Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6869—Methods for sequencing
  • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—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
  • B01L3/502707—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 the manufacture of the container or its components
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—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
  • B01L3/50273—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 the means or forces applied to move the fluids
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—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
  • B01L3/502738—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 integrated valves
• • 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
• • 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
  • B01L2300/0887—Laminated structure
• • 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
  • B01L2400/0481—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure squeezing of channels or 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
  • B01L2400/06—Valves, specific forms thereof
  • B01L2400/0633—Valves, specific forms thereof with moving parts
  • B01L2400/0655—Valves, specific forms thereof with moving parts pinch valves

Definitions

• the present invention relates to methods for high speed, high throughput analysis of polynucleotide sequences and apparatuses for carrying out such methods.
• a multiplicity of DNA fragments are generated from a DNA species which it is intended to sequence. These fragments are incomplete copies ofthe DNA species to be sequenced.
• the aim is to produce a ladder of DNA fragments, each a single base longer than the previous one.
• the target DNA is used as a template for a DNA polymerase to produce a number of incomplete clones.
• the third and final step is the determination ofthe nature ofthe base at the end of each fragment. When ordered by the size ofthe fragments which they terminate, these bases represent the sequence ofthe original DNA species. Automated systems for DNA sequence analysis have been developed, such as discussed in Toneguzzo et al., 6 Biotechniques 460, 1988; Kanbara et al., 6 Biotechnology 816, 1988; and Smith et al., 13 Nuc. Acid. Res. 13: 2399, 1985; U.S. Pat. No. 4,707,237 (1987). However, all these methods still require separation of DNA products by a gel permeation procedure and then detection of their locations relative to one another along the axis of permeation or movement through the gel. These apparatuses used in these methods are not truly automatic sequencers. They are merely automatic gel readers, which require the standard sequencing reactions to be carried out before samples are loaded onto the gel.
• template sequences are determined by priming the template followed by a series of single base primer extension reactions (e.g., as described in WO 93/21340, WO 96/27025, and WO 98/44152). While the basic scheme in these methods no longer require separation of polynucleotides on the gel, they encounter various other problems such as consumption of large amounts of expensive reagents, difficulty in removing reagents after each step, misincorporation due to long exchange times, the need to remove labels from the incorporated nucleotide, and difficulty to detect further incorporation if the label is not removed. Many of these difficulties stem directly from limitations ofthe macroscopic fluidics employed. However, small-volume fluidics have not been available. As a result, these methods have not replaced the traditional gel-based sequencing schemes in practice. The skilled artisans are to a large extent still relying on the gel-based sequencing methods.
• methods for analyzing the sequence of a target polynucleotide include the steps of (a) providing a primed target polynucleotide linked to a microfabricated synthesis channel; (b) flowing a first nucleotide through the synthesis channel under conditions whereby the first nucleotide attaches to the primer, if a complementary nucleotide is present to serve as template in the target polynucleotide; (c) determining presence or absence of a signal, the presence of a signal indicating that the first nucleotide was incorporated into the primer, and hence the identity ofthe complementary base that served as a template in the target polynucleotide; (d) removing or reducing the signal, if present; and (e) repeating steps (b)-(d) with a further nucleotide that is the same or different from the first nucleotide, whereby the further nucleotide attaches to the primer or a nu
• step (a) comprises providing a plurality of different primed target polynucleotides linked to different synthesis channels; step (b) comprises flowing the first nucleotide through each ofthe synthesis channels; and step (c) comprises determining presence or absence of a signal in each ofthe channels, the presence of a signal in a synthesis channel indicating the first nucleotide was incorporated into the primer in the synthesis channel, and hence the identity ofthe complementary base that served as a template in the target polynucleotide in the synthesis channel.
• a plurality of different primed target polynucleotides are linked to each synthesis channels. Some methods include the further steps of flushing the synthesis channel to remove unincorporated nucleotides.
• steps (b)-(d) are performed at least four times with four different types of nucleotides.
• steps (b)-(d) are performed until the identity of each base in the target polynucleotide has been identified.
• the nucleotides are labeled.
• the label can be a fluorescent dye, and the signal can be detected optically.
• the label can also be a radiolabel, and the signal can be detected with a radioactivity detector.
• incorporation of nucleotides is detected by measuring pyrophosphate release.
• the synthesis channel is formed by bonding a micro fluidic chip to a flat substrate.
• the target polynucleotides are immobilized to the interior surface ofthe substrate in the synthesis channel.
• the interior surface is coated with a polyelectrolyte multilayer (PEM).
• PEM polyelectrolyte multilayer
• the microfluidic chip is fabricated with an elastomeric materia such as RTV silicone.
• methods for analyzing a target polynucleotide entails (a) pretreating the surface of a substrate to create surface chemistry that facilitates polynucleotide attachment and sequence analysis; (b) providing a primed target polynucleotide attached to the surface; (c) providing a labeled first nucleotides to the attached target polynucleotide under conditions whereby the labeled first nucleotide attaches to the primer, if a complementary nucleotide is present to serve as template in the target polynucleotide; (d) determining presence or absence of a signal from the primer, the presence of a signal indicating that the labeled first nucleotide was incorporated into the primer, and hence the identity ofthe complementary base that served as a template in the target polynucleotide; and (e) repeating steps (c)-(d) with a labeled further nucleotide that is the same or different from the first
• the substrate is glass and the surface is coated with a polyelectrolyte multilayer (PEM).
• PEM polyelectrolyte multilayer
• the PEM is terminated with a polyanion.
• the polyanion is terminated with carboxylic acid groups.
• the target polynucleotide is biotinylated, and the PEM-coated surface is further coated with biotin and then streptavidin.
• methods of analyzing a target polynucleotide include the steps of (a) providing a primed target polynucleotide; (b) providing a first type of nucleotide of which a fraction is labeled under conditions whereby the first nucleotide attaches to the primer, if a complementary nucleotide is present to serve as template in the target polynucleotide; (c) determining presence or absence of a signal from the primer, the presence of a signal indicating the first nucleotide was incorporated into the primer, and hence the identity ofthe complementary base that served as a template in the target polynucleotide; and (d) repeating steps (b)-(c) with a further type of nucleotide of which a fraction is labeled the same and which is the same or different from the first type of nucleotide, whereby the further nucleotide attaches to the primer or a nucleotide
• the label used is a fluorescent label.
• the removing or reducing step is performed by photobleaching.
• the fraction of labeled nucleotides are less than 10%, less than 1%, less than 0.1%, or less than 0.01%.
• apparatuses for analyzing the sequence of a polynucleotide have (a) a flow cell with at least one microfabricated synthesis channel; and (b) an inlet port and an outlet port which are in fluid communication with the flow cell and which flowing fluids such as deoxynucleoside triphosphates and nucleotide polymerase into and through the flow cell.
• Some ofthe apparatuses additionally have (c) a light source to direct light at a surface ofthe synthesis channel; and (d) a detector to detect a signal from the surface.
• the synthesis channel is formed by bonding a microfluidic chip to a flat substrate.
• the microfluidic chip also contain microfabricated valves and microfabricated pumps in an integrated system with the synthesis channel.
• a plurality of reservoirs for storing reaction reagents are also present, and the microfabricated valve and pump are connected to the reservoirs.
• the detector is a photon counting camera.
• the microfluidic chip is fabricated with an elastomeric material such as RTV silicone.
• the substrate of some ofthe apparatuses is a glass cover slip.
• the cross section ofthe synthesis channel in some ofthe apparatuses has a linear dimension of less than lOO ⁇ m x lOO ⁇ m, less than lO ⁇ m x lOO ⁇ m, less than l ⁇ m x lO ⁇ m, or less than 0.1 ⁇ m x l ⁇ m.
• Fig. 1 is an illustration of a first elastomeric layer formed on top of a micromachined mold.
• Fig. 2 is an illustration of a second elastomeric layer formed on top of a micromachined mold.
• Fig. 3 is an illustration ofthe elastomeric layer of Fig. 2 removed from the micromachined mold and positioned over the top ofthe elastomeric layer of Fig. 1
• Fig. 4 is an illustration corresponding to Fig. 3, but showing the second elastomeric layer positioned on top ofthe first elastomeric layer.
• Fig. 5 is an illustration corresponding to Fig. 4, but showing the first and second elastomeric layers bonded together.
• Fig. 6 is an illustration corresponding to Fig. 5, but showing the first micromachine mold removed and a planar substrate positioned in its place.
• Fig. 7A is an illustration corresponding to Fig. 6, but showing the elastomeric structure sealed onto the planar substrate.
• Figs. 7B is a front sectional view corresponding to Fig. 7A, showing an open flow channel.
• Fig. 7C corresponds to Fig. 7A, but shows a first flow channel closed by pressurization in second flow channel.
• Fig. 8 is an illustration of a first elastomeric layer deposited on a planar substrate.
• Fig. 9 is an illustration showing a first sacrificial layer deposited on top ofthe first elastomeric layer of Fig. 8.
• Fig. 10 is an illustration showing the system of Fig. 9, but with a portion ofthe first sacrificial layer removed, leaving only a first line of sacrificial layer.
• Fig. 11 is an illustration showing a second elastomeric layer applied on top of the first elastomeric layer over the first line of sacrificial layer of Fig. 10, thereby encasing the sacrificial layer between the first and second elastomeric layers.
• Fig. 12 corresponds to Fig. 11, but shows the integrated monolithic structure produced after the first and second elastomer layers have been bonded together.
• Fig. 13 is an illustration showing a second sacrificial layer deposited on top of the integral elastomeric structure of Fig. 12.
• Fig. 14 is an illustration showing the system of Fig. 13, but with a portion of the second sacrificial layer removed, leaving only a second line of sacrificial layer.
• Fig. 15 is an illustration showing a third elastomer layer applied on top ofthe second elastomeric layer and over the second line of sacrificial layer of Fig. 14, thereby encapsulating the second line of sacrificial layer between the elastomeric structure of Fig. 12 and the third elastomeric layer.
• Fig. 16 corresponds to Fig. 15, but shows the third elastomeric layer cured so as to be bonded to the monolithic structure composed ofthe previously bonded first and second elastomer layers.
• Fig. 17 corresponds to Fig. 16, but shows the first and second lines of sacrificial layer removed so as to provide two perpendicular overlapping, but not intersecting, flow channels passing through the integrated elastomeric structure.
• Fig. 18 is an illustration showing the system of Fig. 17, but with the planar substrate thereunder removed.
• Fig. 19 illustrates valve opening vs. applied pressure for various flow channels.
• Fig. 20 illustrates time response of a lOO ⁇ mxlOO ⁇ mxlO ⁇ m RTV microvalve.
• Fig. 21 is a schematic illustration of a multiplexed system adapted to permit flow through various channels.
• Fig. 22A is a plan view of a flow layer of an addressable reaction chamber structure.
• Fig. 22B is a bottom plan view of a control channel layer of an addressable reaction chamber structure.
• Fig. 22C is an exploded perspective view ofthe addressable reaction chamber structure formed by bonding the control channel layer of Fig. 22B to the top ofthe flow layer of Fig. 22 A.
• Fig. 22D is a sectional elevation view corresponding to Fig. 22C, taken along line 28D-28D in Fig. 22C.
• Fig. 23 is a schematic of a system adapted to selectively direct fluid flow into any of an array of reaction wells.
• Fig. 24 is a schematic of a system adapted for selectable lateral flow between parallel flow channels.
• Fig. 25 is a schematic of an integrated system for analyzing polynucleotide sequences.
• Fig. 26 is a schematic of a further integrated system for analyzing polynucleotide sequences.
• Fig. 27 is a schematic diagram of a sequencing apparatus.
• the present invention provides methods and apparatuses for analyzing polynucleotide sequences.
• the sequencing apparatuses comprise a microfabricated flow channel to which polynucleotide templates are attached.
• the apparatuses comprise a plurality of microfabricated channels, and diverse polynucleotide templates can be attached to each channel.
• the apparatuses can also have a plurality of reservoirs for storing various reaction reagents, and pumps and valves for controlling flow ofthe reagents.
• the flow cell can also have a window to allow optical interrogation.
• single stranded polynucleotide templates with primers are immobilized to the surface ofthe microfabricated channel or to the surface of reaction chambers that are disposed along a microfabricated flow channel, e.g., with streptavidin- biotin links.
• a polymerase and one of the four nucleotide triphosphates are flowed into the flow cell, incubated with the template, and flowed out. If no signal is detected, the process is repeated with a different type of nucleotide.
• the microfabricated apparatuses provides parallelization: many synthesis channels can be built on the same substrate. This allows analysis of a plurality of diverse polynucleotide sequences simultaneously. Further, due to the reduction of time and dead volume for exchanging reagents between different steps during the analysis, mismatch incorporation is greatly reduced. Moreover, the read length is also improved because there is less time for the polymerase to incorporate a wrong nucleotide and it is less likely that the polymerase falls off the template. All these advantages result in high speed and high throughput sequence analysis regimes.
• the surface of a substrate is pretreated to create optimal surface chemistry that facilitates polynucleotide template attachment and subsequent sequence analysis.
• the substrate surface is coated with a polyelectrolyte multilayer (PEM).
• PEM polyelectrolyte multilayer
• biotin can be applied to the PEM, and followed by application of streptavidin.
• streptavidin The substrate surface can then be used to attach biotinylated-templates.
• the PEM-coated substrate provides substantial advantages for immobilizing the template polynucleotides and for polymerase extension reaction. First, because PEM can easily be terminated with polymers bearing carboxylic acids, it is easy to attach polynucleotides. Second, the attached template is active for extension by polymerases - most probably, the repulsion of like charges prevents the template from "laying down" on the surface. Finally, the negative charge repels nucleotides, and nonspecific binding is low.
• the extension reaction only a small percentage of each type of nucleotides present in the extension reaction is labeled, e.g., with fluorescent dye.
• fluorescent dye e.g., fluorescent dye
• relatively small numbers of incorporated nucleotides are fluorescently labeled, interference of energy transfer is minimized, and the polymerase is less likely to fall off the template or be "choked” by incorporation of two labeled nucleotides sequentially.
• the incorporated fluorescent signals are extinguished by photobleaching. Employment of photobleaching strategy can reduce the number of steps (e.g., it may not be necessary to perform the removal of label after every extension cycle).
• nucleic acid or “nucleic acid molecule” refer to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, can encompass known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides.
• Nucleoside includes natural nucleosides, including ribonucleosides and 2'- deoxyribonucleosides, as well as nucleoside analogs having modified bases or sugar backbones.
• a “base” or “base-type” refers to a particular type of nucleosidic base, such as adenine, cytosine, guanine, thymine, uracil, 5-methylcytosine, 5-bromouracil, 2-aminopurine, deoxyinosine, N 4 -methoxydeoxycytosine, and the like.
• Oligonucleotide or “polynucleotide” refers to a molecule comprised of a plurality of deoxyribonucleotides or nucleoside subunits.
• the linkage between the nucleoside subunits can be provided by phosphates, phosphonates, phosphoramidates, phosphorothioates, or the like, or by nonphosphate groups as are known in the art, such as peptoid-type linkages utilized in peptide nucleic acids (PNAs).
• PNAs peptide nucleic acids
• the linking groups can be chiral or achiral.
• oligonucleotides or polynucleotides can range in length from 2 nucleoside subunits to hundreds or thousands of nucleoside subunits. While oligonucleotides are preferably 5 to 100 subunits in length, and more preferably, 5 to 60 subunits in length, the length of polynucleotides can be much greater (e.g., up to 100 kb).
• Specific hybridization refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions.
• Stringent conditions are conditions under which a probe can hybridize to its target subsequence, but to no other sequences.
• Stringent conditions are sequence-dependent and are different in different circumstances. Longer sequences hybridize specifically at higher temperatures.
• stringent conditions are selected to be about 5° C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
• T m is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% ofthe probes complementary to the target sequence hybridize to the target sequence at equilibrium.
• stringent conditions include a salt concentration of at least about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides).
• Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide or tetraalkyl ammonium salts.
• destabilizing agents such as formamide or tetraalkyl ammonium salts.
• 5X SSPE 750 mM NaCI, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4
• a temperature of 25-30°C are suitable for allele-specific probe hybridizations. (See Sambrook et al., Molecular Cloning 1989).
• analysis of polynucleotide sequence of a template is meant determining a sequence of at least 3 contiguous base subunits in a sample fragment, or alternatively, where sequence information is available for a single base-type, the relative positions of at least 3 subunits of identical base-types occurring in sequential order in the fragment.
• An example of the latter meaning is a determined sequence "AXXAXA” (5'>3'), where a series of 3 adenine (A) bases are found to be separated by two and then one other base-type in the sample fragment.
• primer refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH).
• the primer is preferably single stranded for maximum efficiency in
• the primer is first treated to separate its strands before being used to prepare extension products.
• the primer is an oligodeoxyribonucleotide.
• the primer must be sufficiently long to prime the synthesis of extension products in the presence ofthe inducing agent. The exact lengths ofthe primers depend on many factors, including temperature, source of primer and the use ofthe method.
• a primer is selected to be "substantially" complementary to a strand of specific sequence ofthe template.
• a primer must be sufficiently complementary to hybridize with a template strand for primer elongation to occur.
• a primer sequence need not reflect the exact sequence ofthe template.
• a non-complementary nucleotide fragment can be attached to the 5' end ofthe primer, with the remainder ofthe primer sequence being substantially complementary to the strand.
• Non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence ofthe template to hybridize and thereby form a template primer complex for synthesis of the extension product of the primer.
• probe refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, which is capable of hybridizing to another oligonucleotide of interest.
• a probe can be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene sequences.
• any probe used in the present invention can be labeled with any "reporter molecule,” so that is detectable in any detection system, including, but not limited to fluorescent, enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), radioactive, quantum dots, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.
• template refers to nucleic acid that is to acted upon, such as nucleic acid that is to be mixed with polymerase. In some cases “template” is sought to be sorted out from other nucleic acid sequences. "Substantially single-stranded template” is nucleic acid that is either completely single-stranded (having no double-stranded areas) or single-stranded except for a proportionately small area of double-stranded nucleic acid (such as the area defined by a hybridized primer or the area defined by intramolecular bonding).
• Substantially double-stranded template is nucleic acid that is either completely double- stranded (having no single-stranded region) or double-stranded except for a proportionately small area of single-stranded nucleic acid.
• the apparatuses comprise microfabricated channels to which polynucleotide templates to be sequenced are attached.
• the apparatuses comprise plumbing components (e.g., pumps, valves, and connecting channels) for flowing reaction reagents.
• the apparatuses can also comprise an array of reservoirs for storing reaction reagents (e.g., the polymerase, each type of nucleotides, and other reagents can each be stored in a different reservoir).
• the microfabricated components ofthe apparatuses all have a basic "flow channel” structure.
• the term "flow channel” or "microfabricated flow channel” refers to recess in a structure which can contain a flow of fluid or gas.
• the polynucleotide templates are attached to the interior surface of microfabricated channels in which synthesis occurs.
• the flow channels are termed "synthesis channel” when referring to such specific use.
• the microfabricated flow channels can also be actuated to function as the plumbing components (e.g., micro-pumps, micro-valves, or connecting channels) ofthe apparatuses.
• microfabricated flow channels are cast on a chip (e.g., a elastomeric chip).
• Synthesis channels are formed by bonding the chip to a flat substrate (e.g., a glass cover slip) which seals the channel.
• a flat substrate e.g., a glass cover slip
• one side ofthe synthesis channel is provided by the flat substrate.
• the polynucleotide templates are attached to the interior surface ofthe substrate within the synthesis channel.
• the plumbing components can be microfabricated as described in the present invention.
• the apparatuses can contain in an integrated system a flow cell in which a plurality of synthesis channels are present, and fluidic components (such as micro- pumps, micro-valves, and connecting channels) for controlling the flow ofthe reagents into and out ofthe flow cell.
• the sequencing apparatuses ofthe present invention utilize plumbing devices described in, e.g., Zdeblick et al., A Microminiature Electric-to- Fluidic Valve, Proceedings ofthe 4th International Conference on Solid State Transducers and Actuators, 1987; Shoji et al., Smallest Dead Volume Microvalves for Integrated
• valves, pumps, and connecting channels are also microfabricated. Unless otherwise specified, the discussion below of microfabrication is applicable to production of all microfabricated components ofthe sequencing apparatuses (e.g., the synthesis channels in which sequencing reactions occur, and the valves, pumps, and connecting channels for controlling reagents flow to the synthesis channels).
• the integrated (i.e., monolithic) microstructures are made out of various layers of elastomer bonded together. By bonding these various elastomeric layers together, the recesses extending along the various elastomeric layers form flow channels through the resulting monolithic, integral elastomeric structure.
• the microfabricated structures e.g., synthesis channels, pumps, valves , and connecting channels
• the microfabricated structures have widths of about 0.01 to 1000 microns, and a width-to- depth ratios of between 0.1 :1 to 100:1.
• the width is in the range of 10 to 200 microns, a width-to-depth ratio of 3:1 to 15:1.
• Various methods can be used to produce the microfabricated components of the sequencing apparatuses ofthe present invention. Fabrication ofthe microchannels, valves, pumps can be performed as described in Unger et al., Science 288:113-116, 2000, which is incorporated herein by reference. In some methods (Figs. 1 to 7B, pre-cured elastomer layers are assembled and bonded to produce a flow channel. As illustrated in Fig.
• a first micro-machined mold 10 is provided.
• Micro-machined mold 10 can be fabricated by a number of conventional silicon processing methods, including but not limited to photolithography, ion-milling, and electron beam lithography.
• the micro-machined mold 10 has a raised line or protrusion 11 extending therealong.
• a first elastomeric layer 20 is cast on
• a second micro-machined mold 12 having a raised protrusion 13 extending therealong is also provided.
• a second elastomeric layer 22 is cast on top of mold 12, as shown, such that a recess 23 can be formed in its bottom surface corresponding to the dimensions of protrusion 13.
• second elastomeric layer 22 is then removed from mold 12 and placed on top of first elastomeric layer 20. As can be seen, recess 23 extending along the bottom surface of second elastomeric layer 22 forms a flow channel 32.
• the separate first and second elastomeric layers 20 and 22 are then bonded together to form an integrated (i.e.: monolithic) elastomeric structure 24.
• elastomeric structure 24 is then removed from mold 10 and positioned on top of a planar substrate 14. As can be seen in Fig. 7A and 7B, when elastomeric structure 24 has been sealed at its bottom surface to planar substrate 14, recess 21 forms a flow channel 30.
• the present elastomeric structures form a reversible hermetic seal with nearly any smooth planar substrate.
• An advantage to forming a seal this way is that the elastomeric structures can be peeled up, washed, and re-used.
• planar substrate 14 is glass.
• a further advantage of using glass is that glass is transparent, allowing optical interrogation of elastomer channels and reservoirs.
• the elastomeric structure can be bonded onto a flat elastomer layer by the same method as described above, forming a permanent and high-strength bond. This can prove advantageous when higher back pressures are used.
• microfabrication involves curing each layer of elastomer "in place" (Figs. 8 to 18).
• flow and control channels are defined by first patterning sacrificial layer on the surface of an elastomeric layer (or other substrate, which can include glass) leaving a raised line of sacrificial layer where a channel is desired.
• a second layer of elastomer is added thereover and a second sacrificial layer is patterned on the second layer of elastomer leaving a raised line of sacrificial layer where a channel is desired.
• a third layer of elastomer is deposited thereover.
• the sacrificial layer is removed by dissolving it out ofthe elastomer with an appropriate solvent, with the voids formed by removal ofthe sacrificial layer becoming the flow channels passing through the substrate.
• a planar substrate 40 is provided.
• a first elastomeric layer 42 is then deposited and cured on top of planar substrate 40.
• a first sacrificial layer 44A is then deposited over the top of elastomeric layer 42.
• a portion of sacrificial layer 44A is removed such that only a first line of sacrificial layer 44B remains as shown.
• a second elastomeric layer 46 is then deposited over the top of first elastomeric layer 42 and over the first line of sacrificial layer 44B as shown, thereby encasing first line of sacrificial layer 44B between first elastomeric layer 42 and second elastomeric layer 46.
• elastomeric layers 46 is then cured on layer 42 to bond the layers together to form a monolithic elastomeric substrate 45.
• a second sacrificial layer 48 A is then deposited over elastomeric structure 45. Referring to Fig.
• second sacrificial layer 48 A is removed, leaving only a second sacrificial layer 48B on top of elastomeric structure 45 as shown.
• a third elastomeric layer 50 is then deposited over the top of elastomeric structure 45 (comprised of second elastomeric layer 42 and first line of sacrificial layer 44B) and second sacrificial layer 48B as shown, thereby encasing the second line of sacrificial layer 48B between elastomeric structure 45 and third elastomeric layer 50.
• third elastomeric layer 50 and elastomeric structure 45 (comprising first elastomeric layer 42 and second elastomeric layer 46 bonded together) is then bonded together forming a monolithic elastomeric structure 47 having sacrificial layers 44B and 48B passing therethrough as shown.
• sacrificial layers 44B and 48B are then removed (for example, by an solvent ) such that a first flow channel 60 and a second flow channel 62 are provided in their place, passing through elastomeric structure 47 as shown.
• planar substrate 40 can be removed from the bottom of the integrated monolithic structure.
• Soft lithographic bonding can be used to construct an integrated system which contains multiple flow channels.
• a heterogenous bonding can be used in which different layers are of different chemistries.
• the bonding process used to bind respective elastomeric layers together can comprise bonding together two layers of RTV 615 silicone.
• RTV 615 silicone is a two-part addition-cure silicone rubber. Part A contains vinyl groups and catalyst; part B contains silicon hydride (Si-H) groups. The conventional ratio for RTV 615 is 10A:1B.
• one layer can be made with 30A:1B (i.e. excess vinyl groups) and the other with 3 A: IB (i.e. excess Si-H groups). Each layer is cured separately.
• a homogenous bonding can also be used in which all layers are ofthe same chemistry.
• elastomeric structures are formed utilizing Sylgard 182, 184 or 186, or aliphatic urethane diacrylates such as (but not limited to) Ebecryl 270 or Irr 245 from UCB Chemical.
• two-layer elastomeric structures were fabricated from pure acrylated Urethane Ebe 270.
• a thin bottom layer was spin coated at 8000 rpm for 15 seconds at 170°C.
• the top and bottom layers were initially cured under ultraviolet light for 10 minutes under nitrogen utilizing a Model ELC 500 device manufactured by Electro lite corporation.
• the assembled layers were then cured for an additional 30 minutes. Reaction was catalyzed by a 0.5%) vol/vol mixture of Irgacure 500 manufactured by Ciba-Geigy Chemicals.
• the resulting elastomeric material exhibited moderate elasticity and adhesion to glass.
• two-layer elastomeric structures were fabricated from a combination of 25% Ebe 270 / 50% Irr245 / 25% isopropyl alcohol for a thin bottom layer, and pure acrylated Urethane Ebe 270 as a top layer.
• the thin bottom layer was initially cured for 5 min, and the top layer initially cured for 10 minutes, under ultraviolet light under nitrogen utilizing a Model ELC 500 device manufactured by Electrolite corporation.
• the assembled layers were then cured for an additional 30 minutes. Reaction was catalyzed by a 0.5% vol/vol mixture of Irgacure 500 manufactured by Ciba-Geigy Chemicals.
• the resulting elastomeric material exhibited moderate elasticity and adhered to glass.
• bonding of successive elastomeric layers can be accomplished by pouring uncured elastomer over a previously cured elastomeric layer and any sacrificial material patterned thereupon. Bonding between elastomer layers occurs due to interpenetration and reaction ofthe polymer chains of an uncured elastomer layer with the polymer chains of a cured elastomer layer. Subsequent curing ofthe elastomeric layer creates a bond between the elastomeric layers and create a monolithic elastomeric structure.
• first elastomeric layer 20 can be created by spin-coating an RTV mixture on microfabricated mold 12 at 2000 rpm's for 30 seconds yielding a thickness of approximately 40 microns.
• Second elastomeric layer 22 can be created by spin-coating an RTV mixture on microfabricated mold 11. Both layers 20 and 22 can be separately baked or cured at about 80°C for 1.5 hours. The second elastomeric layer 22 can be bonded onto first elastomeric layer 20 at about 80°C for about 1.5 hours.
• Micromachined molds 10 and 12 can be patterned sacrificial layer on silicon wafers.
• a Shipley SJR 5740 sacrificial layer was spun at 2000 rpm patterned with a high resolution transparency film as a mask and then developed yielding an inverse channel of approximately 10 microns in height. When baked at approximately 200°C for about 30 minutes, the sacrificial layer reflows and the inverse channels become rounded.
• the molds can be treated with trimethylchlorosilane (TMCS) vapor for about a minute before each use in order to prevent adhesion of silicone rubber.
• TMCS trimethylchlorosilane
• elastomers in general as polymers existing at a temperature between their glass transition temperature and liquefaction temperature. Elastomeric materials exhibit elastic properties because the polymer chains readily undergo torsional motion to permit uncoiling ofthe backbone chains in response to a force, with the backbone chains recoiling to assume the prior shape in the absence ofthe force. In general, elastomers deform when force is applied, but then return to their original shape when the force is removed. The elasticity exhibited by elastomeric materials can be characterized by a Young's modulus.
• Elastomeric materials having a Young's modulus of between about 1 Pa - 1 TPa, more preferably between about 10 Pa - 100 GPa, more preferably between about 20 Pa - 1 GPa, more preferably between about 50 Pa - 10 MPa, and more preferably between about 100 Pa - 1 MPa are useful in accordance with the present invention, although elastomeric materials having a Young's modulus outside of these ranges could also be utilized depending upon the needs of a particular application.
• the systems ofthe present invention can be fabricated from a wide variety of elastomers.
• elastomeric layers 20, 22, 42, 46 and 50 can preferably be fabricated from silicone rubber.
• microstructures ofthe present systems are fabricated from an elastomeric polymer such as GE RTV 615 (formulation), a vinyl-silane crosslinked (type) silicone elastomer (family).
• An important requirement for the preferred method of fabrication is the ability to bond multiple layers of elastomers together.
• layers of elastomer are cured separately and then bonded together. This scheme requires that cured layers possess sufficient reactivity to bond together.
• the layers can be ofthe same type, and are capable of bonding to themselves, or they can be of two different types, and are capable of bonding to each other.
• Other possibilities include the use an adhesive between layers and the use of thermoset elastomers.
• Common elastomeric polymers include, but are not limited to, polyisoprene, polybutadiene, polychloroprene, polyisobutylene, poly(styrene-butadiene-styrene), the polyurethanes, and silicones.
• elastomeric materials which can be utilized in connection with the present invention: polyisoprene, polybutadiene, polychloroprene, polyisobutylene, poly(styrene-butadiene-styrene), the polyurethanes, and silicone polymers; or poly(bis(fluoroalkoxy)phosphazene) (PNF, Eypel-F), poly(carborane- siloxanes) (Dexsil), poly(acrylonitrile-butadiene) (nitrile rubber), poly(l-butene), poly(chlorotrifluoroethylene-vinylidene fluoride) copolymers (Kel-F), poly(ethyl vinyl ether), poly(vinylidene fluoride), poly(vinylidene fluoride - hexafluoropropylene) copolymer (Viton), elastomeric compositions o/polyvin
• polymers incorporating materials such as chlorosilanes or methyl-, ethyl-, and phenylsilanes, and polydimethylsiloxane (PDMS) such as Dow Chemical Corp. Sylgard 182, 184 or 186, or aliphatic urethane diacrylates such as (but not limited to) Ebecryl 270 or Irr 245 from UCB Chemical can also be used.
• PDMS polydimethylsiloxane
• elastomers can also be "doped" with uncrosslinkable polymer chains ofthe same class.
• RTV 615 can be diluted with GE SF96-50 Silicone Fluid. This serves to reduce the viscosity ofthe uncured elastomer and reduces the Young's modulus ofthe cured elastomer.
• the crosslink-capable polymer chains are spread further apart by the addition of "inert” polymer chains, so this is called “dilution”.
• RTV 615 cures at up to 90% dilution, with a dramatic reduction in Young's modulus.
• doping of elastomer material can include the introduction of electrically conducting or magnetic species.
• doping with fine particles of material having an index of refraction different than the elastomeric material is also contemplated as a system for altering the refractive index of the material. Strongly absorbing or opaque particles can be added to render the elastomer colored or opaque to incident radiation. This can conceivably be beneficial in an optically addressable system.
• Some flow channels (30, 32, 60 and 62) preferably have width-to-depth ratios of about 10:1.
• a non-exclusive list of other ranges of width-to-depth ratios in accordance with the present invention is 0.1 :1 to 100:1, more preferably 1 :1 to 50:1, more preferably 2:1 to 20: 1 , and most preferably 3 : 1 to 15 : 1.
• flow channels 30, 32, 60 and 62 have widths of about 1 to 1000 microns.
• a non-exclusive list of other ranges of widths of flow channels in accordance with the present invention is 0.01 to 1000 microns, more preferably 0.05 to 1000 microns, more preferably 0.2 to 500 microns, more preferably 1 to 250 microns, and most preferably 10 to 200 microns.
• Exemplary channel widths include 0.1 ⁇ m, 1 ⁇ m, 2 ⁇ m, 5 ⁇ m, 10 ⁇ m, 20 ⁇ m, 30 ⁇ m, 40 ⁇ m, 50 ⁇ m, 60 ⁇ m, 70 ⁇ m, 80 ⁇ m, 90 ⁇ m, 100 ⁇ m, 110 ⁇ m, 120 ⁇ m, 130 ⁇ m, 140 ⁇ m, 150 ⁇ m, 160 ⁇ m, 170 ⁇ m, 180 ⁇ m, 190 ⁇ m, 200 ⁇ m, 210 ⁇ m, 220 ⁇ m, 230 ⁇ m, 240 ⁇ m, and 250 ⁇ m.
• Flow channels 30, 32, 60, and 62 have depths of about 1 to 100 microns.
• a non-exclusive list of other ranges of depths of flow channels in accordance with the present invention is 0.01 to 1000 microns, more preferably 0.05 to 500 microns, more preferably 0.2 to 250 microns, and more preferably 1 to 100 microns, more preferably 2 to 20 microns, and most preferably 5 to 10 microns.
• Exemplary channel depths include including 0.01 ⁇ m, 0.02 ⁇ m, 0.05 ⁇ m, 0.1 ⁇ m, 0.2 ⁇ m, 0.5 ⁇ m, 1 ⁇ m, 2 ⁇ m, 3 ⁇ m, 4 ⁇ m, 5 ⁇ m, 7.5 ⁇ m, 10 ⁇ m, 12.5 ⁇ m, 15 ⁇ m, 17.5 ⁇ m, 20 ⁇ m, 22.5 ⁇ m, 25 ⁇ m, 30 ⁇ m, 40 ⁇ m, 50 ⁇ m, 75 ⁇ m, 100 ⁇ m, 150 ⁇ m, 200 ⁇ m, and 250 ⁇ m.
• the flow channels are not limited to these specific dimension ranges and examples given above, and can vary in width in order to affect the magnitude of force required to deflect the membrane as discussed at length below in conjunction with Fig. 21.
• extremely narrow flow channels having a width on the order of 0.01 ⁇ m can be useful in optical and other applications, as discussed in detail below.
• Elastomeric structures which include portions having channels of even greater width than described above are also contemplated by the present invention, and examples of applications of utilizing such wider flow channels include fluid reservoir and mixing channel structures.
• Elastomeric layer 22 can be cast thick for mechanical stability.
• layer 22 is 50 microns to several centimeters thick, and more preferably approximately 4 mm thick.
• a non-exclusive list of ranges of thickness ofthe elastomer layer in accordance with other embodiments ofthe present invention is between about 0.1 micron to 10 cm, 1 micron to 5 cm, 10 microns to 2 cm, 100 microns to 10 mm. ⁇ Accordingly, membrane 25 of Fig.
• 7B separating flow channels 30 and 32 has a typical thickness of between about 0.01 and 1000 microns, more preferably 0.05 to 500 microns, more preferably 0.2 to 250, more preferably 1 to 100 microns, more preferably 2 to 50 microns, and most preferably 5 to 40 microns.
• the thickness of elastomeric layer 22 is about 100 times the thickness of elastomeric layer 20.
• Exemplary membrane thicknesses include 0.01 ⁇ m, 0.02 ⁇ m, 0.03 ⁇ m, 0.05 ⁇ m, 0.1 ⁇ m, 0.2 ⁇ m, 0.3 ⁇ m, 0.5 ⁇ m, 1 ⁇ m, 2 ⁇ m, 3 ⁇ m, 5 ⁇ m, 7.5 ⁇ m, 10 ⁇ m, 12.5 ⁇ m, 15 ⁇ m, 17.5 ⁇ m, 20 ⁇ m, 22.5 ⁇ m, 25 ⁇ m, 30 ⁇ m, 40 ⁇ m, 50 ⁇ m, 75 ⁇ m, 100 ⁇ m, 150 ⁇ m, 200 ⁇ m, 250 ⁇ m, 300 ⁇ m, 400 ⁇ m, 500 ⁇ m, 750 ⁇ m, and 1000 ⁇ m
• first elastomeric layer 42 can have a preferred thickness about equal to that of elastomeric layer 20 or 22
• second elastomeric layer 46 can have a preferred thickness about equal to that of elastomeric layer 20
• third elastomeric layer 50 can
• Figs. 7B and 7C together show the closing of a first flow channel by pressurizing a second flow channel, with Fig. 7B (a front sectional view cutting through flow channel 32 in corresponding Fig. 7A), showing an open first flow channel 30; with Fig. 7C showing first flow channel 30 closed by pressurization ofthe second flow channel 32.
• first flow channel 30 and second flow channel 32 are shown.
• Membrane 25 separates the flow channels, forming the top of first flow channel 30 and the bottom of second flow channel 32. As can be seen, flow channel 30 is "open".
• pressurization of flow channel 32 causes membrane 25 to deflect downward, thereby pinching off flow F passing through flow channel 30. Accordingly, by varying the pressure in channel 32, a linearly actuable valving system is provided such that flow channel 30 can be opened or closed by moving membrane 25 as desired.
• dead volumes and areas consumed by the moving membrane are approximately two orders of magnitude smaller than known conventional microvalves.
• valves and switching valves are contemplated in the present invention, and a non-exclusive list of ranges of dead volume includes 1 aL to 1 uL, 100 aL to 100 nL, 1 fL to 10 nL, 100 fL to 1 nL, and 1 pL to 100 pL
• the extremely small volumes capable of being delivered by pumps and valves in accordance with the present invention represent a substantial advantage. Specifically, the smallest known volumes of fluid capable of being manually metered is around 0.1 ⁇ l. The smallest known volumes capable of being metered by automated systems is about ten-times larger (1 ⁇ l). Utilizing pumps and valves ofthe present invention, volumes of liquid of 10 nl or smaller can routinely be metered and dispensed. The accurate metering of extremely small volumes of fluid enabled by the present invention would be extremely valuable in a large number of biological applications, including diagnostic tests and assays.
• Figs. 21a and 21b illustrate valve opening vs. applied pressure for a 100 ⁇ m wide first flow channel 30 and a 50 ⁇ m wide second flow channel 32.
• the membrane of this device was formed by a layer of General Electric Silicones RTV 615 having a thickness of approximately 30 ⁇ m and a Young's modulus of approximately 750 kPa.
• Figs. 21a and 21b show the extent of opening ofthe valve to be substantially linear over most ofthe range of applied pressures.
• Air pressure was applied to actuate the membrane ofthe device through a 10 cm long piece of plastic tubing having an outer diameter of 0.025" connected to a 25 mm piece of stainless steel hypodermic tubing with an outer diameter of 0.025" and an inner diameter of 0.013". This tubing was placed into contact with the control channel by insertion into the elastomeric block in a direction normal to the control channel. Air pressure was applied to the hypodermic tubing from an external LHDA miniature solenoid valve manufactured by Lee Co.
• valves ofthe present invention is almost perfectly linear over a large portion of its range of travel, with minimal hysteresis. While valves and pumps do not require linear actuation to open and close, linear response does allow valves to more easily be used as metering devices. In some applications, the opening ofthe valve is used to control flow rate by being partially actuated to a known degree of closure. Linear valve actuation makes it easier to determine the amount of actuation force required to close the valve to a desired degree of closure. Another benefit of linear actuation is that the force required for valve actuation can be easily determined from the pressure in the flow channel.
• 150A, 150B, 150C and 150D have labeled nucleotides A, C, T and G respectively disposed therein.
• Four flow channels 30A, 30B, 30C and 30D are connected to reservoirs 150A, 150B, 150C and 150D.
• Four control lines 32A, 32B, 32C and 32D (shown in phantom) are disposed thereacross with control line 32A permitting flow only through flow channel 30A (i.e.: sealing flow channels 30B, 30C and 30D), when control line 32A is pressurized.
• control line 32B permits flow only through flow channel 30B when pressurized.
• the selective pressurization of control lines 32A, 32B, 32C and 32D sequentially selects a desired nucleotide (A, C, T or G) from a desired reservoir (150A, 150B, 150C or 150D).
• the fluid then passes through flow channel 120 into a multiplexed channel flow controller 125, which in turn directs fluid flow into one or more of a plurality of synthesis channels or reaction chambers 122A, 122B, 122C, 122D or 122E in which solid phase synthesis can be carried out.
• Fig. 26 illustrates a further extension ofthe system shown in Fig. 25. It has a plurality of reservoirs Rl to R13. These reservoirs can contain the labeled nucleotides, nucleotide polymerase, or reagents for coating the surface ofthe synthesis channel and attaching polynucleotide templates (see below for further discussion).
• the reservoirs are connected to systems 200 as set forth in Figs. 25. Systems 200 are connected to a multiplexed channel flow controller 125, which is in turn connected to a plurality of synthesis channels or reaction chambers.
• An advantage of this system is that both of multiplexed channel flow controllers 125 and fluid selection systems 200 can be controlled by the same pressure inputs 170 and 172 , provided a "close horizontal” and a “close vertical” control lines (160 and 162, in phantom) are also provided.
• Some apparatuses comprise a plurality of selectively addressable reaction chambers that are disposed along a flow channel.
• the polynucleotide templates can be attached to the surface ofthe reaction chambers instead ofthe surface of flow channels.
• An exemplary embodiment of such apparatuses is illustrated in Figs. 22A,
• FIG. 22B shows a top view of a flow channel 30 having a plurality of reaction chambers 80A and 80B disposed therealong.
• flow channel 30 and reaction chambers 80A and 80B are formed together as recesses into the bottom surface of a first layer 100 of elastomer.
• Fig. 22B shows a bottom plan view of another elastomeric layer 110 with two control lines 32A and 32B each being generally narrow, but having wide extending portions 33 A and 33B formed as recesses therein.
• elastomeric layer 110 is placed over elastomeric layer 100.
• Layers 100 and 110 are then bonded together, and the integrated system operates to selectively direct fluid flow F (through flow channel 30) into either or both of reaction chambers 80A and 80B, as follows.
• Pressurization of control line 32A will cause the membrane 25 (i.e.: the thin portion of elastomer layer 100 located below extending portion 33 A and over regions 82A of reaction chamber 80A) to become depressed, thereby shutting off fluid flow passage in regions 82A, effectively sealing reaction chamber 80 from flow channel 30.
• extending portion 33 A is wider than the remainder of control line 32A. As such, pressurization of control line 32A will not result in control line 32A sealing flow channel 30.
• control lines 32 A and 32B can be actuated at once.
• sample flow in flow channel 30 will enter neither of reaction chambers 80A or 80B.
• Figs. 22 The concept of selectably controlling fluid introduction into various addressable reaction chambers disposed along a flow line (Figs. 22) can be combined with concept of selectably controlling fluid flow through one or more of a plurality of parallel flow lines (Fig. 21) to yield a system in which a fluid sample or samples can be sent to any particular reaction chamber in an array of reaction chambers.
• An example of such a system is provided in Fig. 23, in which parallel control channels 32 A, 32B and 32C with extending portions 34 (all shown in phantom) selectively direct fluid flows FI and F2 into any ofthe array of reaction wells 80A, 80B, 80C or 80D as explained above; while pressurization of control lines 32C and 32D selectively shuts off flows F2 and FI, respectively.
• fluid passage between parallel flow channels is possible. Referring to Fig. 24, either or both of control lines 32 A or 32D can be depressurized such that fluid flow through lateral passageways 35 (between parallel flow channels 30A and
• control lines 32C and 32D would shut flow channel 30A between 35A and 35B, and would also shut lateral 3 passageways 35B. As such, flow entering as flow FI would sequentially travel through 30A, 35A and leave 30B as flow F4.
• Non-elastomer based apparatuses As discussed above, while elastomers are preferred materials for fabricating the sequencing apparatuses ofthe present invention, non-elastomer based microfluidic devices can also be used in the apparatuses ofthe present invention. In some applications, the sequencing apparatuses utilize microfluidics based on conventional micro-electromechanical system (MEMS) technology. Methods of producing conventional MEMS microfluidic systems such as bulk micro-machining and surface micro-machining have been described, e.g., in Terry et al., A Gas Chromatographic Air Analyzer Fabricated on a Silicon Wafer, IEEE Trans, on Electron Devices, v. ED-26, pp. 1880-1886, 1979; and Berg et al., Micro Total Analysis Systems, New York, Kluwer, 1994.
• MEMS micro-electromechanical system
• Bulk micro-machining is a subtractive fabrication method whereby single crystal silicon is lithographically patterned and then etched to form three-dimensional structures.
• bulk micromachining technology which includes the use of glass wafer processing, silicon-to-glass wafer bonding, has been commonly used to fabricate individual microfluidic components. This glass-bonding technology has also been used to fabricate microfluidic systems.
• Surface micro-machining is an additive method where layers of semiconductor-type materials such as polysilicon, silicon nitride, silicon dioxide, and various metals are sequentially added and patterned to make three-dimensional structures. Surface micromachining technology can be used to fabricate individual fluidic components as well as microfluidic systems with on-chip electronics.
• hermetic channels can be built in a relatively simple manner using channel walls made of polysilicon (see, e.g., Webster et al., Monolithic Capillary Gel Electrophoresis Stage with On-Chip Detector, in International Conference on Micro Electromechanical Systems, MEMS 96, pp. 491-496, 1996), silicon nitride (see, e.g., Mastrangelo et al., Vacuum-Sealed Silicon Micromachined Incandescent Light Source, in Intl. Electron Devices Meeting, IDEM 89, pp. 503-506, 1989), and silicon dioxide.
• polysilicon see, e.g., Webster et al., Monolithic Capillary Gel Electrophoresis Stage with On-Chip Detector, in International Conference on Micro Electromechanical Systems, MEMS 96, pp. 491-496, 1996)
• silicon nitride see, e.g., Mastrangelo et al., Vacuum-S
• electrokinetic flow based microfluidics can be employed in the sequencing apparatuses ofthe present invention. Briefly, these systems direct reagents flow within an interconnected channel and/or chamber containing structure through the application of electrical fields to the reagents.
• the electrokinetic systems concomitantly
• An exemplary electrokinetic flow based microfluidic device can have a body structure which includes at least two intersecting channels or fluid conduits, e.g., interconnected, enclosed chambers, which channels include at least three unintersected termini.
• the intersection of two channels refers to a point at which two or more channels are in fluid communication with each other, and encompasses "T" intersections, cross intersections, "wagon wheel” intersections of multiple channels, or any other channel geometry where two or more channels are in such fluid communication.
• An unintersected terminus of a channel is a point at which a channel terminates not as a result of that channel's intersection with another channel, e.g., a "T" intersection.
• At least three intersecting channels having at least four unintersected termini are present.
• controlled electrokinetic transport operates to direct reagent flow through the intersection, by providing constraining flows from the other channels at the intersection.
• Simple electrokinetic flow of this reagent across the intersection could be accomplished by applying a voltage gradient across the length ofthe horizontal channel, i.e., applying a first voltage to the left terminus of this channel, and a second, lower voltage to the right terminus of this channel, or by allowing the right terminus to float (applying no voltage).
• the apparatus comprises a microfabricated flow cell with external mini-fluidics.
• a microfabricated flow cell with external mini-fluidics Such an apparatus is illustrated in Fig. 27.
• the glass cover slip can be anodically bonded to the surface ofthe flow cell.
• the interrogation region is lOO ⁇ m x lOO ⁇ m x lOO ⁇ m, while the input and output channels are lOO ⁇ m x lOO ⁇ m x lOO ⁇ m. Holes for the attachment of plumbing are etched at the ends of the channels.
• the fluidics can be external. Plumbing can be performed with standard HPLC components, e.g., from Upchurch and Hamilton.
• the polynucleotide template is attached to the surface with standard avidin-biotin chemistry.
• templates can be attached to the apparatus.
• the radius of gyration is approximately 0.2 ⁇ m. Therefore, about 10 5 molecules can be attached while preventing the molecules from touching.
• Reagent switching can be accomplished with, e.g., an Upchurch six port injection valve and driven by, e.g., a Thar Designs motor. Fluid can be pumped with a syringe pump.
• the detection system can be an
• the polynucleotides to be analyzed are first cloned in single-stranded Ml 3 plasmid (see, e.g., Current Protocols In Molecular Biology, Ausubel, et al., eds., John Wiley & Sons, Inc. 1995; and Sambrook, et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press, 1989).
• the single stranded plasmid is primed by 5'-biotinylated primers (see, e.g., U.S. Patent No. 5,484,701), and double stranded plasmid can then be synthesized.
• templates of around 100 bp in length are analyzed.
• templates to be sequenced are about 1 kb in length.
• templates that can be analyzed have a length of about 3 kb, 10 kb, or 20 kb.
• Primer annealing is performed under conditions which are stringent enough to achieve sequence specificity yet sufficiently permissive to allow formation of stable hybrids at an acceptable rate.
• the temperature and length of time required for primer annealing depend upon several factors including the base composition, length and concentration ofthe primer, and the nature ofthe solvent used, e.g., the concentration of DMSO, formamide, or glycerol, and counter ions such as magnesium.
• hybridization with synthetic polynucleotides is carried out at a temperature that is approximately 5 to 10°C below the melting temperature ofthe target-primer hybrid in the annealing solvent.
• the annealing temperature is in the range of 55 to 75°C. and the primer concentration is approximately 0.2 ⁇ M. Under these preferred conditions, the annealing reaction can be complete in only a few seconds.
• the single stranded polynucleotide templates to be analyzed can be DNA or RNA. They can comprise naturally occurring and or non-naturally occurring nucleotides. Templates suitable for analysis according to the present invention can have various sizes. For example, the template can have a length of 100 bp, 200 bp, 500 bp, 1 kb, 3 kb, 10 kb, or 20 kb.
• the templates are immobilized to the surface ofthe synthesis channels (e.g., 122A-122E in Fig. 25). By immobilizing the templates, unincorporated nucleotides can be removed from the synthesis channels by a washing step.
• the templates are immobilized to the surface ofthe synthesis channels (e.g., 122A-122E in Fig. 25).
• ⁇ 2 can be immobilized to the surface prior to hybridization to the primer.
• the templates can also be hybridized to the primers first and then immobilize to the surface.
• the primers are immobilized to the surface, and the templates are attached to the synthesis channels through hybridization to the primers.
• Various methods can be used to immobilize the templates or the primers to the surface ofthe synthesis channels or reaction chambers.
• the immobilization can be achieved through direct or indirect bonding ofthe templates to the surface. The bonding can be by covalent linkage. See, Joos et al., Analytical Biochemistry 247:96-101, 1997; Oroskar et al., Clin. Chem 42:1547-1555, 1996; and Khandjian, Mole. Bio.
• the bonding can also be through non-covalent linkage.
• Biotin-streptavidin Teylor et al., J. Phys. D. Appl. Phys. 24:1443, 1991
• digoxigenin and anti-digoxigenin Smith et al., Science 253: 1122, 1992
• the bonding can be achieved by anchoring a hydrophobic chain into a lipidic monolayer or bilayer.
• the templates are biotinylated, and one surface ofthe synthesis channels are coated with streptavidin. Since streptavidin is a tetramer, it has four biotin binding sites per molecule. Thus, in order to coat a surface with streptavidin, the surface can be biotinylated first, and then one ofthe four binding sites of streptavidin can be used to anchor the protein to the surface, leaving the other sites free to bind the biotinylated template (see, Taylor et al., J. Phys. D. Appl. Phys. 24:1443, 1991).
• Such treatment leads to a high density of streptavidin on the surface ofthe synthesis channel, allowing a correspondingly high density of template coverage.
• Reagents for biotinylating a surface can be obtained, for example, from Vector laboratories.
• the substrate or synthesis channel is pretreated to create surface chemistry that facilitates attachment ofthe polynucleotide templates and subsequent synthesis reactions.
• the surface is coated with a polyelectrolyte multilayer (PEM). Attachment of templates to PEM-coated surface can be accomplished by light- directed spatial attachment (see, e.g., U.S. Patent Nos. 5,599,695, 5,831,070, and 5,959,837). Alternatively, the templates can be attached to PEM-coated surface entire chemically (see below for detail). In some methods, non-PEM based surface chemistry can be created prior to template attachment.
• Such a surface can be used as a substrate that is to be bond to a microfluidic chip and form the synthesis channel.
• Primers with two domains, a priming domain and a capture domain can be used to anchor templates to the substrate.
• the priming domain is complementary to the target template.
• the capture domain is present on the non-extended side ofthe priming sequence. It is not complementary to the target template, but rather to a specific oligonucleotide sequence present on the substrate.
• the target templates can be separately hybridized with their primers, or (if the priming sequences are different) simultaneously hybridized in the same solution. Incubation ofthe primer/template duplexes in the flow channel under hybridization conditions allows attachment of each template to a unique spot. Multiple synthesis channels can be charged with templates in this fashion simultaneously.
• Another method for attaching multiple templates in a single channel is to sequentially activate portions ofthe substrate and attach template to them. Activation ofthe substrate can be achieved by either optical or electrical means. Optical illumination can be used to initiate a photochemical deprotection reaction that allows attachment ofthe template to the surface (see, e.g., U.S. Patent Nos. 5,599,695, 5,831,070, and 5,959,837).
• the substrate surface can be derivatized with "caged biotin", a commercially available derivative of biotin that becomes capable of binding to avidin only after being exposed to light. Templates can then be attached by exposure of a site to light, filling the channel with avidin solution, washing, and then flowing biotinylated template into the channel.
• Another variation is to prepare avidinylated substrate and a template with a primer with a caged biotin moiety; the template can then be immobilized by flowing into the channel and illumination of the solution above a desired area. Activated template/primer duplexes are then attached to the first wall they diffused to, yielding a diffusion limited spot.
• Electrical means can also be used to direct template to specific points in the channel.
• a field gradient can be created which drives the template to a single electrode, where it can attach (see, e.g., U.S. Patent Nos. 5,632,957, 6,051,380, and 6,071,394).
• it can be achieved by electrochemically activating regions ofthe surface and changing the voltage applied to the electrodes.
• the surface of synthesis channels are coated with PEM prior to attachment ofthe templates (or primers).
• Such attachment scheme can be both an ex-situ process or an in situ process.
• the surface ofthe flat substrate is coated with PEM first, followed by attachment ofthe templates.
• the elastomeric microfluidic chip is then bonded to the substrate to form and seal the synthesis channel.
• the microfluidic chip is attached to the flat substrate first, and a PEM is then constructed in the channels.
• the templates are then attached inside the channels.
• the microfluidic chip can be bonded to the flat substrate at any point in the template attachment process, and the remaining steps can be completed inside the microfluidic channels.
• the in-situ protocol is used.
• the method described here leads to low nonspecific binding of labeled (e.g., with fluorescent dye) nucleotides and good seal of the microfluidic components and the synthesis channels.
• a good seal between the microfluidic components and the synthesis channels allows the use of higher pressures, which in turn increases flow rates and decreases exchange times.
• the various methods for attaching the templates to the surface ofthe synthesis channel are discussed in detail below.
• An exemplified scheme ofthe ex situ protocol is as follows. First, the surface of a glass cover slip is cleaned and then coated with a polyelectrolyte multilayer (PEM). Following biotinylation ofthe carboxyhc acid groups, streptavidin is then applied to generate a surface capable of capturing biotinylated molecules. Biotinylated polynucleotide templates are then added to the coated glass cover slip for attachment.
• the surface chemistry thus created is particularly suited for sequencing by synthesis with fluorescent nucleotides, because it generates a strong negatively-charged surface which repels the negatively-charged nucleotides. Detailed procedures for cleaning the cover slips, coating of polyelectrolyte multilayer, and attachment ofthe templates are described below.
• PEM formation proceeds by the sequential addition of polycations and polyanions, which are polymers with many positive or negative charges, respectively. Upon addition of a polycation to a negatively-charged surface, the polycation deposits on the
• Carboxyhc acid groups are negatively charged at pH 7, and are a common target for covalent bond formation. By terminating the surface with carboxyhc acid groups, a surface which is both strongly negatively-charged and chemically reactive can be generated In particular, amines can link to them to form amide bonds, a reaction that can be catalyzed by carbodiimides. A molecule with biotin at one end, a hydrophilic spacer, and an amine at the other end is used to terminate the surface with biotin.
• An avidin molecule is capable of binding up to four biotin molecules. This means that avidin, and its derivative Streptavidin, is capable of converting a biotin-terminated surface to a surface capable of capturing biotin. Streptavidin, which carries a slight negative charge, is used to attach the polynucleotide templates to be analyzed to the surface by using a biotinylated primer. A buffer with a high concentration of multivalent salt is used in order to screen the repulsion ofthe negatively charged surface for the negatively-charged DNA.
• the glass cover slips are first cleaned with HP H 2 O (H 2 O deionized to 18.3 MOhm-cm and filtered to 0.2 ⁇ m) and a RCA Solution (6:4:1 mixture of HP H 2 O, (30% HjOH), and (30% H 2 O 2 )). The cover slips are then sonicated in 2% Micro 90 detergent for 20 minutes. After rinse thoroughly with HP H 2 O, the cover slips are stirred in gently boiling RCA solution for at least 1 hour, and rinsed again with HP H 2 O.
• HP H 2 O H 2 O deionized to 18.3 MOhm-cm and filtered to 0.2 ⁇ m
• a RCA Solution (6:4:1 mixture of HP H 2 O, (30% HjOH), and (30% H 2 O 2 )
• the cover slips are then sonicated in 2% Micro 90 detergent for 20 minutes. After rinse thoroughly with HP H 2 O, the cover slips are stirred in gently boiling RCA solution for at least 1 hour, and rinsed again with HP H
• the glass cover slips are submerged in PAH solution (Poly(allylamine) (PAH, +): 2 mg/ml in HP H 2 O, adjusted to pH 7.0) and agitate for at least 10 minutes.
• PAH solution Poly(allylamine) (PAH, +): 2 mg/ml in HP H 2 O, adjusted to pH 7.0
• PAcr solution Poly(acrylic acid) (PAcr, -): 2 mg/ml in HP H O, adjusted to pH
• the PEM coated glass is incubated with a EDC/BLCPA solution for 30 minutes.
• the EDC/BLCPA solution is prepared by mixing equal amounts of 50 mM EDC solution (in MES buffer) and 50 mM BLCPA (in MES buffer) and diluting to 5mM in MES buffer.
• the glass is then rinsed with 10 mM Tris-NaCl and incubated with 0.1 mg/ml streptavidin solution for 1 hour. After washing with 10 mM Tris-NaCl, the glass is incubated with a solution containing the polynucleotide template (10 "7 M in Tris 100 mM MgCl 2 ) for 30 minutes. The glass is again rinsed thoroughly with 10 mM Tris-NaCl.
• the microfluidic substrate is bonded to the glass cover slip by HCl-assisted bonding.
• the chips are first washed with a surfactant (e.g., first with HP H 2 O, then in 0.1% Tween 20, then rinse again with HP H 2 O).
• the washed microfluidic chips are then put on the glass cover slips with a few microliters of dilute HC1 (e.g., 1% HC1 in HP H 2 O), followed by baking at 37° C for 1-2 hours.
• dilute HC1 e.g., 1% HC1 in HP H 2 O
• Such treatment enhances the bond strength to glass (e.g., >20 psi pressure) without increasing nonspecific adsorption.
• Coating the microchannel surface with the PEM technique is significant for analyzing polynucleotide sequences according to the present invention.
• the method used to attach the template to the surface should fulfill several requirements in order to be useful in a sequencing-by-synthesis application. First, it must be possible to attach reasonable quantities of polynucleotide templates. In addition, the attached templates should remain active for polymerase action. Further, nonspecific binding of fluorescent nucleotides should be very low.
• nucleotides When the nucleotides are fluorescently labeled, they generally have relatively strong nonspecific binding to many surfaces because they possess both a strongly polar
• a surface bearing positively-charged groups invariably has a very high nonspecific binding due to the attraction ofthe triphosphate group (which is strongly negatively charged) to the positively-charged amines.
• Neutral surfaces generally have strong nonspecific binding due to the action ofthe fluorescent nucleotide as a surfactant (i.e. assembling with nonpolar moiety towards the uncharged (more hydrophobic) surface and polar end in the aqueous phase).
• a surface bearing negative charges can repel the negatively charged fluorescent nucleotides, so it has the lowest nonspecific binding.
• a polyelectrolyte multilayer terminated with carboxyhc acid-bearing polymer fulfills all three criteria. First, it is easy to attach polynucleotide to because carboxyhc acids are good targets for covalent bond formation. Second, the attached template is active for extension by polymerases - most probably, the repulsion of like charges prevents the template from "laying down” on the surface. Finally, the negative charge repels the fluorescent nucleotides, and nonspecific binding is low.
• the attachment scheme described here is easy to generalize on. Without modification, the PEM/biotin streptavidin surface that is produced can be used to capture or immobilize any biotinylated molecule. A slight modification can be the use of another capture pair, i.e. substituting digoxygenin (dig) for biotin and labeling the molecule to be immobilized with anti-digoxygenin (anti-dig). Reagents for biotinylation or dig-labeling of amines are all commercially available. Another generalization is that the chemistry is nearly independent ofthe surface chemistry ofthe support. Glass, for instance, can support PEMs terminated with either positive or negative polymer, and a wide variety of chemistry for either.
• the templates can be attached to microbeads, which can be arranged within the microfluidic system. For instance, commercially-available latex microspheres with pre-defined surface chemistry can be used.
• the polynucleotide templates can be attached either before or after the microbeads are inducted into the microfluidic system. Attachment of template before beads are added allows a reduction in system complexity and setup time (as many templates can be attached to different aliquots of beads simultaneously).
• Attachment of template to beads in situ can allow easier manipulation of surface chemistry (as bead surface chemistry can be manipulated in bulk and externally to the microfluidic device). Beads should be held in place within the flow system for this technique to be effective. Methods to achieve this include, e.g., flowing the beads into orifices too small for them to flow through (where they would become “wedged in"), the creation of "microscreens” (i.e. barriers in the channel with apertures too small for beads to pass through), and insertion ofthe beads into hollows in the channels where they are affixed by simple Van der Waals forces.
• primer extension reaction Once templates are immobilized to the surfaces of synthesis channels, primer extension reactions are performed (E. D. Hyman, Anal. Biochem., 174, p. 423, 1988). If part ofthe template sequence is known, a specific primer can be constructed and hybridized to the template. Alternatively, a linker can be ligated to the template of unknown sequence in order to allow for hybridization of a primer. The primer can be hybridized to the template before or after immobilization ofthe template to the surface ofthe synthesis channel.
• the primer is extended by a nucleic acid polymerase in the presence of a single type of labeled nucleotide.
• Label is incorporated into the template/primer complex only if the labeled nucleotide added to the reaction is complementary to the nucleotide on the template adjacent the 3' end ofthe primer.
• the template is subsequently washed to remove any unincorporated label, and the presence of any incorporated label is determined.
• a radioactive label can be determined by counting or any other method known in the art, while fluorescent labels can be induced to fluoresce, e.g., by excitation.
• a combination of labeled and non-labeled nucleotides are used in the analysis. Because there are multiple copies of each template molecule immobilized on the surface ofthe synthesis channel, a small percentage of labeled nucleotides is sufficient for detection by a detection device (see below). For example, for fluorescently labeled nucleotides, the percentage of labeled nucleotide can be less than 20%, less than 10%, less than 5%, less than 1%, less than 0.1%, less than 0.01%, or even less than 0.001% ofthe total labeled and unlabeled nucleotides for each type ofthe nucleotides.
• At least one and usually all types ofthe deoxyribonucleotides (dATP, dTTP, dGTP, dCTP, dUTP/dTTP) or nucleotides (ATP, UTP, GTP, and CTP) are labeled.
• Various labels which are easily detected include radioactive labels, optically detectable labels, spectroscopic labels and the like.
• fluorescent labels are used.
• the different types of nucleotides can be labeled with the same kind of labels. Alternatively, a different kind of label can be used to label each different type of nucleotide.
• fluorescent labels are used, the fluorescent label can be selected from any of a number of different moieties.
• the preferred moiety is a fluorescent group for which detection is quite sensitive.
• fluorescein- or rhodamine-labeled nucleotide triphosphates are available (e.g., from NEN DuPont).
• Fluorescently labeled nucleotide triphosphates can also be made by various fluorescence-labeling techniques, e.g., as described in Kambara et al. (1988) "Optimization of Parameters in a DNA Sequenator Using Fluorescence Detection," Bio/Technol. 6:816-821; Smith et al. (1985) Nucl. Acids Res, 13:2399-2412; and Smith et al. (1986) Nature 321:674- 679. Fluorescent labels exhibiting particularly high coefficients of destruction can also be useful in destroying nonspecific background signals.
• a chain elongation inhibitor can be employed in the reaction (see, e.g., Dower et al., U.S. Patent No. 5,902,723.
• Chain elongation inhibitors are nucleotide analogues which either are chain terminators which prevent further addition by the polymerase of nucleotides to the 3' end ofthe chain by becoming incorporated into the chain themselves.
• the chain elongation inhibitors are dideoxynucleotides.
• chain elongation inhibitors are incorporated a y into the growing polynucleotide chain, they should be removed after incorporation ofthe labeled nucleotide has been detected, in order to allow the sequencing reaction to proceed using different labeled nucleotides.
• a blocking agent or blocking group can be employed on the 3' moiety ofthe deoxyribose group ofthe labeled nucleotide to prevent nonspecific incorporation.
• the blocking agent should be removable under mild conditions (e.g., photosensitive, weak acid labile, or weak base labile groups), thereby allowing for further elongation ofthe primer strand with a next synthetic cycle.
• the blocking agent also contains the fluorescent label, the dual blocking and labeling functions are achieved without the need for separate reactions for the separate moieties.
• the labeled nucleotide can be labeled by attachment of a fluorescent dye group to the 3' moiety ofthe deoxyribose group, and the label is removed by cleaving the fluorescent dye from the nucleotide to generate a 3' hydroxyl group.
• the fluorescent dye is preferably linked to the deoxyribose by a linker arm which is easily cleaved by chemical or enzymatic means.
• blocking agents include, among others, light sensitive groups such as 6-nitoveratryloxycarbonyl (NVOC), 2-nitobenzyloxycarbonyl (NBOC), . ⁇ ,. ⁇ - dimethyl-dimethoxybenzyloxycarbonyl (DDZ), 5-bromo-7-nitroindolinyl, o-hydroxy-2- methyl cinnamoyl, 2-oxymethylene anthraquinone, and t-butyl oxycarbonyl (TBOC).
• NVOC 6-nitoveratryloxycarbonyl
• NBOC 2-nitobenzyloxycarbonyl
• DDZ dimethyl-dimethoxybenzyloxycarbonyl
• TBOC t-butyl oxycarbonyl
• Other blocking reagents are discussed, e.g., in U.S. Ser. No
• RNA polymerase or DNA polymerases can be used in the primer extension.
• DNA polymerases are available.
• suitable DNA polymerases include, but are not limited to, Sequenase 2.0.RTM., T4 DNA polymerase or the Klenow fragment of DNA polymerase 1, or Vent polymerase.
• polymerases which lack 3' - 5' exonuclease activity can be used (e.g., T7 DNA polymerase (Amersham) or Klenow fragment of DNA polymerase I ⁇ T (New England Biolabs)).
• polymerases lacking 3' -» 5' exonuclease activity are not used.
• thermostable polymerases such as ThermoSequenaseTM (Amersham) or TaquenaseTM (ScienTech, St Louis, MO) are used.
• the nucleotides used in the methods should be compatible with the selected polymerase. Procedures for selecting suitable nucleotide and polymerase combinations can be adapted from Ruth et al. (1981) Molecular Pharmacology 20:415-422; Kutateladze, T., et al. (1984) Nuc.
• the polymerase can be stored in a separate reservoir in the apparatus and flowed into the synthesis channels prior to each extension reaction cycle.
• the enzyme can also be stored together with the other reaction agents (e.g., the nucleotide triphosphates).
• the polymerase can be immobilized onto the surface ofthe synthesis channel along with the polynucleotide template.
• the nucleotide on the template adjacent the 3' end ofthe primer can be identified. Once this has been achieved, the label should be removed before repeating the process to discover the identity ofthe next nucleotide. Removal ofthe label can be effected by removal ofthe labeled nucleotide using a 3 '-5' exonuclease and subsequent replacement with an unlabeled nucleotide. Alternatively, the labeling group can be removed from the nucleotide. In a further alternative, where the label is a fluorescent label, it is possible to neutralize the label by bleaching it with radiation.
• Photobleaching can be performed according to methods, e.g., as described in Jacobson et al., "International Workshop on the Application of Fluorescence Photobleaching Techniques to Problems in Cell Biology", Federation Proceedings, 42:72-79, 1973; Okabe et al., J Cell Biol 120:1177-86, 1993; and Close et al., Radiat Res 53:349-57, 1973.
• chain terminators or 3' blocking groups should be removed before the next cycle can take place.
• 3' blocking groups can be removed by chemical or enzymatic cleavage ofthe blocking group from the nucleotide. For example, chain terminators are removed with a 3'-5' exonuclease, e.g., exonuclease III. Once the label and terminators/blocking groups have been removed, the cycle is repeated to discover the identity ofthe next nucleotide.
• Removal ofthe blocking groups can be unnecessary if the labels are removable.
• the chains incorporating the blocked nucleotides are permanently terminated and no longer participate in the elongation processes. So long as these blocked nucleotides are also removed from the labeling process, a small percentage of permanent loss in each cycle can also be tolerated.
• nucleotide incorporation is monitored by detection of pyrophosphate release (see, e.g., WO98/13523, WO98/28440, and Ronaghi et al., Science 281:363, 1998).
• a pyrophosphate-detection enzyme cascade is included in the reaction mixture in order to produce a chemoluminescent signal.
• nucleotide analogues are used which are capable of acting as substrates for the polymerase but incapable of acting as substrates for the pyrophosphate-detection enzyme. Pyrophosphate is released upon incorporation of a deoxynucleotide or dideoxynucleotide, which can be detected enzymatically. This method employs no wash steps, instead relying on continual addition of reagents.
• Methods for visualizing single molecules of DNA labeled with an intercalating dye include, e.g., fluorescence microscopy as described in Houseal et al., BiophysicalJournal
• the detection system for the signal or label can also depend upon the label used, which can be defined by the chemistry available.
• a combination of an optical fiber or charged couple device (CCD) can be used in the detection step.
• CCD charged couple device
• the matrix is itself transparent to the radiation used, it is possible to have an incident light beam pass through the substrate with the detector located opposite the substrate from the polynucleotides.
• CCD charged couple device
• various forms of spectroscopy systems can be used.
• Various physical orientations for the detection system are available and discussion of important design parameters is provided, e.g., in Jovin, Adv. in Biochem. Bioplyms.
• Incorporated signals can be detected by scanning the synthesis channels.
• the synthesis channels can be scanned simultaneously or serially, depending on the scanning method used.
• the signals can be scanned using a CCD camera (TE/CCD512SF, Princeton Instruments, Trenton, N.J.) with suitable optics (Ploem, J. S., in Fluorescent and Luminescent Probes for Biological Activity, Mason, T. W., Ed., Academic Press, London, pp. 1-11, 1993), such as described in Yershov et al. (Proc. Natl. Acad. Sci. 93:4913, 1996), or can be imaged by TV monitoring (Khrapko et al., DNA Sequencing 1:375, 1991).
• a phosphorimager device can be used (Johnston et al., Johnston, R. F., et al., Electrophoresis 11:355, 1990; and Drmanac et al., Drmanac, R., et al., Electrophoresis
• the synthesis channels can be serially scanned one by one or row by row using a fluorescence microscope apparatus, such as described in U.S. Patent Nos. 6,094,274, 5,902,723, 5,424,186, and 5,091,652.
• a fluorescence microscope apparatus such as described in U.S. Patent Nos. 6,094,274, 5,902,723, 5,424,186, and 5,091,652.
• standard low- light level cameras such as a SIT and image intensified CCD camera, are employed (see, Funatsu et al., Nature 374, 555, 1995).
• An ICCD can be preferable to a cooled CCD camera because of its better time resolution.
• These devices are commercially available (e.g., from Hammamatsu).
• only the intensifier unit from Hammamatsu or DEP are used and incorporated into other less expensive or home built cameras.
• the intensifier can be cooled.
• CCD camera can be purchased from Phillips, who offer a low priced, low noise (40 electron readout noise per pixel) model.
• a home built camera allows greater flexibility in the choice of components and a higher performance device.
• the advantage of using a camera instead of an avalanche photodiode is that one can image the whole field of view.
• This extra spatial information allows the development of new noise reduction techniques. For example, one can use the fact that signals are expected from certain spatial locations (i.e. where the polynucleotide template is attached) in order to reject noise.
• fluorescent excitation is exerted with a Q-switched frequency doubled Nd YAG laser, which has a KHz repetition rate, allowing many samples to be taken per second.
• a wavelength of 532 nm is ideal for the excitation of rhodamine. It is a standard device that has been used in the single molecule detection scheme (Smith et al., Science 253 : 1122, 1992).
• a pulsed laser allows time resolved experiments, which are useful for rejecting extraneous noise.
• excitation can be performed with a mercury lamp and signals from the incorporated nucleotides can be detected with an inexpensive CCD camera (see, e.g., Unger et al., Biotechniques 27:1008, 1999.
• the scanning system should be able to reproducibly scan the synthesis channels in the apparatuses. Where appropriate, e.g., for a two dimensional substrate where the synthesis channels are localized to positions thereon, the scanning system should positionally define the synthesis channels attached thereon to a reproducible coordinate system. It is important that the positional identification of synthesis channels be repeatable in successive scan steps.
• Various scanning systems can be employed in the apparatuses ofthe present invention. For example, electrooptical scanning devices described in, e.g., U.S. Pat. No. 5,143,854, are suitable for use with the apparatuses ofthe present invention. The system could exhibit many ofthe features of photographic scanners, digitizers or even compact disk reading devices. For example, a model no.
• PM500-A1 x-y translation table manufactured by Newport Corporation can be attached to a detector unit.
• the x-y translation table is connected to and controlled by an appropriately programmed digital computer such as an IBM PC/AT or AT compatible computer.
• the detection system can be a model no. R943-02 photomultiplier tube manufactured by Hamamatsu, attached to a preamplifier, e.g., a model no. SR440 manufactured by Stanford Research Systems, and to a photon counter, e.g., an SR430 manufactured by Stanford Research System, or a multichannel detection device.
• the stability and reproducibility ofthe positional localization in scanning determine, to a large extent, the resolution for separating closely positioned polynucleotide clusters on a 2 dimensional substrate. Since the successive monitoring at a given position depends upon the ability to map the results of a reaction cycle to its effect on a positionally mapped cluster of polynucleotides, high resolution scanning is preferred. As the resolution increases, the upper limit to the number of possible polynucleotides which can be sequenced on a single matrix also increases. Crude scanning systems can resolve only on the order of
• refined scanning systems can resolve on the order of 100 ⁇ m, more refined systems can resolve on the order of about 10 ⁇ m, and with optical magnification systems a resolution on the order of 1.0 ⁇ m is available.
• the limitations on the resolution can be diffraction limited and advantages can arise from using shorter wavelength radiation for fluorescent scanning steps.
• the time required to fully scan a matrix can increased and a compromise between speed and resolution can be selected.
• Parallel detection devices which provide high resolution with shorter scan times are applicable where multiple detectors are moved in parallel.
• the reliability of a signal can be pre-selected by counting photons and continuing to count for a longer period at positions where intensity of signal is lower. Although this decreases scan speed, it can increase reliability ofthe signal determination.
• Various signal detection and processing algorithms can be incorporated into the detection system. In some methods, the distribution of signal intensities of pixels across the region of signal are evaluated to determine whether the distribution of intensities corresponds to a time positive signal.
• nucleotide incorporation is also contemplated in the present invention, including the use of mass spectrometry to analyze the reaction products, the use of radiolabeled nucleotides, and detection of reaction products with "wired enzymes".
• mass spectrometry is employed to detect nucleotide incorporation in the primer extension reaction.
• a primer extension reaction consumes a nucleotide triphosphate, adds a single base to the primer/template duplex, and produces pyrophosphate as a by-product.
• Mass spectrometry can be used to detect pyrophosphate in the wash stream after a nucleotide has been incubated with the template and polymerase.
• radiolabeled nucleotides are used. Nucleotides can be radiolabeled either in the sugar, the base, or the triphosphate group. To detect radioactivity, small radioactivity sensor can be incorporated in the substrate on which the microfluidic chip is mounted. A CCD pixel, for instance, serves as a good detector for some radioactive decay processes. Radiolabeling ofthe sugar or base produces an additive signal: each incorporation increases the amount of radiolabel in the primer-template duplex. If the nucleotide is labeled in the portion that is released as pyrophosphate (e.g. dNTP with ⁇ - or ⁇ - 3 P), the radioactive pyrophosphate can be detected in the wash stream.
• pyrophosphate e.g. dNTP with ⁇ - or ⁇ - 3 P
• This radioactivity level is not additive, but rather binary for each attempted nucleotide addition, so subsequent addition poses no read length limit. Due to the small reagent consumption and contained nature of microfluidics, the total radioactivity used in such a system is relatively minimal, and containment is relatively simple. In some methods, non-optical detection of pyrophosphate release makes use of
• enzymes are covalently linked to a hydrogel matrix containing redox active groups capable of transporting charge.
• the analyte to be detected is either acted on directly by a redox enzyme (either releasing or consuming electrons) or consumed as a reagent in an enzymatic cascade that produces a substrate that is reduced or oxidized by a redox enzyme.
• the production or consumption of electrons is detected at a metal electrode in contact with the hydrogel.
• pyrophosphatase For the detection of pyrophosphate, an enzymatic cascade using pyrophosphatase, maltose phosphorylase, and glucose oxidase can be employed. Pyrophosphatase converts pyrophosphate into phosphate; maltose phosphorylase converts maltose (in the presence of phosphate) to glucose 1 -phosphate and glucose. Then, glucose oxidase converts the glucose to gluconolactone and H2O2; this final reaction is the redox step which gives rise to a detectable current at the electrode. Glucose sensors based on this principle are well known in the art, and enzymatic cascades as described here have been demonstrated previously. Other enzymatic cascades besides the specific example given here are also contemplated the present invention. This type of detection scheme allows direct electrical readout of nucleotide incorporation at each reaction chamber, allowing easy parallelization.
• polynucleotide sequences are analyzed with a fluorescent photobleaching method.
• fluorescently labeled nucleotides are used in the primer extension. Signals from the incorporated nucleotides are removed by photobleaching before next extension cycle starts.
• the polynucleotide templates can be prepared as described above (e.g., cloning in single-stranded Ml 3 plasmid). Biotinylated templates are attached to surface of the synthesis channel that has been pretreated with the PEM technique as discussed above. After the primed, single stranded DNA is immobilized to the synthesis channel in the flow cell.
• a polymerase and one nucleotide triphosphate e.g.
• dATP are flowed into the flow cell.
• a high fidelity polymerase with no exonuclease proofreading ability is preferred.
• only a fraction (e.g., less than 10%, 5%, 1%, 0.1%, 0.01%, or 0.001%) of each type ofthe nucleotide triphosphates is fluorescently labeled (e.g., rhodamine-labeled nucleotide triphosphates from NEN DuPont).
• fluorescently labeled e.g., rhodamine-labeled nucleotide triphosphates from NEN DuPont.
• the reagents are then flowed out ofthe flow cell, and the fluorescence ofthe DNA is measured. If no fluorescence is detected, the procedure is repeated with one ofthe other nucleotide triphosphates. If fluorescence is detected, the identity ofthe first base in the sequence has been determined. The fluorescence signal is photobleached and extinguished before the procedure is then repeated for the next base in the template sequence.
• the fluorescence can be excited with, e.g., a Q-switched frequency doubled Nd YAG laser (Smith et al., Science 253: 1122, 1992).
• a Q-switched frequency doubled Nd YAG laser Smith et al., Science 253: 1122, 1992.
• This is a standard device used in the single molecule detection scheme that measures the fluorescent spectrum and lifetime of a single molecule before it photobleached. It has a kHz repetition rate, allowing many samples to be taken per second.
• the wavelength can be. e.g., 532 nm that is ideal for the excitation of rhodamine.
• a pulsed laser allows time resolved experiments and is useful for rejecting extraneous noise.
• Detection ofthe incorporated label can be performed with a standard low-light level cameras, such as a SIT or a image intensified CCD camera (Funatsu et al, supra).
• An Intensified CCD (ICCD) camera is preferable to a cooled CCD camera because of its better time resolution.
• ICCD Intensified CCD
• These devices are available from, e.g., Hammamatsu.
• a detection device can be made by building just the intensifier unit from Hammamatsu into a CCD camera.
• the intensifier can be cooled.
• the CCD camera is available from Phillips, e.g., a low priced, low noise model (40 electron readout noise per pixel).
• a customarily built camera allows greater flexibility in the choice of components and a higher performance device.
• the advantage of using a camera instead of an avalanche photodiode is that the whole field of view can be imaged.
• This extra spatial information allows the development of new noise reduction techniques. For example, the fact that signals are expected from certain spatial locations (i.e. where the DNA is attached) can be used to reject noise.
• a combination of factors affect the read length and throughput ofthe sequencing analysis according to the present invention.
• the present invention teaches how to determine acceptable flow rate of fluids in the apparatuses.
• flow rate in the apparatuses with microfabricated flow channels having a depth of 100 ⁇ m is typically 0.1-1 cm/sec.
• the flow rate is usually in the range of 1-10 cm/sec.
• the limiting velocity is in the order of 1 cm/sec for a lOO ⁇ m channel depth. For microchannels with a depth of 10 ⁇ m, the limit is 10 cm/sec.
• the ultimate limit on the rate at which fluid can be exchanged is determined by the effect of drag and shear flows on the polynucleotide template and the polymerase.
• the velocity profile of constrained flow is parabolic (v( ⁇ )-v ave (l-( ⁇ /R) )), causing a shear force.
• RNA polymerase Another consideration is to prevent the polymerase from falling off the template or becoming damaged.
• RNA polymerase it has been shown that the stalling force for RNA polymerase, at which it might receive irreversible damage, is 14 pN (Yin et al., Science 270:1653, 1995). Since one the drag coefficient of a DNA polymerase can be estimated from its size, a similar calculation as for the DNA shear leads to a maximum velocity of 500 cm/sec.
• the time to remove all ofthe free nucleotides can be calculated by including the effects of diffusion into hydrodynamic calculation ofthe fluid flow.
• a six port valve from Upchurch with electric motor from Thar Designs has a dead volume of 2 ⁇ l and switching time of 166 msec.
• 4 ⁇ l of material should be exchanged for each step in the process.
• a syringe or peristaltic pump can give very high flow rates, the limiting factor is low Reynolds number.
• the dead volume is reduced and throughput increased.
• the valves can provide an essentially zero dead volume and 10 msec switching time. This and the reduced dimensions ofthe device leads to a drastic increase of throughput: the time to flush the reagents (e.g., nucleotides) from the system is reduced to 0.8 sec.
• the overall throughput is approximately 1 base per second. Table 1 summarizes the various factors affecting throughput of apparatuses with microfabricated flow channels having a depth of 100 ⁇ m or 10 ⁇ m.
• the limiting factor is the fluid velocity that causes turbulent flow.
• shear forces on the DNA also becoming limiting.
• the reagent exchange time is expected to improve by a factor of 100 in apparatuses II.
• the DNA polymerases can fall off of the DNA. If enzyme is replenished, it takes time for the enzyme to find and bind to a free DNA site. This could affect throughput ofthe apparatuses.
• the attrition rate ofthe polymerase can be determined according to methods described in the art. For example, using the kinetics ofthe T4 DNA polymerase as nominal values (Taylor et al., J. Phys. D. Appl. Phys. 24:1443, 1991), an on-rate of 11 ⁇ M "1 sec "1 was obtained. Hence a 1 ⁇ M concentration of enzyme gives an on rate of 11 sec "1 , and after 1 second, 99.3% ofthe DNA have polymerase bound.
• the polymerase falls off of the DNA with a time constant of 0.2 sec "1 (Yin et al., Science 270:1653, 1995). In other words, after 5 seconds without nucleotides, this can become a source of attrition. It can be compensated for by the addition of fresh polymerase with every sequencing cycle ofthe device.
• the reagent exchange is fast enough that polymerase falling off has no significant effect on the throughput.
• the rate of incorporation of nucleotides by the polymerase is typically about 300 bases per second. This is not a rate limiting factor for the device throughput.
• Read length ofthe sequencing analysis can be affected by various factors. However, photobleaching is unlikely to cause any chemical changes to the polynucleotide template that prevent the attachment ofthe next base. During the photobleaching, the dye molecule is held off from the DNA on a linker arm, and it gives off so few photons that the interaction cross section is negligible. Any attrition ofthe labeled nucleotides also does not present any significant problem. The statistics ofthe photobleaching scheme are robust enough to allow sequencing to continue in spite of any attrition ofthe labeled nucleotides. For example, if 0.1% ofthe bases are labeled, then after 3000 bases the attrition is 95% if incorporation of a labeled nucleotide terminates strand extension completely.
• Misincorporation efficiencies have been measured to be three to five orders of magnitude below the efficiency for proper nucleotide incorporation (Echols et al., Ann. Rev. Biochem 60:477, 1991). Misincorporation can be minimized by only exposing the DNA polymerase- DNA complexes to nucleotides for as much time as is needed to incorporate the proper nucleotide. For a high fidelity DNA polymerase, misincorporation happens with a frequency of about 10 "4 . If dephasing due to misincorporation is treated as total attrition, the attrition is only 25% after 3 kb, i.e, the signal is reduced to 75% of its original. Thus, misincorporation does not hinder a 3 kb or perhaps longer read length.

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