface is molded, hi one embodiment, said first surface is elastomeric.
- said first element is a microchip, hi one embodiment, said first PCR primers consist of forward PCR primers.
- said second PCR primers consist of reverse PCR primers
- the present invention contemplates the array created by the above-described method (as a composition), hi one embodiment, the contacting prior to step c) is under conditions such that at least a portion of said template is amplified, hi one embodiment, the contacting after step c) is under conditions such that at least a portion of said template is amplified, hi a preferred embodiment, the template amplified in any one sealed region is homogeneous (i.e.
- the method further comprises the step d) separating said first element from said second element.
- the present invention contemplates a method for making an array, comprising: a) providing: i) a first element comprising a first surface, said first surface comprising a plurality of indentations; ii) a second element comprising a second surface; iii) a third element; and iv) a solution comprising molecules selected from the group consisting of biomolecules and anchoring molecules; b) contacting said first surface with said solution under conditions such that at least a portion of said molecules attach to at least a portion of said first surface so as to create a modified surface comprising attached molecules; c) positioning said second surface on top of said modified surface, so as to create a plurality of first regions defined by said indentations, said first regions comprising unmasked attached molecules, and second regions comprising masked attached molecules, thereby making an array; and d) positioning said third element on top of said second element, thereby sandwiching said second element between said first element and said third element, hi one embodiment, said second element is
- said second element is a sheet of elastomer.
- said sheet is a film.
- said molecules are PCR primers, in which case it is preferred that said solution comprises nucleic acid template and reagents for PCR (e.g. polymerase, dNTPs, buffer, etc.).
- said template has been diluted to a concentration such that less than one hundred molecules on average (more preferably, less than 10, still more preferably, less than 1 molecule) of template are in contact with any one indentation
- said template of said second solution has been diluted to a concentration such that less than one hundred molecules (more preferably, less than 10, still more preferably, less than 1 molecule) of template are within any one region
- said template has been diluted to a concentration such that less than three on average - but more than 1 molecule - of template are in contact with any one indentation (or are within any one region).
- the present invention contemplates a method for making an array, comprising: a) providing: a first element comprising a surface; a second element comprising bottom and top surfaces, said second element further comprising a plurality of channels extending from said bottom to said top surfaces; and a solution comprising molecules selected from the group consisting of biomolecules and anchoring molecules; b) contacting the surface of said first element with said solution under conditions such that at least a portion of said molecules attach to at least a portion of said surface (more preferably over the entire surface at a substantially even density) so as to create a modified surface comprising attached molecules; and c) positioning said bottom surface of said second element on top of said modified surface, so as to create first regions defined by said channels, said first regions comprising unmasked attached molecules, and second regions comprising masked attached molecules, thereby making an array.
- the masking the above-described method differs from other masking processes in that the molecules are attached and thereafter masked (rather than using the mask to control where the molecules
- the present invention contemplates the array made by the above-described process.
- the present invention also contemplates the device comprising said first and second elements, as described above. It is not intended that the present invention be limited to how the solution is brought into contact with the surface in step b).
- the surface is dipped or immersed in the solution.
- the solution is poured or pipetted onto the surface.
- said positioning of step c) causes at least a portion of said solution (regardless of the manner by which it was introduced) on said surface of said first element to move off (or run off) of said surface. It is preferred that at least a portion of said solution remain on said surface after said positioning of step c).
- the method further comprises: d) providing a third element comprising a surface; and e) bringing the surface of said third element into contact with the top surface of said second element, thereby sandwiching said second element between the surfaces of said first and third elements, so as to create a device comprising sealed first regions.
- the present invention contemplates this device, comprising first, second and third elements, said second element positioned between said first and third elements, said first element comprising masked and unmasked attached molecules. It is not intended that the present invention be limited to the nature of the first, second or third elements. While these elements may be porous, it is preferred that they are non-porous. Importantly, the nature of each element may be different.
- the first element is a membrane (e.g. nylon, nitrocellulose, etc.).
- one or more elements are made from one or more polymers.
- said second element comprises a polymer film.
- said first and third elements are made from silicon, quartz or glass.
- said first and third elements are glass plates.
- said first and third elements are microscope slides.
- said first and third elements are microchips or silicon wafers.
- the surfaces may be flat or substantially flat (e.g. flat over 90% of the surface or comprising less than 10% deviation from flat).
- the surfaces may be curved or raised (or partially curved or partially raised).
- one or more surfaces may have depressions or protrusions.
- the depressions can take the form of wells or pits.
- the form of the protrusions can be of any type (e.g. conical or cylindrical shape and a cross-sectional configuration of a polygon, circle, ellipse or a combination thereof) and need not be identical.
- the protrusions may be identical in size and shape.
- crystalline silicon may not be the ideal material for biological compatibility.
- deposition of a metal e.g. gold
- deposition of a water-impermeable layer is contemplated on one or more elements.
- the impermeable layer is made by a sequence of three plasma enhanced vapor depositions: silicon oxide, silicon nitride, and silicon oxide.
- the surface comprises an alkyne derivatized surface and said biomolecules are azido-labeled.
- the surface is treated with a poly(ethylene glycol) such as a synthetic acridinyl poly(ethylene glycol) (APEG).
- APEG synthetic acridinyl poly(ethylene glycol)
- the surface is treated with protein such as BSA.
- the surface is pretreated with a "hydrophilicity-enhancing compounds" which are those compounds or preparations that enhance the hydrophilicity of the surface.
- the definition is functional, rather than structural. For example, Rain-X anti-fog is a commercially available reagent containing glycols and siloxanes in ethyl alcohol.
- the fact that it renders a glass or silicon surface more hydrophilic is more important than the reagent's particular formula.
- the surface (or portion thereof) is treated with a "hydrophobic reagents" which are compounds used to make “hydrophobic regions.” It is not intended that the present invention be limited to particular hydrophobic reagents.
- the present invention contemplates hydrophobic polymer molecules that can be grafted chemically to the silicon oxide surface. Such polymer molecules include, but are not limited to, polydimethylsiloxane.
- the present invention contemplates the use of silanes to make hydrophobic regions, including but not limited to halogenated silanes and alkylsilanes.
- the present invention be limited to particular silanes; the selection of the silane is only limited in a functional sense, i.e. that it render the surface hydrophobic.
- the second element or portion thereof is dipped in a hydrophobic reagent prior to bringing it into contact with the surface of the first element, so as to create hydrophobic regions around hydrophilic regions defined by said channels.
- n-octadecyltrichlorosilane OTS
- OTS octadecyldimethylchlorosilane is employed.
- the channels are etched.
- said second element comprises a polymer film treated with a laser to create said channels. While a variety of dimensions are possible, it is generally preferred that the regions defined by the channels have a width of between approximately 10 and 1000 ⁇ m (or greater if desired), and more preferably between approximately 100 and 500 ⁇ m.
- said channels are approximately 10-50 microns in diameter (most preferably 20 microns) and are spaced approximately 10-100 microns apart (most preferably 30 microns).
- thermoplastic or elastomeric materials can be used for this purpose, such materials typically comprising one or more polymers, hi one embodiment, polymers such as polyvinyl and polyurethane are employed.
- polypropylene is employed.
- polypropylene can be modified with amino groups by treatment with plasma in a mixture of nitrogen and hydrogen (1:2 V /V). Thereafter, using the method described above, oligonucleotides may be attached or synthesized in-situ.
- polystyrene e.g. a polystyrene thin film
- amino-modified DNA is attached to the surface by reaction with succinimide ester groups bound to the polystyrenes.
- polyethylenimine is used.
- said polymer comprises a polyimide.
- poly-methyl methacrylate PMMA
- polydimethylsiloxane (PDMS) material is employed. Mixtures of two or polymers may also be employed.
- an epoxy- based photosensitive resist e.g. SU-8 may be used. It is not intended that the present invention be limited by the nature of the biomolecules used in the above-described method.
- Biomolecules such as enzymes (e.g., polymerases, nucleases, etc.) and nucleic acids (both RNA and DNA) are contemplated, hi addition, biomolecules such as proteins (e.g. antibodies) and lipids (e.g. glycosphingolipids) are contemplated.
- the present invention contemplates oligonucleotides, primers (e.g. for PCR), probes (e.g. for RCA) and the like, hi some embodiments, nucleic acids having particular designs (e.g. hairpins) are desired.
- nucleic acid template is introduced into the device.
- said template has been diluted to a concentration such that less than one hundred molecules (more preferably, less than 10, still more preferably, less than 1 molecule) of template are in contact with any one channel. In a preferred embodiment, said template has been diluted to a concentration such that less than one hundred molecules on average (more preferably, less than 10, still more preferably, less than 1 molecule) of template are within any one region.
- the template amplified in any one sealed region is homogeneous (i.e. only one type of template was amplified), hi a preferred embodiment, different (diluted) template is introduced into at least two different sealed regions such that each of said different template is simultaneously amplified and each of said amplified product is homogeneous.
- said template has been diluted to a concentration such that less than three on average - but more than 1 molecule - of template are in contact with any one channel (or are within any one region).
- the amplified product within a region may not be homogeneous.
- anchoring molecules can be used, including chemical moieties (epoxide groups, ester groups, amino groups, etc.) and biomolecules that function as anchoring molecules (e.g. biotin, avidin, etc.). Molecules function as anchoring molecules when they permit the attachment and immobilization of other molecules.
- the present invention contemplates a method for making an array, comprising: a) providing: a first element comprising a surface; a second element comprising bottom and top surfaces, said second element further comprising a plurality of channels extending from said bottom to said top surfaces; and a solution comprising forward and reverse PCR primers; b) positioning said bottom surface of said second element on top of said surface of said first element, so as to create unmasked regions defined by said channels, and masked regions; and c) contacting the surface of said first element with said solution under conditions such that at least a portion of said PCR primers attach to at least a portion of said surface so as to create a modified surface comprising attached molecules, said attached molecules positioned in said unmasked regions, thereby making an array, hi one embodiment, said masked regions are free of attached molecules.
- said contacting of step c) comprises introducing said solution into said channels.
- said solution further comprises template, polymerase and dNTPs (or a second solution containing these components is introduced), and at least a portion of said PCR primers amplify at least a portion of said template.
- said template has been diluted to a concentration such that less than one hundred molecules on average (more preferably, less than 10, still more preferably, less than 1 molecule) of template are in contact with any one channel (or within any one unmasked region).
- the template amplified in any one unmasked region is homogeneous (i.e. only one type of template was amplified).
- different (diluted) template is introduced into at least two different unmasked regions such that each of said different template is simultaneously amplified and each of said amplified product is homogeneous.
- said template has been diluted to a concentration such that less than three on average - but more than 1 molecule - of template are in contact with any one channels (or are within any one region).
- the amplified product may not be homogeneous in every instance.
- the method further comprises: providing a third element comprising a surface; and bringing the surface of said third element into contact with the top surface of said second element, thereby sandwiching said second element between the surfaces of said first and third elements, so as to create a device comprising sealed unmasked regions.
- the present invention contemplates this device, comprising first, second and third elements, said second element positioned between said first and third elements, said first element comprising unmasked attached molecules. Again, it is not intended that the present invention be limited by the nature or topography of the elements.
- said first element is substantially flat
- the bottom surface of said second element is substantially flat
- the surface of said third element is substantially flat.
- these surfaces may be curved or raised (or partially curved or raised), smooth or rough, with or without depressions or protrusions.
- said first and third elements comprise glass (e.g. glass plates, microscope slides, etc.). In one embodiment, said first and third elements are microchips.
- the present invention contemplates a method for making an array, comprising: a) providing: first and third elements each comprising a substantially flat surface; a second element comprising substantially flat bottom and top surfaces, said second element further comprising a plurality of substantially evenly spaced channels extending from said bottom to said top surfaces; and a solution comprising molecules selected from the group of biomolecules and anchoring molecules; b) contacting the substantially flat surface of said first element with said solution under conditions such that at least a portion of said molecules attach over substantially the entire surface at a substantially even density so as to create a modified surface comprising attached molecules; c) positioning said bottom surface of said second element on top of said modified surface, so as to create first regions defined by said channels, said first regions comprising unmasked attached molecules, and second regions comprising masked attached molecules, thereby making an array; and d) bringing the surface of said third element into contact with the top surface of said second element, thereby sandwiching said second element between the surfaces of said first and third elements, so as to create
- said first and third elements comprise glass (e.g. glass plates, microscope slides, etc.).
- said first and third elements are microchips.
- said second element comprises a polymer film (e.g. polyimide or other suitable polymer as discussed previously) treated (e.g. with a laser or by another process) to create said channels.
- said channels of said second element are evenly spaced.
- said channels are approximately 10-50 microns (preferably 20 microns) in diameter and are spaced approximately 10-100 microns (preferably 30 microns) apart.
- the present invention be limited to the extent a surface is covered with attached molecules.
- said attached molecules after step b) are attached over substantially the entire exposed surface at a substantially even density.
- the present invention be limited by the nature of the anchoring molecules or biomolecules.
- the preferred biomolecule comprises nucleic acid molecules.
- Particularly useful nucleic acid molecules include (but are not limited to) probes for rolling circle amplification (RCA), primers for PCR, and oligonucleotides for sequencing by synthesis. With regard to the latter, a preferred oligonucleotide comprises one or more hairpins.
- template is introduced into the device (e.g. for amplification).
- said template has been diluted to a concentration such that less than one hundred molecules on average (more preferably, less than 10, still more preferably, less than 1 molecule) of template are in contact with any one channel (or within any one region).
- the template amplified in any one sealed region is homogeneous (i.e. only one type of template was amplified).
- different types of template was amplified.
- (diluted) template is introduced into at least two different sealed regions such that each of said different template is simultaneously amplified and each of said amplified product is homogeneous.
- said template has been diluted to a concentration such that less than three on average - but more than 1 molecule - of template are in contact with any one channel (or within any one region).
- the amplified product may not be homogeneous in every instance.
- the present invention contemplates a method of amplifying nucleic acid template, comprising: a) providing: first and third elements, each comprising a surface; a second element comprising bottom and top surfaces, said second element further comprising a plurality of channels extending from said bottom to said top surfaces; a first solution comprising primers; and a second solution comprising template, polymerase and dNTPs; b) contacting at least a portion of said surface of said first element with said first solution under conditions such that at least a portion of said primers attach so as to create a modified surface comprising attached primers; c) positioning said bottom surface of said second element on top of said modified surface, so as to create first regions defined by said channels, said first regions comprising unmasked attached primers, and second regions comprising masked attached primers, thereby making an array; d) introducing said second solution into said
- said treating of step (f) comprises thermally cycling the device of step (e).
- said first solution comprises forward primers for PCR.
- said first solution further comprises reverse primers for PCR.
- said template has been diluted to a concentration such that less than one hundred molecules on average (more preferably, less than 10, still more preferably, less than 1 molecule) of template are in contact with any one channel (or within any one sealed region).
- the template amplified in any one sealed region is homogeneous (i.e. only one type of template was amplified).
- different (diluted) template is introduced into at least two different sealed regions such that each of said different template is simultaneously amplified and each of said amplified product is homogeneous.
- said template has been diluted to a concentration such that less than three on average - but more than 1 molecule - of template are within any one region. In such a case, the amplified product may not be homogeneous in every instance.
- said surfaces of said first and third elements are substantially flat.
- said first and third elements comprise glass.
- said first and second elements are microscope slides.
- said first and second elements are microchips.
- said third element comprises a polymer film treated with a laser to create said channels.
- said channels are approximately 20 microns in diameter and are spaced approximately 30 microns apart.
- the present invention contemplates the device (as a composition) with the elements assembled as described above.
- the present invention contemplates a method of amplifying nucleic acid template, comprising: a) providing: i) first and third elements, each comprising a surface; ii) a second element comprising bottom and top surfaces, said second element further comprising a plurality of channels extending from said bottom to said top surfaces; iii) a first solution comprising primers; and iv) a second solution comprising template, polymerase and dNTPs; b) positioning said bottom surface of said second element on top of said surface of said first element, so as to create unmasked regions defined by said channels, and masked regions; c) contacting at least a portion of said surface of said first element with said first solution under conditions such that at least a portion of said primers attach in said unmasked regions so as to create a modified surface comprising attached primers; d) introducing said second solution into said unmasked regions; e) bringing the surface of said third element into contact with the top surface of said second element, thereby
- said first solution comprises forward primers for PCR.
- said first solution further comprises reverse primers for PCR.
- said template has been diluted to a concentration such that less than one hundred molecules on average (more preferably, less than 10, still more preferably, less than 1 molecule) of template are in contact with any one channel (or within any one sealed unmasked region), hi a preferred embodiment, the template amplified in any one sealed unmasked region is homogeneous (i.e. only one type of template was amplified), hi a preferred embodiment, different (diluted) template is introduced into at least two different sealed unmasked regions such that each of said different template is simultaneously amplified and each of said amplified product is homogeneous.
- said template has been diluted to a concentration such that less than three on average - but more than 1 molecule - of template are within any one unmasked region).
- said surfaces of said first and third elements are substantially flat, hi one embodiment, said first and third elements comprise glass, hi one embodiment, said first and second elements are microscope slides, hi one embodiment, said first and second elements are microchips.
- said third element comprises a polymer film treated with a laser to create said channels. In one embodiment, said channels are approximately 20 microns in diameter and are spaced approximately 30 microns apart.
- the present invention contemplates the device with the elements assembled as described above.
- the present invention contemplates a method of amplifying nucleic acid template, comprising: a) providing: i) first and third elements, each comprising a surface; ii) a second element comprising bottom and top surfaces, said second element further comprising a plurality of channels extending from said bottom to said top surfaces; iii) a first solution comprising a plurality of forward PCR primers; iv) a second solution comprising a plurality of reverse PCR primers; and v) a third solution comprising template, polymerase and dNTPs; b) contacting at least a portion of said surface of said first element with said first solution under conditions such that at least a portion of said forward primers attach so as to create a first modified surface comprising attached forward primers; c) contacting at least a portion of said surface of said third element with said second solution under conditions such that at least a portion of said reverse primers attach so as to create a second modified surface comprising attached reverse primers; d
- step (diluted) template is introduced into at least two different sealed regions such that each of said different template is simultaneously amplified and each of said amplified product is homogeneous.
- said template has been diluted to a concentration such that less than three on average - but more than 1 molecule - of template are in contact with any one channel (or are within any one region).
- said treating of step (g) comprises thermally cycling the device of step (f).
- the present invention contemplates the device with the elements assembled as described above.
- said first and third elements comprise glass.
- said first and third elements are microscope slides.
- said first and third elements are microchips.
- said second element comprises a polymer film.
- said polymer film is treated with a laser to create said channels.
- both the forward and reverse primers are attached to the same surface in the same or different concentrations.
- amplification causes the generation of DNA strands attached to the surface by either the forward or reverse primer.
- a separate sequencing primer is then used to determine the sequence of the forward single strands; then, a re-priming step with a different sequencing primer is used to determine the sequence of the reverse single strands. In this manner, one can get sequence information from both ends. To maximize the efficiency of generating both forward and reverse strands on a solid surface, it is useful to have the amplification occur in two phases under different conditions.
- the supply of solution-based primers is regulated based on the reaction and/or structural conditions (i.e. melting temperature, hairpins, cleavable blocker, etc).
- solution-based reverse primers For example, if one inhibits the annealing of solution-based reverse primers, but allow for the annealing of an abundance of solution-based forward primers, then we can drive the amplification of fragments that are only attached through the reverse primers on the solid surface (only reverse primers that are available are on the surface). However, if required, a small quantity of active reverse primer could added to improve efficiency. As this reaction progresses, the solution-based forward primers will be exhausted. During a second phase of the reaction, the solution-based (inactive) reverse primers will be activated by some means (change in temperature, removal of blocking group, etc) and amplification occurs with strands being attached through the forward primers affixed to the solid surface.
- primer activation could include, by are not limited to, a photocleavable blocking group or by changing the PCR annealing temperature allowing initially dormant reverse primers designed with or without hairpins to become active in the second phase of a PCR reaction.
- the present invention contemplates a method of amplifying nucleic acid template, comprising: a) providing i) a population of nucleic acid template molecules and ii) a plurality of regions, said regions defined by a plurality of indentations in a first surface, said first surface in contact with a second surface, wherein each of said regions comprises one or more PCR primers; and b) amplifying, in said regions, at least a portion of said population of nucleic acid template molecules.
- the template amplified in any one sealed region is homogeneous (i.e. only one type of template was amplified).
- different (diluted) template is introduced into at least two different sealed regions such that each of said different template is simultaneously amplified and each of said amplified product is homogeneous.
- said template has been diluted to a concentration such that less than three on average - but more than 1 molecule - of template are within any one region.
- the amplified product may not be homogeneous but may be substantially homogeneous (i.e. the product of not more than three different templates).
- said PCR primers are attached to either said first surface or said second surface.
- said amplifying in step b) is performed in a solution comprising said template molecules, one or more polymerases, and all four dNTPs.
- said solution further comprises unattached PCR primers.
- the concentration of said template molecules in said solution is such that there is less than one template molecule on average per region. In one embodiment, the concentration of said template molecules in said solution is such that there is greater than one template molecule on average per region. In one embodiment, the concentration of said template molecules in said solution is such that there are greater than two template molecules on average per region. In one embodiment, the concentration of said template molecules in said solution is such that there are not greater than three template molecules on average per region.
- the present invention contemplates a method of amplifying nucleic acid template, comprising: a) providing a population of nucleic acid template molecules and a device comprising a first surface comprising attached PCR primers in contact with a second surface comprising attached PCR primers, either said first surface or said second surface comprising a plurality of indentations, said indentations defining a plurality of regions, said regions comprising unmasked PCR primers; and b) amplifying at least a portion of said population of nucleic acid template molecules with said unmasked PCR primers in one or more of said regions.
- said attached PCR primers of said first and second surfaces that are outside said regions are masked by said contact.
- said amplifying in step b) is performed in a solution comprising said template molecules, one or more polymerases, and all four dNTPs.
- said solution further comprises unattached PCR primers.
- the concentration of said template molecules in said solution is such that there is less than one template molecule on average per region. In one embodiment, the concentration of said template molecules in said solution is such that there is greater than one template molecule on average per region. In one embodiment, the concentration of said template molecules in said solution is such that there are greater than two template molecules on average per region. In one embodiment, the concentration of said template molecules in said solution is such that there are not greater than three template molecules on average per region.
- the present invention contemplates a method, comprising: a) providing: I) a first element comprising a surface; ii) a second element comprising bottom and top surfaces, said bottom surface comprising a plurality of indentations; iii) a first solution comprising a plurality of first PCR primers; iv) a second solution comprising a plurality of second PCR primers; and v) a third solution comprising template at a known concentration; b) contacting the surface of said first element with said first solution under conditions such that at least a portion of said first PCR primers attach to at least a portion of said surface so as to create a first modified surface comprising attached molecules; c) contacting the bottom surface of said second element with said second solution under conditions such that at least a portion of said second PCR primers attach to at least a portion of said bottom surface so as to create a second modified surface comprising attached molecules; and positioning said second modified surface on top of said first modified surface, so as to create a first modified
- the method further comprises introducing said third solution to said array under conditions such that at least a portion of said template is amplified, wherein the known concentration is such that less than one template molecule is present on average in each of said plurality of first regions. In one embodiment, the method further comprises introducing said third solution to said array under conditions such that at least a portion of said template is amplified, wherein the known concentration is such that greater than one template molecule is present on average in each of said plurality of first regions. In one embodiment, the method further comprises introducing said third solution to said array under conditions such that at least a portion of said template is amplified, wherein the known concentration is such that greater than two template molecules are present on average in each of said plurality of first regions. In one embodiment, said introducing of said third solution is prior to step d). In one embodiment, said third solution further comprises at least one polymerase and all four dNTPs. In one embodiment, said third solution further comprises PCR primers.
- the method further comprises the step e) separating said first element from said second element.
- the present invention contemplates beads or particles disposed within indentations on a surface.
- the beads comprise biomolecules (e.g. nucleic acid template, oligonucleotides, primers, and the like) on the bead surface.
- the beads are uniform in spherical shape, sized within a narrow range (preferably within +/-10% of the mean diameter for better fit into the indentations or microwells), and of low fluorescence (to keep background down to a minimum).
- microspheres useful in performing the invention are: silica, polyglycidyl, poly-methyl methacrylate (PMMA), Polymethyl acrylate, polystyrene, polystyrene/divinylbenzene copolymers, polystyrene/acrylate copolymers, polystyrene/maleic acid copolymers, and cycloolef ⁇ n polymer/copolymer microspheres.
- the microspheres have paramagnetic/magnetic properties.
- the microspheres can be prepared by emulsion polymerization and the functional groups on the surface can be incorporated through the polymerization by the choice of monomers and crosslinking parameters or created/grafted post polymerization.
- PMMA microspheres which have low fluorescence in all channels, they exhibit excellent shape and size uniformity, they have carboxylic acid methyl ester groups on the surface which can be easily accessed and converted into carboxylic or amine groups, they can be inexpensively manufactured by emulsion polymerization process.
- the present invention contemplates PMMA microspheres with functionalized groups (e.g. carboxylic functional groups, amine groups, etc.) for attachment of biomolecules (e.g. primers, oligos, nucleic acid templates, and the like).
- the present invention contemplates the modification of the PMMA with polyacrylate oligomer on the surface of PMMA microspheres.
- the present invention contemplates such microspheres or beads disposed at high density into microwells or indentations on a surface. It is not intended that the present invention be limited by the nature of the surface or the method of fabrication. Nonetheless, in one embodiment, the present invention contemplates methods of fabrication to generate beads on slides at high density.
- the method relies on the use of a hot embossing technique as schematically shown in Figure 8.
- the process employs a stamp (80) having projections (81) that will create desired features (83) of desired dimensions when pressed into the polymer (82).
- the pressing step (B) is typically done with heat and pressure.
- the stamp is removed and the polymer is cooled (step C).
- microspheres (84) containing biomolecules (85) are loaded into the microwells (86).
- the method relies on the use of injection molding technique.
- polymers used in performing the hot embossing or molding process can be used including but not limited to: PMMA (polymethyl methacrylate), COP (cycloolefine polymer), and COC (cycloolefine copolymer).
- PMMA polymethyl methacrylate
- COP cycloolefine polymer
- COC cycloolefine copolymer
- these groups can be grafted on the surface by performing ozonation, oxidation, corona discharge treatment, surface plasma or UV treatment or combination thereof.
- These fabrication methods allow to generate substrates with varying features/wells density. Using standard size microscope slides casted out of PMMA or COP polymers one can create wells with 20 um, 5 um, and 1 um diameters.
- the slides with approximately 5 um features contain about 40 million microwells per slide, while the 1 um feature slide contains about 1 billion features per slide.
- a single slide with such features permits a variety of high throughput, robust assays (e.g. sequencing by synthesis, hybridization, etc.).
- Nucleic acid fragments representing a large portion of a genome (e.g. human genome) or even an entire genome can be placed on a single slide or handful of slides, and then assayed sequentially or simultaneously.
- U.S. Patent No. 6,664,079 describes a parallel sequencing approach which can be used with the high density bead/slide array embodiments described herein.
- the biomolecule-containing microspheres are chemically attached to the microwells of the slide surface.
- the surfaces of microspheres and the substrates are chemically treated to create interactions that result in affinity and attraction of both surfaces.
- the surface treatment involves attaching polyanionic molecules to one of the surfaces and polycationic molecules to the other surface to create electrostatic interactions.
- specific affinity between biotin and streptavidin is used.
- an antigen - antibody interactions are used. This initial interaction serves to localize both surfaces in close proximity.
- the present invention contemplates a next step wherein a chemical crosslinking agent is introduced to secure microspheres onto the surface.
- the chemical crosslinking requires the availability of chemically compatible reactive groups that are capable of forming covalent bonds upon the introduction of chemical crosslinker or chemical activator.
- these compatible chemical groups include, but are not limited to: amine/carboxyl, sulfhydryl/iodo- acetamide, sulfhydryl/maleimide, alkyne/azide, ketone/hydroxylamine.
- the biomolecule-containing microspheres are not chemically attached and are instead retained within the microwell by physical means (e.g. the microsphere is sized so that it fits into the microwell with minimum space for movement).
- microspheres that are approximately 5 um (5.0 to 5.7 um) in diameter are placed in approximately 5 um (4.8 to 5.4) diameter micro wells (although in some embodiments the bead diameter can exceed the microwell diameter, e.g. by 0.3 to 0.5 um).
- microspheres that are approximately 3 um in diameter are placed in approximately 3 um microwells.
- microspheres that are approximately 1 um in diameter are placed in approximately 1 um microwells.
- microspheres are used herein interchangeably.
- micro simply indicates more information, i.e. that the sphere or well is on the order of microns (um) in size.
- the present invention contemplates a plurality (e.g. millions to billions) of beads (preferably 5 um in diameter, or 3 um in diameter, or 1 um in diameter) disposed within (and either chemically attached or not attached to) a plurality (e.g. millions to billions) of wells (preferably 5 um in diameter, or 3 um in diameter, or 1 um in diameter) on a surface (preferably a hot embossed polymer), each of said beads comprising a homogenous population of amplified template, wherein different beads have different template.
- said amplified template is the amplified product of a single nucleic acid template.
- each well has one bead (and no more than one bead).
- the present invention contemplates a method, comprising: providing a plurality (e.g. millions to billions) of wells (preferably 5 um in diameter, or 3 um in diameter, or 1 um in diameter) and a plurality (e.g. millions to billions) of beads (preferably 5 um in diameter, or 3 um in diameter, or 1 um in diameter), each of said beads comprising a homogenous population of amplified template, wherein different beads have different template; and introducing said beads into said wells, said wells on a surface (preferably a hot embossed polymer), wherein each well has no more than one bead.
- each well has one bead.
- said amplified template is the amplified product of a single nucleic acid template.
- the present invention contemplates a method, comprising: providing a solution comprising nucleic acid template (e.g. collectively reflecting an entire genome), a plurality (e.g. millions to billions) of beads (preferably 5 um in diameter, or 3 urn in diameter, or 1 um in diameter) and a surface (e.g. embossed polymer) comprising a plurality (e.g.
- indentations preferably 5 um in diameter, or 3 um in diameter, or 1 um in diameter
- said beads comprising one or more primers; contacting said beads to said solution comprising nucleic acid template under conditions such that at least a portion of said primers are extended on said beads, so as to create treated beads; and introducing said treated beads into said indentations of said surface.
- said solution is diluted such that said contacting is at a concentration of between 1 and 3 molecules of template per bead.
- said introducing results in no more than one bead per indentation (and ideally one bead in each indentation).
- Figure 1 shows one embodiment of a device for amplification on a surface.
- Figure IA shows the three elements of the device (a bottom piece, a top piece, and a middle piece) not yet combined.
- a solution typically containing biomolecules, such as nucleic acid template and reagents for PCR
- Figure IB shows the assembled device wherein the middle piece (in this case, an elastomeric sheet) acts as a seal, trapping fluid in the wells.
- the middle piece in this case, an elastomeric sheet
- the middle piece will mask the primers on the surface interface, but leave the primers attached to the wells unmasked and functional for amplification.
- a portion of the solution positioned on the bottom surface typically runs off the bottom surface when the middle piece is applied and the device is assembled. After the device is used (e.g. nucleic acid within the wells is amplified by thermocycling the device, or portion thereof), it can be taken apart, resulting in the three separated elements.
- Figure 2 shows another embodiment, wherein the device is characterized by channels created by top and bottom pieces (without indentations) separated by a third piece (which can be, in one embodiment, a perforated polymeric gasket).
- Figure 2A shows the assembled device. Where primers have been attached to the surface of the bottom piece prior to assembly (as shown in 2A), the middle piece will mask the primers at the point of contact, but leave the primers in the channels unmasked and functional for amplification.
- the device e.g. nucleic acid template within the channels is amplified by thermocycling the device, or portion thereof
- it can be taken apart (as shown in 2B), resulting in a surface comprising discrete regions comprising amplified product, i.e. an array.
- Figure 3 shows one embodiment of the method of utilizing one embodiment of the device of the present invention to create amplified product.
- Figure 4 shows a Poisson probability distribution with varying average molecule densities per well (m).
- Figure 5 shows a perforated gasket with 30 micron holes at 50 micron spacing through a 25 micron thick polyimide film.
- Figure 6 is a scanned fluorescent image from single base extension reactions on DNA templates bound to the surface of a glass slide using a patterned ("MIT") prototype chip.
- the integrated chip was formed from a glass slide and a molded PDMS piece that had 40 micron holes at about a 200 micron spacing.
- the scanner resolution was 5 microns.
- Figure 7 is a fluorescent scanner image of a glass slide showing PCR amplification of primers bound to a slide.
- Figure 8 is a schematic showing one embodiment of a hot embossing technique (steps B and C) for creating indentations on a surface, such indentations being useful for, among other things, holding biomolecule-containing beads or microspheres (step D).
- Figure 9 is a photograph showing images of a 3 um bead/slide array embodiment: A) before microspheres deposition; B) after microspheres deposition.
- Figure 10 is a photograph showing an image of 1 um slide array features.
- Figure 11 is a photograph showing images of 5 um bead/slide array embodiment: A) before microspheres deposition; B) after microspheres deposition.
- the present invention relates to methods and devices for amplifying nucleic acid, and, in particular, amplifying so as to generate products on a surface without the use of emulsions.
- a plurality of groups of amplified product are generated on the surface, each group positioned in different (typically predetermined) locations on said surface so as to create an array.
- each group is homogeneous.
- each group consists of amplified product of a single nucleic acid template.
- the method comprises performing limiting dilution PCR within closed compartments (e.g. sealed regions) created by two surfaces coming together. Performing a limiting dilution PCR on a surface (e.g.
- the present invention contemplates that the device a) isolates each region (e.g. reaction site) from one another and b) contains them for thermal cycling. In one embodiment, the device is disposable.
- Figure IA shows an embodiment wherein the top surface (10) of the bottom piece (11) has a plurality of indentations (9); however, in other embodiments, the bottom surface (12) of the top piece (13) has indentations, or both pieces have indentations.
- Figure IB shows an assembled three piece embodiment (17); however, in some embodiments, the middle piece (14) is eliminated and the bottom surface (12) of the top piece (13) is simply brought into contact with the top surface (10 of the bottom piece (11). This will also cause a portion of the solution (15) to run off the bottom piece (11), although a portion (16) will remain in the indentations (9), i.e. they will be fluid-filled (although they need not be completely filled).
- the bottom surface (12) of the top piece (13) in this embodiment may or may not have biomolecules (e.g. primers) attached thereto (not shown). Whether the middle piece (14) is used or not, biomolecules on the contact points (8) (i.e. at the interface between the surfaces) will be masked, while biomolecules (e.g. primers) within the indentations (9) will be unmasked and functional.
- Template in the solution (15) can be diluted to a concentration whereby each indentation (9) on average contains between 1 and 100 molecules, and more preferably, 1 and 3 molecules of template at the point the device is assembled (or, if desired, less than 1 molecule on average).
- Figure 2 A shows an embodiment of an assembled three-piece device (20) wherein the primers (21) are only on the top surface (22) of the bottom piece (23).
- the middle piece (18) masks the primers where it contacts the top surface (22).
- the result is amplified product (24) in discrete regions (25) on one surface, i.e. one array (26).
- both surfaces comprise primers; for example, the bottom surface (27) of the top piece (28) can also comprise primers (not shown) and the result is amplified product on both surfaces, i.e. two arrays (one being the mirror image of the other).
- Such arrays can be used for standard biological assays (e.g. hybridization, sequencing, etc.).
- Figure 2 A shows a molecule of template (29) suspended in solution.
- the solution can be diluted to maximize the chance of having one (or a few) starting molecules in each chamber.
- Template (29) in the solution can be diluted to a concentration whereby each channel (30) on average contains between 1 and 100 molecules, and more preferably, 1 and 3 molecules of template at the point the device is assembled. Or, if desired, the solution can be diluted to maximize the chance of have no more than one starting molecules in each chamber.
- template (29) in solution can be diluted to a concentration whereby each channel (30) on average contains less than 1 molecule).
- FIG. 2 has been illustrated with reference to primers, other biomolecules are contemplated.
- enzymes might be attached the bottom surface (or both surfaces) and masked by the middle piece so as to create reaction channels.
- Substrate could be processed in the channels and the result captured in an array format.
- antibodies, receptors and the like can be similarly arrayed.
- Figure 3 shows one embodiment of the method of utilizing one embodiment of the device of the present invention to create amplified product.
- Step 1 comprises coating a surface (31) with a biomolecule to create attached biomolecules (32) (e.g. attached primer(s)).
- Step 2 comprises a) masking a portion of the attached biomolecules (32) using a middle piece (or pieces) at the interface (19) of the coated surface (31) and the middle piece (33), b) leaving a portion of the attached biomolecules (32) unmasked in discrete regions (34) (e.g. channels), and c) introducing a solution (35) comprising unattached biomolecules (e.g. template, polymerase, etc.).
- a solution (35) comprising unattached biomolecules (e.g. template, polymerase, etc.).
- Step 3 comprises sealing the channels (34) with a top piece (36) to create sealed regions (37) (e.g. sealed compartments, sealed chambers, etc.) wherein the unmasked attached biomolecules (32) are functional.
- Step 4 comprises initiating a reaction (e.g. PCR by thermocycling the assembled device (38)) so as to create product (39) (e.g. amplified product from PCR).
- Step 5 involves taking the assembled device (38) apart, thereby removing the top piece (36) and the middle piece or pieces (33) so as to provide a surface (31) with product (39) in a plurality of discrete regions (40).
- Step 6 (optional) comprises washing to ensure the removal of all unattached biomolecules (41).
- the present invention contemplates using a limiting dilution technique to provide conditions such that PCR products may be generated from a single molecule (i.e., for example, a DNA template or nucleic acid fragment).
- a limiting dilution technique to provide conditions such that PCR products may be generated from a single molecule (i.e., for example, a DNA template or nucleic acid fragment).
- Poisson distribution governs the distribution of fragments in wells: where P (a) is the probability of a well having some integer number of molecules (a) based upon a per well average number of available molecules (m). In one embodiment, (m) is equivalent to a specific dilution level. Performing PCR on a set of highly dilute wells will then generate some number of wells with copies of identical molecules and a few other wells with different templates. Indeed, the present invention contemplates in one embodiment methods and devices wherein different template are placed in the same indentation or channel.
- a dilution level may be chosen that maximizes the number templates that will be amplified, but does not use too many templates to provide useful results (i.e., for example, more than four).
- each well has between 1 and 3 templates on average.
- limiting dilution may result in any particular well i) being empty; ii) consisting of a single DNA template; or iii) comprising two or more different DNA templates.
- universal primers may be employed to amplify the single molecule to saturation.
- PCR under limiting dilution conditions will start more slowly than a standard PCR assay since it will be more difficult for the polymerase to "find" the single DNA template. It is further believed that it may take up to 60 cycles in a thermal cycler to reach saturation.
- the present invention contemplates methods wherein a first DNA template is amplified faster than a second DNA template in a multiplexed amplification configuration.
- FIG. 5 shows one embodiment of a prototype gasket fabricated from 25 micron thick polyimide film (42) with 30 micron holes (43) drilled at 50 micron spacing several hundred at a time by an Excimer laser system.
- Example 2 In this example, a prototype molded piece of polydimethylsiloxane (PDMS) material was employed which had 40 micron wells as small reaction chambers. The piece was molded using a micromachined silicon wafer as the negative mold for the PDMS. When cured, PDMS is a flexible polymer which is frequently used to create microchannel fluidic systems for a variety of applications. The PDMS piece had a pattern of 40 micron holes at 200 micron spacing. It was clamped against an epoxide activated microarray slide (Corning, Corning, NY) which was covered with a solution of oligonucleotide templates with an amine group on the 5' end and a hairpin on the 3' end (used for extension priming).
- PDMS polydimethylsiloxane
- the reaction chamber was formed around the bound forward primers by a one cm square adhesive gasket and a thin plastic barrier.
- the PCR reaction solution was added inside the gasket and then sealed,with the barrier.
- the slide (45) was sandwiched between metal plates and placed onto a Bio-Rad PTC-200 thermal cycler with a heated lid. The entire sandwich was cycled through three temperatures of 95, 50, and 72°C for 30 cycles. Following the completion of thermal cycling, the sandwich was disassembled and the slide was washed for one hour in PCR buffer and water. It was then dried and imaged on a ScanArray 4000 microarray scanner. The bound, labeled amplicons, Figure 7, show the PCR reaction was successful.
- two surfaces e.g. glass slides
- a middle piece e.g. perforated polymeric gasket
- the perforated film between the slides has potentially millions of holes at close spacing and is about 50 microns thick.
- the surface of at least one of the slides which faces the gasket has at least one of the PCR primers (forward or reverse, or both) attached to it ( Figure 3, step (I)).
- the reaction cocktail of templates, primers, dNTPs, polymerase and buffer is added, the gasket is placed over the coated surface, and then the second surface is placed on top of the entire assembly ( Figure 3 steps (2 and 3)).
- any extra fluid is allowed to escape out the sides of the sandwich and the entire assembly is clamped together.
- the gasket may be treated on one or two sides with an adhesive material to either eliminate the clamping or help to facilitate sealing.
- the entire secured assembly is then subjected to thermal cycling and PCR amplification occurs in each of the chambers created by the gasket holes and the two slides ( Figure 3 step (4)).
- PCR amplification occurs in each of the chambers created by the gasket holes and the two slides (Figure 3 step (4)).
- PCR amplification occurs in each of the chambers created by the gasket holes and the two slides (Figure 3 step (4)).
- a number of amplicons are formed and some are attached to the surface through extension of the primers which were attached to the surface prior to amplification. No amplicons are attached to the primers which are masked by the gasket material (not in the chambers).
- the result will be a surface with a plurality of discrete groups comprising DNA amplicon attached in an array of the same pattern as the holes in the gasket which were used ( Figure 3 step (6)).
- the top glass slide and gasket may also be made from one piece which both provides the chambers for PCR and creates a seal against the bottom slide.
- both the forward and reverse primers are attached to the same surface.
- amplification causes the generation of forward single strands and reverse single strands.
- a separate sequencing primer is then used to determine the sequence of the forward single strands; then, a re-priming step with a different sequencing primer is used to determine the sequence of the reverse single strands. In this manner, one can get sequence information from both ends.
- the present invention contemplates, activatable (temporarily inactive) oligonucleotide primers are employed.
- the second sequencing primer is inactive.
- an activatable primer has a phosphorylated 3 '-terminal phosphate that prevents the primers from themselves being extended.
- the phosphate group is removed by treatment with a phosphatase enzyme, thereby "activating" the primer.
- a phosphatase enzyme is alkaline phosphatase.
- PMMA microspheres ( ⁇ 6 umoles of COOMe groups) were placed in the 15 ml disposable conical tube and 10 ml of 0.1 N NaOH solution was added. The tube was closed and placed on shaker for 12 hrs. The suspension of microspheres was then centrifuged at 5,000 rpm for 10 minutes and supernatant removed. The microspheres were then resuspended in water and centrifuged again and this process was repeated until the supernatant had neutral pH. The beads were then washed with 3 x 10 ml of dry acetonitrile using centrifugation again.
- the beads were then washed with acetonitrile (3 x 10 ml), 20% ethanol (3 x 10 ml) and finally resuspended in 20% ethanol as 10% w/v stock.
- the beads exhibit positive reaction with ninhydrin indicating the presence of primary amino groups and can be modified by reaction with NHS activated dyes.
- Amino modified microspheres ( ⁇ 2g in 20% ethanol suspension) were washed with 3 x 10 ml of 0.1 M N-Methyl imidazole solution in water. After last wash they were resuspended in 5 ml of 0.1 M N-Methyl imidazole solution in water, to which 1 ml of polyacrylic acid stock solution (0. Im in 0.1 M N-Methyllmidazole) was added. The pH of the resulting solution was adjusted if necessary with additional N-Methylaimidazole to pH ⁇ 7 and then EDC (38 mg) dissolved in 2 ml of water was added to the beads suspension.
- the mixture was incubated overnight and washed with 3 x 10ml of 0.1 M N- methylimidazole, and 3 x 10 ml of 20% ethanol.
- the beads were stored as 10% stock in 20% ethanol.
- a reaction with a solution of succinic anhydride (Ig in 10ml) is performed by incubating for 2 hrs at room temperature.
- the microspheres at this stage are ninhydrin negative indicating coupling to the polyacrylic acid and conversion of the amino functions into carboxyls.
- PMMA slides either flat pieces or embossed were placed in a container and then a solution of 0.1 N NaOH was added. The slides were then incubated at room temperature overnight, washed and dried.
- COP slides prepared using either hot embossing or molding technique are washed in 0. IN NaOH, water and dried. The slides are then loaded into ozonation chamber and the chamber is then connected to the ozone generator. The ozonation is performed overnight and the slides are then washed with 0.1N NaOH, rinsed with water and dried.
- Example 10 Conjugation of polyamines to the carboxyl terminated slides
- Carboxyl terminated slides were immersed in a 20 ml of a solution of EDC (100 mM) in 200 mM N-Methyl-Imidazole and incubated for 10 minutes.
- EDC 100 mM
- N-Methyl-Imidazole 200 mM N-Methyl-Imidazole
- 1 ml of a solution of polyethyleneimine or poly-allylamine was added, the contents of the tube mixed well and incubated overnight on a tumbler. The slides were then washed with water and dried.
- the carboxyl terminated microspheres with DNA attached as described in EXAMPLE 11 were washed with water (3x) and the droplet deposited onto embossed and polyamine modified slide and dried. The beads were then spread across the slide and any excess removed using rubber spreader. The slide was then submerged in a solution containing 40 mM of EDC in 0.1 N N-methyl imidazole and incubated overnight, washed with 1 x SSC, and Thermopol buffer and used for sequencing.
- the carboxyl terminated microspheres with DNA attached as described in EXAMPLE 11 were washed with water (3x) and the droplet deposited onto embossed carboxyl modified slide and dried. The beads were then spread across the slide and any excess removed using rubber spreader. The slide was then washed with 1 x SSC, and Thermopol buffer and used for sequencing.
- 5 um diameter PMMA beads coated with polyacrylic acid as described above were deposited in the 5 um slide prepared using hot embossing technique and treated to generate carboxylic groups on the surface as described above. 3 regions of the slide were then marked and imaged using optical microscope and CCD camera. The slide was then loaded onto the prototype sequencer and varying numbers of sequencing cycles were performed after which the regions of interest on the slide were re-imaged and the beads counted. The retention percent was then calculated and averaged between three regions and the average loss of beads per cycle was computed. The results are shown in the table below.
- Sequencing is performed using appropriate self priming DNA template or templates generated using PCR reactions that were immobilized onto the microspheres.
- the slides containing the microspheres with immobilized templates were mounted into the gaskets forming wells.
- Next to the wells were added the solution containing fluorescently labeled terminating nucleotides at 1 uM concentration, DNA polymerase capable of incorporating terminating nucleotides (such as Therminator II, New Englan Biolabs) at 50 ug/ml, 10 mM magnesium chloride, 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl and 0.1 % w/v Triton X-100.
- DNA polymerase capable of incorporating terminating nucleotides such as Therminator II, New Englan Biolabs
- the mixture was then incubated at 65 deg C for 20 minutes, the slide was washed with 5 x SSC/0.1% Tween, 1 x Thermopol and imaged using prototype imager.
- the fluorescent signal detected identified the nucleotide incorporated into the DNA.
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US20020039728A1 (en) * | 2000-02-10 | 2002-04-04 | Robert Kain | Alternative substrates and formats for bead-based array of arrays |
US6585939B1 (en) * | 1999-02-26 | 2003-07-01 | Orchid Biosciences, Inc. | Microstructures for use in biological assays and reactions |
US20040258832A1 (en) * | 2003-06-17 | 2004-12-23 | Barklund Anna M. | Method of chemical analysis using microwells patterned from self-assembled monolayers and substrates |
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US6585939B1 (en) * | 1999-02-26 | 2003-07-01 | Orchid Biosciences, Inc. | Microstructures for use in biological assays and reactions |
US20020039728A1 (en) * | 2000-02-10 | 2002-04-04 | Robert Kain | Alternative substrates and formats for bead-based array of arrays |
US20040258832A1 (en) * | 2003-06-17 | 2004-12-23 | Barklund Anna M. | Method of chemical analysis using microwells patterned from self-assembled monolayers and substrates |
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