Patent Application
20070068573
The section headings used herein are solely for organization purposes and are not to be construed as limiting the subject matter described in any way.
Large scale sequencing projects can involve cloning DNA fragments in bacteria, picking and amplifying those fragments, and performing individual sequencing reactions on each clone. Standard sequencing reactions can often be performed in 5 μl to 20 μl reaction volumes, even though only a small fraction of the sequencing product can be analyzed. Such cloning and sequencing protocols can be time consuming and can use relatively large sample and reagent volumes. The relatively large volumes can be wasteful in terms of expensive consumable reagents.
Various embodiments of the present teachings relate to systems, apparatus, and/or methods for sample preparation that can be used for biochemical or molecular biology procedures involving different volumes, for example, small volumes such as micro-liter sized volumes or smaller.
According to the present teachings, the system can comprise an apparatus for generating discrete volumes of at least a first fluid in contact with a second fluid, wherein the first and second fluids are immiscible with each other, for example, discrete volumes of an aqueous liquid (herein “aqueous immiscible-fluid-discrete-volumes”), spaced-apart from one another by a spacing fluid that is immiscible with the immiscible-fluid-discrete-volumes. An immiscible-fluid-discrete-volume can be a partitioned segment in which molecular biology procedures can be performed. As used herein, an immiscible-fluid-discrete-volume can be one of many structures, three of which are: a fluid segment, a slug, and an emulsified droplet. In some embodiments, immiscible-fluid-discrete-conduits are formed and/or processed in a conduit.
This paragraph defines a conduit as it is used herein. A conduit can be any device in which an immiscible-fluid-discrete-volume can be generated, conveyed, and/or flowed. For example, a conduit as defined herein can comprise any of a duct, a tube, a pipe, a channel, a capillary, a hole or another passageway in a solid structure, or a combination of two or more of these, as long as the spaces defined by the respective solid structures are in fluid communication with one another. A conduit can comprise two or more tubes or other passageways connected together, or an entire system of different passageways connected together. An exemplary conduit can comprise an immiscible-fluid-discrete-volume-forming tube, thermal spirals, valve passageways, a processing conduit, junctions, and the like components all connected together to form one or more fluid communications therethrough, which system is also referred to herein as a main processing conduit. Examples of solid structures with holes or passageways therein that can function as conduits are manifolds, T-junctions, Y-junctions, rotary valves, and other valves. Thus, when connected to conduits, such structures can be considered part of a conduit as defined herein.
This paragraph defines a fluid segment, as it is used herein. A fluid segment is a discrete volume that has significant contact with one or more conduit wall(s), such that a cross-sectional area of the fluid segment is the same size and shape as the cross-sectional area of the conduit it contacts. At least a portion of a fluid segment fully fills the cross-sectional area of the conduit, such that the immiscible fluid adjacent it in the conduit can not flow past the fluid segment. The entire longitudinal length of the fluid segment may not contact the conduit walls.
This paragraph defines a slug as used herein. A slug is a discrete volume that has at least a portion of which has approximately the same cross-sectional shape as the conduit in which it exists, but a smaller size. The smaller size is due to the insignificant contact, if any, of the slug with the conduit wall(s). A slug can have a cross-sectional dimension between approximately 0.5 and approximately 1.0 times the maximum dimension of a cross sectional area of the conduit. If the conduit has a circular cross section, the cross-sectional area of a slug can be concentric with the conduit's cross-sectional area, but it does not have to be, such as, for example, when the conduit is horizontal and, due to different specific gravities, one fluid rises toward the top of the cross-sectional area of the conduit under the influence of gravity. A slug can be free of contact with the conduit walls. When not moving relative to the conduit, a slug can have “feet” that appear as nibs or bumps along an otherwise smoothly appearing round surface. It is theorized that the feet at the bottom of the slug may have contact with the conduit wall. In contrast to a fluid segment, the contact a slug can have with the conduit wall(s) still permits the immiscible fluid adjacent it in the conduit to flow past the slug.
The “slugs” formed by the teachings herein, separated by spacing fluid, can merge together to form larger slugs of liquid, if contacted together. The ability of the slugs, for example, aqueous slugs, described and taught herein, to merge together with each other, facilitates the downstream addition of aqueous reagents to the slugs.
This paragraph defines an emulsified droplet, as used herein. An emulsified droplet is a discrete volume that has no contact with the walls of the conduit. The size of an emulsified droplet is not necessarily constrained by the conduit, and examples of emulsified droplets described in the prior art range in size from about 1 femtoliter to about 1 nanoliter. The shape of an emulsified droplet is not constrained by the conduit, and due to the difference in surface-energies between it and the continuous phase liquid in which it is dispersed, it is generally spherical. It can have a maximum dimension that is not equal to, nor approximately equal to, but much less than the maximum dimension of the cross-sectional area of the conduit, for example, 20%, 10%, 5% or less. An emulsified droplet will not merge upon contact with another emulsified droplet to form a single, larger discrete volume, without external control. Put another way, an emulsified droplet is a stable discontinuous phase in a continuous phase.
A conduit can contain more than one emulsified droplet, but not more than one slug or fluid segment, at any cross-sectional location. Thus, a first emulsified droplet does not necessarily impede the movement of a second emulsified droplet past it, where as a fluid segment and a slug necessarily do not permit the passage of another fluid segment or slug past them, respectively. If two fluid segments are separated by a fluid with which the first and second of the two fluids is each immiscible, then the immiscible fluid also forms a discrete volume. It is likely that it has significant contact with the conduit walls and thus is another fluid segment.
Whether two immiscible fluids, when present in a conduit, form fluid segments of the first and second of the two immiscible fluids, slugs of the first immiscible fluid, or emulsified droplets of the first immiscible fluid depends on at least the method of introduction of each fluid into the conduit, the relative surface energies of the first immiscible fluid, the second immiscible fluid, and the conduit material, the contact angle each forms with the other two materials, respectively, and the volume of the discrete volume of the first immiscible fluid. Thus, it is recognized that these definitions are merely reference points on a continuum, the continuum of the shape and size of discrete volumes of a first immiscible fluid in a conduit, and discrete volumes will exist that, when described, fall between these definitions.
The molecular biology procedures can, for example, utilize proteins or nucleic acids. Procedures with nucleic acids can comprise, for example, a PCR amplification and/or nucleic acid analysis of an amplification product. The PCR amplification and/or nucleic acid analysis of an amplification product can comprise an integrated DNA amplification/DNA sequencing method.
Using the apparatus, methods, and/or systems provided in this application, a polymerase chain reaction (PCR) amplification of single DNA molecules can be performed, for example, to obtain amplicons. The amplified DNA or amplicons can then be used in a sequencing reaction and then be sequenced in small volumes. Other manipulations of nucleic acids or proteins can also be accomplished, for example, DNA hybridization reactions or antibody-antigen binding assays.
The apparatus, system and/or methods described herein can also be used in conjunction with U.S. Provisional Patent Application No. 60/710,167 entitled “Sample Preparation for Sequencing” to Lee et al., filed Aug. 22, 2005 (Attorney Docket No. 5841P), U.S. Provisional Patent Application No. 60/731,133 entitled “Method and System for Spot Loading a Sample” to Schroeder et al., filed Oct. 28, 2005 (Attorney Docket No. 5010-288), and systems described in U.S. Provisional Patent Application No. 60/818,197 filed June 30, 2006, each of which are incorporated herein in their entireties by reference.
An exemplary type of sample preparation can be used for genotyping, gene-expression, methylation analysis, and/or directed medical sequencing (VariantSEQr™, for example, an Applied Biosystems product comprising primers for resequencing genes and detecting variations) that requires multiple liquids to be brought together in an aqueous discrete volume. For example, in a gene-expression application, each aqueous discrete volume can contain individual primer sets. The sample to be analyzed, for example, complementary DNA (cDNA), can be added to each aqueous discrete volume. In the VariantSEQrm application, for example, an aqueous discrete volume can comprise a primer set and genomic DNA can be added to that discrete volume. According to various embodiments, a system and method are provided that are able to generate discrete volumes with unique content. According to various embodiments of the present teachings, sipping, other aspirating, or other techniques to generate immiscible-liquid, discrete volumes can be used. According to various embodiments, an immiscible-liquid, discrete volume of at least an aqueous sample fluid can be generated in a tube by alternately drawing into the tube the aqueous sample fluid and spacing fluid, with which the aqueous sample fluid is immiscible, from a single container or well containing both fluids or from different containers or wells each containing one of the two fluids.
Using the apparatus, methods, and/or systems provided in this application, one can control the positioning of immiscible-fluid, discrete volumes during processing. These processes can include, for example, polymerase chain reaction (PCR) amplification of single DNA molecules to obtain, for example, amplicons. The amplified DNA or amplicons can then be used in a sequencing reaction and be sequenced using small volumes. Other manipulations of nucleic acids or proteins can also be accomplished, for example, DNA hybridization reactions or antibody-antigen binding assays.
In some embodiments, flow rates for moving aqueous discrete volumes can comprise rates of from about 1 picoliter/sec. to about 200 microliters/sec., and can be selected based on the inner diameter of the conduits through which the liquids are to be pumped. Within that broad range, in some embodiments, the aqueous discrete volumes comprising 50% reagents, for example, PCR reagents, and 50% other reagents, for example, sample fluid, in contact with oil can flow at 0.5 microliter/sec, or in a range from about 0.1 microliter/sec. to 2 microliters/sec. In some embodiments, aqueous volumes comprising reagents, for example, exo SAP or sequencing mix, can flow at a rate of about 1/3 microliters/sec., or in a range from about 0.1 microliters/sec. to 1 microliters/sec. In some embodiments, when aspirating aqueous fluid into a conduit as discrete volumes, flow rates of up to about 0.1 microliters/sec. max can be used. In some embodiments, when outputting an aqueous discrete volume, a flow rate of about 1/3 to about 1/2 ul/sec can be used, or in a range from about 0.1 microliters/sec. to about 2 microliters/sec. can be used.
Tubing that can be used with the 1 picoliter/sec. to 200 microliter/sec. flow rate can comprise an inner diameter of from about 250 microns to about 1000 microns. In other embodiments, the inner diameter of the inner tube can be from about 10 microns to about 2000 microns, while the inner diameter of the outer tube can be from about 20 microns to about 5000 microns, for example, from about 35 microns to about 500 microns. Other diameters, however, can be used based on the characteristics of the slug processing system desired. In some embodiments, a tube having a 10 micron inner diameter is used with a flow rate of from about 8 to about 10 picoliters/second. In some embodiments, a tube having a 5000 micron inner diameter is used with a flow rate of from about 25 to about 200 microliters/second. In some embodiments, a tube having a 500 micron inner diameter is used with a flow rate of from about 0.25 to about 2.0 microliters/second.
The aqueous sample liquid, which in some embodiments can form discrete volumes in contact with a fluid with which it is immiscible, can comprise a plurality of target nucleic acid sequences, wherein at least one of the discrete volumes comprises at least one target nucleic acid sequence. In some embodiments, after formation, at least 37% of the plurality of the discrete volumes in the inner conduit can each comprise a single target nucleic acid sequence. In various other embodiments, less than about 37% of the plurality of discrete volumes in the conduit can each comprise a single target nucleic acid sequence. In other embodiments, at least 1% or more, 5% or more, 10% or more, or 20% or more can have a single target nucleic acid sequence, for example, upon formation of the discrete volumes.
According to various embodiments, each of the plurality of discrete volumes in the inner conduit can comprise one or more respective oligonucleotide primers. Oligonucleotide primers can be chosen as determined by one of skill in the art to accomplish the desired objective. For example, universal primers can be used.
In some embodiments, further downstream processing of the prepared immiscible-fluid-discrete-volumes can be integrated with the system, of which embodiments are described herein. Such downstream processing can include amplifying the at least one target nucleic acid sequence in the first discrete volume in the conduit to form an amplicon, and thereafter subjecting the amplicon to a nucleic acid sequencing reaction. For such purposes, and in some embodiments, the discrete volumes or immiscible-fluid-discrete-volumes can comprise reaction components, for example, oligonucleotide primers. Various embodiments of downstream processing can include universal PCR, or can comprise up-front multiplexed PCR followed by decoding, for example, see WO 2004/051218 to Andersen et al., U.S. Pat. No. 6,605,451 to Marmaro et al., U.S. patent application Ser. No. 11/090,830 to Andersen et al., and U.S. patent application Ser. No. 11/090,468 to Lao et al., all of which are incorporated herein in their entireties by reference. Details of real time PCR can be found in Higuchi et al., U.S. Pat. No. 6,814,934 B1, which is incorporated herein by reference in its entirety.
Further devices, systems, and methods that can be used with or otherwise implement the present teachings include those described, for example, in U.S. patent application Ser. No. ______, filed Aug. 22, 2006, entitled “Apparatus, System, and Method Using Immiscible-Fluid-Discrete-Volumes,” to Lee et al. (attorney docket number 5010-362), in U.S. patent application Ser. No. ______, entitled “Device and Method for Making Discrete Volumes of a First Fluid in Contact With a Second Fluid, Which are Immiscible With Each Other,” to Cox et al. (attorney docket number 5010-363), and in U.S. patent application Ser. No. ______, filed Aug. 22, 2006, entitled “Apparatus and Method for Depositing Processed Immiscible-Fluid-Discrete-Volumes,” to Schroeder et al. (attorney docket number 5010-365), which are herein incorporated in their entireties by reference.
The skilled artisan will understand that the drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way. In the drawings:
It is to be understood that the following descriptions are exemplary and explanatory only. The accompanying drawings are incorporated in and constitute a part of this application and illustrate several exemplary embodiments with the description. Reference will now be made to various embodiments, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers are used in the drawings and the description to refer to the same or like parts.
Throughout the application, descriptions of various embodiments use “comprising” language, however, it will be understood by one of skill in the art, that in some specific instances, an embodiment can alternatively be described using the language “consisting essentially of” or “consisting of.”
For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, it will be clear to one of skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms “a,” “an” and “at least one” are used interchangeably in this application.
Unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. In some instances, “about” can be understood to mean a given value+5%. Therefore, for example, about 100 nl, could mean 95-105 nl. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Reference to “nucleotide” should be understood to mean a phosphate ester of a nucleotide, as a monomer unit or within a nucleic acid. Nucleotides are sometimes denoted as “NTP”, or “dNTP” and “ddNTP” to particularly point out the structural features of the ribose sugar. “Nucleotide 5′-triphosphate” can refer to a nucleotide with a triphosphate ester group at the 5′ position. The triphosphate ester group may include sulfur substitutions for the various oxygens, for example, α-thio-nucleotide 5′-triphosphates. Nucleotides can comprise a moiety of substitutes, for example, see, U.S. Pat. No. 6,525,183 B2 to Vinayak et al., incorporated herein by reference in its entirety.
The terms “polynucleotide” or “oligonucleotide” or “nucleic acid” can be used interchangeably and include single-stranded or double-stranded polymers of nucleotide monomers, including 2′-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by internucleotide phosphodiester bond linkages, or internucleotide analogs, and associated counter ions, for example, H+, NH4+, trialkylammonium, Mg2+, Na+ and the like. A polynucleotide may be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or chimeric mixtures thereof. Polynucleotides may be comprised of nucleobase and sugar analogs. Polynucleotides typically range in size from a few monomeric units, for example, 5-40 when they are frequently referred to in the art as oligonucleotides, to several thousands of monomeric nucleotide units. Unless denoted otherwise, whenever a polynucleotide sequence is represented, it will be understood that the nucleotides are in 5′ to 3′ order from left to right and that “A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotes thymidine, unless otherwise noted. A labeled polynucleotide can comprise modification at the 5′terminus, 3′terminus, a nucleobase, an internucleotide linkage, a sugar, amino, sulfide, hydroxyl, or carboxyl. See, for example, U.S. Pat. No. 6,316,610 B2 to Lee et al. which is incorporated herein by reference. Similarly, other modifications can be made at the indicated sites as deemed appropriate.
The term “reagent,” should be understood to mean any reaction component that in any way affects how a desired reaction can proceed or be analyzed. The reagent can comprise a reactive or non-reactive component. It is not necessary for the reagent to participate in the reaction. The reagent can be a recoverable component comprising for example, a solvent and/or a catalyst. The reagent can comprise a promoter, accelerant, or retardant that is not necessary for a reaction but affects the reaction, for example, affects the rate of the reaction. A reagent can comprise, for example, one member of a binding pair, a buffer, or a DNA that hybridizes to another DNA. The term “reagent” is used synonymous with the term “reaction component.”
Methods, apparatuses and systems described herein can use fluids immiscible in each other. Fluids can be said to be immiscible in each other when they can be maintained as separate fluid phases under conditions being used. Immiscible fluids can also be said to be incapable of mixing with each other or attaining a solution with each other. An aqueous liquid and a non-aqueous liquid such as oil can be said to be immiscible with each other. Throughout the specification, reference is made to aqueous slugs. This is merely exemplary and does not necessarily preclude the use or manufacture of non-aqueous liquid slugs in combination with an immiscible liquid.
While oil and aqueous liquids are immiscible in each other, such a combination does not necessarily form aqueous immiscible-fluid-discrete-volumes in the oil when the two liquids are mixed or placed together. For example, oil may form the disperse phase in a continuous aqueous liquid in a larger volume, as it does in certain salad dressings. For another example, oil and aqueous liquids may merely form aqueous droplets or microdroplets in a larger volume of oil, but not necessarily aqueous immiscible-fluid-discrete-volumes. Aqueous immiscible-fluid-discrete-volumes can form, however, using an apparatus such as, for example, those described in U.S. patent application Ser. No. ______, entitled “Device and Method for Making Immiscible-Fluid-Discrete-Volumes,” to Cox et al. (attorney docket number 5010-363).
Aqueous solutions and oil from separate sources can be combined to form a continuous flowing liquid stream comprising aqueous immiscible-fluid-discrete-volumes separated from one another by the oil. Because the aqueous immiscible-fluid-discrete-volumes entirely or almost entirely fill the cross-sectional area of the conduit or tube in which they are formed, the resulting stream of aqueous immiscible-fluid-discrete-volumes in oil can exhibit a banded appearance. According to various embodiments, such a pattern can be exhibited by combining any two immiscible fluids with one another. The pattern can be formed throughout the length of the conduit. In various embodiments, a first aqueous immiscible-fluid-discrete-volume can contain different reagents than a second aqueous immiscible-fluid-discrete-volume. In other words, not all aqueous immiscible-fluid-discrete-volumes throughout the conduit need to contain the same reagents.
An aqueous immiscible-fluid-discrete-volume can be spaced apart from an adjacent aqueous immiscible-fluid-discrete-volume by the oil. In various embodiments, liquids other than oil can act as a spacing fluid, provided that the spacing fluid and aqueous fluid are immiscible with respect to each other and provided that they can form individual aqueous immiscible-fluid-discrete-volumes spaced apart from one another by the spacing fluid. In various embodiments, gas can be used as a spacing fluid.
According to various embodiments, methods are provided that refer to processes or actions involved in sample preparation and analysis. It will be understood that in various embodiments a method can be performed in the order of processes as presented, however, in related embodiments, the order can be altered as deemed appropriate by one of skill in the art in order to accomplish a desired objective.
According to various embodiments, an apparatus is provided that can be used as a front-end sample preparation device for high-throughput sequencing, or other applications requiring preparation and/or processing of a plurality of small samples. The sample liquid that can become an immiscible-fluid-discrete-volume can comprise, for example, nucleic acids, proteins, polypeptides, carbohydrates, or the like. The apparatus can be part of an integrated system and/or be adapted to function with other pieces of equipment adapted for further sample processing of samples, for example, an ABI 310, ABI 3130, ABI 3130x1, ABI 3700, ABI 3730, or ABI 3730x1 capillary electrophoresis analyzer (available from Applied Biosystems, Foster City, Calif.) that can be used for sequencing. In some embodiments, the apparatus can be part of an integrated system and/or be adapted to function with other pieces of equipment adapted for further sample processing of samples, for example, a PCR detector. Exemplary detectors that can be used include real-time sequence detection systems and real-time PCR detectors, for example, the ABI 7900, available from Applied Biosystems, Foster City, Calif.
The apparatus, system and/or methods described herein can also be used in conjunction with downstream processing of immiscible-fluid-discrete-volumes in conduits as described, for example, in
Generally, system 10 can be configured to perform different types of assays on fluids introduced thereinto. The amounts and types of fluids introduced into system 10 can be varied depending on a particular assay to be performed. Exemplary assays can include, for example, de novo nucleic acid sequencing reactions, and nucleic acid resequencing reactions, as discussed herein. An exemplary type of sample preparation can be used for genotyping, gene-expression, methylation analysis, and/or directed medical sequencing (VariantSEQr™, for example) that requires multiple liquids to be brought together in an aqueous discrete volume. For example, in a gene-expression application, each aqueous discrete volume can contain individual primer sets. The sample to be analyzed, for example, complementary DNA (cDNA), can be added to each aqueous discrete volume. In the VariantSEQr™ application, for example, an aqueous discrete volume can comprise a primer set, and genomic DNA can be added to that discrete volume
According to various embodiments, one or more samples 22, 24, can be introduced to system 10. Samples 22 and 24, for example, can comprise a nucleic acid containing fluid. According to some embodiments, the nucleic acid contained in a sample can be, for example, a single copy of a genomic DNA sequence of an organism, or complementary DNA from an organism.
In some embodiments, a plurality of fluids can be received by fluid processing system 10 through an immiscible-fluid-discrete-volume-forming tube 12, which is a part of main conduit system 50. Other types of conduits may be used instead of a tube to accept the introduction of fluids into fluid processing system 10. Immiscible-fluid-discrete-volume-forming tube 12 can be part of a system that can comprise, for example, a pump or another apparatus adapted to produce controlled intake of fluids through intake tip 13 into immiscible-fluid-discrete-volume-forming tube 12. Motive force providers for moving fluids into and along a tube or other type of conduit include differential pressure, displacement, electowetting, optoelectrowetting, magnetohydrodynamic “pumps” among others. The immiscible-fluid-discrete-volume-forming conduit 12 can be adapted to control an introduction unit to introduce alternate volumes of aqueous sample fluid and spacing fluid that together form discrete volumes of aqueous sample fluid in contact with spacing fluid, i.e., aqueous sample immiscible-fluid-discrete-volumes, in the at least one conduit wherein each aqueous sample immiscible-fluid-discrete-volume can comprise a maximum outer dimension that is equal to or slightly less than the maximum inner cross-sectional dimension of immiscible-fluid-discrete-volume-forming conduit 12. One of skill in the art will understand that the maximum inner cross-sectional dimension of a tube is the inner diameter of the tube if the tube has a circular cross-section.
According to various embodiments, immiscible-fluid-discrete-volume-forming tube 12 can comprise a tip 13. Tip 13 can interface with fluids to be drawn into system 10. Tip 13 can comprise an angled surface or have any suitable geometry such that the creation of air bubbles in immiscible-fluid-discrete-volume-forming tube 12 is minimized or eliminated when tip 13 contacts and draws in a fluid. Immiscible-fluid-discrete-volume-forming tube 12 can be robotically controlled, or manually controlled. Robotic configurations can comprise, for example, stepper motors 14, 16, and 18, which can move immiscible-fluid-discrete-volume-forming tube 12 in X-axis, Y-axis, and Z-axis directions, respectively. In some embodiments, tube 12 can be moved in the Z-axis direction by a stepper motor 18, and a fluid container can be moved in the X-axis and Y-axis directions by stepper motors 14 and 16, respectively. In some embodiments, tube 12 can be stationary and a fluid container can be moved in the X-axis, Y-axis, and Z-axis directions by stepper motors 14, 16, and 19, respectively. Motive force providers other than stepper motors can be used.
According to various embodiments, a variety of fluids can be introduced into fluid processing system 10, in a number of different combinations, depending on the particular type of assay to be performed. The fluids can reside in or on any suitable fluid retaining device, for example, in the wells of a multi-well plate 20, an opto-electrowetting plate, a tube of preformed slugs, a tube of stable emulsified nanodroplets, individual tubes, strips of tubes, vials, flexible bags or the like.
According to some embodiments, fluid processing system 10 can comprise a number of different fluid conduits and fluid control devices. The following description applies to the embodiment as illustrated in
Main conduit system 50 can provide a fluid communication between T-junction 52 and output tube 54. From T-junction 52, conduit system 50 comprises two pathways that intersect at cross-junction 68 and at T 66. A first pathway can take a fluid sequentially through holding tubes 56, 60 and 64, and T-junction 66, before reaching cross-junction 68. A second pathway can take a fluid sequentially through holding tubes 63, and 65, and through either T-junction 66, to cross-junction 68, or directly to cross-junction 68. Both the first pathway and the second pathways are configured to hold fluids for later analysis and are configured to interface with pumps for moving fluids along the conduits as discussed below.
From cross-junction 70, fluids can move sequentially to thermal-spiral 74, cross-junction 76, thermal-spiral 80, and T-junction 84. At T-junction 84 fluids can sequentially move either through cross-junction 86, thermal-spiral 90, and output tube 54, or through cross-junction 88, thermal-spiral 92, and an output tube 54.
According to some embodiments fluid processing system 10 can comprise pumps 39 and 40. Pump 40 can be configured to remove or add oil to main conduit system 50, and thereby move fluids located therein. Pump 39 can be configured to remove or add oil to main conduit system 50 to move fluids located therein. All of the pumps described herein can create positive and/or negative pressures in the various conduits of system 10.
According to various embodiments, a T-junction can comprise any junction having three discrete pathways extending from, for example, either a Y-junction or a T-junction. In various embodiments, the junction can comprise a valve-less junction where a stream of aqueous sample fluid and a stream of non-aqueous spacing fluid can meet and form at least discrete volumes of the aqueous sample fluid in contact with the non-aqueous spacing fluid. For example, microfabrication technology and the application of electrokinetics or magneto hydrodynamics can achieve fluid pumping in valve-less, electronically controlled systems. Components comprising shape-optimized channel turns, optimal injection methods, micromixers, and/or high flow rate electroosmotic pumps can be used in such a valve-less system.
According to some embodiments, system 10 can comprise discrete volume detectors D-1, D-2, D-3, D-4, D-5, D-6, D-7, D-8, D-9, D-10, D-11, D-12, D-13, D-14, D-15, D-16, D-17, D-18, D-19, and D-20, and detector 98. The discrete-volume detectors can comprise, for example, fluorescent or infra-red, refractive-index detectors, and possibly capacitive and absorption detectors. In
According to various embodiments, the system can comprise a thermal-cycling device or thermal cycler, adapted to thermally cycle an aqueous immiscible-fluid, discrete volume in a conduit disposed thereon or therein. In some embodiments, the conduit can contact the thermal cycler in a single straight-line segment, or a coil around the external perimeter of thermal cycler, or a spiral of decreasing radius on one surface, or a serpentine pattern across one or more surfaces of thermal cycler. The thermal-cycling device can comprise a heat source, for example, a radiant heat source, a non-radiant heat source, a peltier device, or the like, and a cooling source, for example, a fan, an air jet, or a liquid-circulating system in a thermal block. The thermal-cycling device can comprise one or more temperature sensors and one or more control units for controlling heating and cooling according to a desired or programmed thermal cycle.
The conduits of the present teachings can comprise capillary tubes having an inner diameter and the inner diameter can be, for example, about 1000 microns or less, for example, about 800 microns or less, or about 500 microns or less. In some embodiments, the tube has a minimum inner dimension, or diameter, of from about 1.0 micron to about 100 microns, or from about 50 microns to about 75 microns. In other embodiments, the tube can have an inner diameter greater than about 300 microns. In some embodiments, main conduit system 50 can comprise tubing with a 635 micron inner diameter. In some embodiments, thermal spirals after T-84 can comprise tubing with a 480 micron inner diameter. In some embodiments conduit system 50 can comprise tubing with an inner diameter in a range of from about 380 microns to about 635 microns, with the smallest diameters at the ends of the system. Other details about the thermal-cycling device, capillary channel, and other system components will become apparent in view of the teachings herein.
System 10 can comprise an single molecule amplification fluid (“SMAF”) conduit system 51. SMAF tube system 51 can supply sample fluid to a T-junction through positive pressure rather than by aspiration. SMAF conduit system 51 can comprise a supply conduit connected to and in fluid communication with a supply of single molecule amplification fluid. The SMAF can comprise a solution or mixture of target nucleic acids diluted to a degree such that there is an average of less than about one target nucleic acid per volume of single molecule amplification fluid that is used to make an immiscible-fluid-discrete-volume. An exemplary concentration of target molecules can be 0.4 molecule per volume used to make an immiscible-fluid-discrete-volume. SMAF conduit system 51 can comprise conduits connecting a SMAF reservoir 69 sequentially to valve V-18 and T-junction 67. SAMF conduit system 51 can comprise conduits that connect T-junction 67 to main conduit system 50 and a rotary valve 71.
Fluid processing system 10 can comprise rotary valves 71, 73, 75, 77, and 79. Each rotary valve can function to direct the flow of metered amounts of different reagents from different respective reagent reservoirs connected thereto, as described below, to main conduit system 50. Syringe pumps 58, 66, 78, and 82 can be in fluid communication with rotary valves 73, 75, 77 and 79, respectively. Pumps 42, 43, 44, and 45 can be in fluid communication with rotary valves 73, 75, 77 and 79, respectively.
Fluid processing system 10 can comprise a first waste tube system 81. Waste tube system 81 can comprise conduits connecting the following components: valves V-17, V-20, V-21, V-22, V-23, V-24, V-25, and a waste reservoir 83. Waste tube system 81 can provide a fluid communication between and cross-junctions 68, 70, 76, 86, and 88 and waste reservoir 83.
Fluid processing system 10 can comprise a second waste tube system 48. Second waste tube system 48 can comprise conduits connecting a pump 87, a waste reservoir 85, and a valve V-26, that interface with output tube 54. Second waste tube system 48 can be used to remove liquids from output tube 54.
Fluid processing system 10 can comprise reagent reservoirs 89, 91, 93, 95, 97, and 99, which can be in fluid communication with rotary valves 75, 77, 77, 79, 79, and 73, respectively. Reagent reservoir 89 can contain, for example, an exo-nuclease and shrimp alkaline phosphatase. Reagent reservoir 91 can contain, for example, nucleic acid amplification reaction forward primers. Reagent reservoir 93 can contain, for example, nucleic acid amplification reaction chain terminating dyes. Reagent reservoir 95 can contain, for example, nucleic acid amplification reaction reverse primers. Reagent reservoir 97 can contain, for example, nucleic acid amplification reaction chain terminating dyes. Reagent reservoir 99 can contain, for example, a nucleic acid amplification reaction master mix comprising, for example, reactive single base nucleotides, buffer, a polymerase, and the like, for example, to carry out a polymerase chain reaction.
According to various embodiments, fluid processing system 10 can comprise a rinse system 30. Rinse system 30 can provide a fluid communication between a rinse fluid reservoir 28, rotary valve 73, rotary valve 75, rotary valve 77, rotary valve 79, and immiscible-fluid-discrete-volume-forming tube 12. Rinse fluid reservoir 28 can contain a rinse fluid 26. Rinse fluid 26 can comprise microbiologic grade water, for example, distilled, de-ionized water.
Rinse fluid 26 can be used to remove residual sample, for example, from immiscible-fluid-discrete-volume-forming tube 12. Rinse fluid can be provided to multi-well plate 20, by way of rinse tube system 30. In some embodiments, rinse fluid 26 can be added to immiscible-fluid, discrete volumes to adjust the volume or concentration thereof, in conjunction with an addition station, as described in
According to various embodiments, fluid processing system 10 can comprise a spacing fluid tube system 36. Spacing fluid tube system 36 can provide a fluid communication between a spacing fluid reservoir 34, vacuum pump 41, and multi-well plate 20. Spacing fluid reservoir 34 can contain an oil 32 or other spacing fluid that is immiscible with an immiscible-fluid-discrete-volume-forming fluid, for example, an aqueous slug fluid.
In some embodiments, the spacing fluid can be non-aqueous. The spacing fluid can comprise an organic phase, for example, a polydimethylsiloxane oil, a mineral oil (e.g., a light white mineral oil), a silicon oil, a hydrocarbon oil (e.g., decane), a fluorinated fluid or a combination thereof.
Fluorinated compounds such as, for example, perfluoroooctyl bromide, perfluorodecalin, perfluoro-1,2-dimethylcyclohexane, FC 87, FC 72, FC 84, FC 77, FC 3255, FC 3283, FC 40, FC 43, FC 70, FC 5312 (all “FC” compounds are available from 3M, St. Paul, Minn.), the Novec® line of HFE compounds (also available from 3M, St. Paul, Minn.), such as, for example, HFE-7000, HFE-7100, HFE-7200, HFE-7500, and perfluorooctylethane can also be used as the spacing fluid. Combinations, mixtures, and solutions of the above materials can also be used as the spacing fluid.
In some embodiments, fluorinated alcohols, such as, for example, 1H, 1H, 2H, 2H-perfluoro-decan-1-ol, 1H, 1H, 2H, 2H-perfluoro-octan-1-ol, and 1H, 1H-perfluoro-1-nonanol can be added to a fluorinated compound, such as those listed above, to improve the stability of aqueous discrete volumes within the spacing fluid, but still maintain the ability to coalesce upon contact. In some embodiments, fluorinated alcohols can be added in a range of approximately 0.1% to approximately 5% by weight. In some embodiments, the fluorinated alcohol additive can be approximately 0.1%, 0.2%, 0.5%, 1.0%, 1.5%, 2.0%, 3.0%, 4.0% or 5% by weight of the fluorinated compound. In some embodiments, the fluorinated alcohol additive can be from approximately 1% to approximately 10% by volume of the fluorinated compound. In some embodiments, the fluorinated alcohol additive may comprise approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% by volume of the spacing fluid. In some embodiments, F-alkyl dimorpholinophosphates can be added as surfactants to fluorinated compounds.
In some embodiments, the organic phase can include non ionic surfactants such as sorbitan monooleate (Span 80 (no. S-6760, Sigma)), polyoxyethylenesorbitan monooleate (Tween 80 (no. S-8074, Sigma)), sorbitan monostearate (Span 60), octylphenoxyethoxyethanol (Triton X-100 (no. T9284, Sigma)). In some embodiments, Span 80 can be added in an amount ranging from about 1.0% to about 5.0%, or about 3.0% to about 4.5%. In some embodiments, adding surfactants in the quantities of 4.5% Span 80, 0.40% Tween 80, and 0.05% Triton X-100 to mineral (no. M-3516, Sigma) can result in the creation of stable emulsified droplets.
In some embodiments, the organic phase can include ionic surfactants, such as sodium deoxycholate, sodium cholate, and sodium taurocholate. In some embodiments, the organic phase can include chemically inert silicone-based surfactants, such as, for example, polysiloxane-polycetyl-polyethylene glycol copolymer.
In some embodiments, the non-aqueous, spacing fluid can have a viscosity between approximately 0.5 to approximately 0.75 centistokes. In some embodiments, the non-aqueous spacing fluid can have a viscosity between approximately 0.75 centistokes to about 2.0 centistokes. In some embodiments, the non-aqueous spacing fluid can have a viscosity greater than 2.0 centistokes. In some embodiments, the non-aqueous spacing fluid can have a viscosity between 0.5 to greater than about 2.0 centistokes. In some embodiments, the non-aqueous spacing fluid can have a viscosity greater than 2.0 centistokes. In some embodiments, the non-aqueous, spacing fluid can have a boiling point greater than or equal to 100 C.
Spacing fluid 32 can function to separate discrete volumes of an immiscible-fluid-discrete-volume-forming fluid, for example, an aqueous sample fluid, before, during, or after the immiscible-fluid-discrete-volume-forming fluid has been introduced into system 10. Spacing fluid can be provided to multi-well plate 20, from a spacing fluid reservoir 34, by way of a spacing fluid tube system 36.
According to some embodiments, a de novo nucleic acid sequencing method is provided that uses system 10. The de novo sequencing method can be used to sequence an entire genome or portions thereof. The de novo sequencing method can be especially useful when the sequence of the organism is unknown.
In some embodiments, a de novo sequencing method comprises pre-processing a sample, separating the sample into a set of immiscible-fluid, discrete volumes, optionally adding amplification reagents to each discrete volume of the set, amplifying nucleic acids in the set of immiscible-fluid, discrete volumes to form a set of amplified immiscible-fluid, discrete volumes, optionally detecting, and removing, discrete volumes without amplified sample molecules therein, adding primer and dNTP deactivation agents to each discrete volume in the set, or optionally, to only those with amplified sample molecules, incubating the set of amplified immiscible-fluid, discrete volumes with primer and dNTP deactivation agents, subjecting the resulting nucleic acids to sequencing conditions to form detectable products, and detecting the detectable products.
In some embodiments, the method can comprise pre-processing a sample before it is input into system 10. The pre-processing of a sample can comprise fragmenting the nucleic acid present in the sample. The fragmentation can be accomplished by any suitable method known in the art. For example, the nucleic acid can be fragmented by enzymatic digestion, or physical disruption methods, for example, hydro-shearing or sonication. In some embodiments the nucleic acid can be fragmented to an average size of about 500 B, 750 B, 850 B, 1 KB, 2 KB, or 3 KB, for example.
According to some embodiments, the pre-processing of sample can comprise ligating sequences to a sample. In universal sequences can be used to facilitate universal nucleic acid amplification. Universal sequences can be artificial sequences that generally have no homology with the target nucleic acids. Universal sequences can be designed to resist the formation of dimers between themselves. Universal sequences can be designed to bind with analogous primers with a consistent efficiency.
According to some embodiments, the present teachings can encompass a de novo sequencing method wherein universal sequences can be ligated to the 5′ and 3′ ends of the DNA fragments in a sample by, for example, T-4 DNA ligase, thereby forming a universal tail. The universal tail sequences can function as sites of complementarity for zip code primers. Details of universal tail procedures can be found in U.S. Pat. App. No. 2004/0185484, to Costa et al., which is incorporated herein, in its entirety, by reference.
According to various embodiments, the amplifying of a nucleic acid can comprise a thermal cycling nucleic acid sequence amplification process or an isothermal nucleic acid sequence amplification process. If a thermal cycling nucleic acid sequence amplification process is used, the process can comprise, for example, a polymerase chain reaction (PCR). The nucleic acid sequence amplification reaction can comprise an exponential amplification process, for example, PCR, or a linear amplification process, as can occur during, for example, during Sanger cycle sequencing. In various embodiments, other nucleic acid amplification processes can be used, for example, ligase chain reaction (LCR), nucleic acid sequence based amplification (NASBA), Q-beta replicase (QB) amplification, or strand displacement amplification (SDA). These alternatives, as well as others known to one skilled in the art, can be used either by themselves or in combination with PCR to amplify nucleic acids.
According to various embodiments, nucleic acid sequence processing methods comprising a first type of nucleic acid amplification reaction followed by one or more of a second different type of amplification reaction, and/or detection assay reaction, can be carried out, for example, as described in U.S. Patent Application No. 60/699,782 to Faulstich et al., filed Jul. 15, 2005, (Attorney Docket No. 5010-297), which is incorporated herein in its entirety by reference.
According to some embodiments, the present teaching can comprise a method of de novo sequencing wherein pre-processing of sample can comprise adding zip code primers to a sample of nucleic acid having universal tail sequences ligated therein. Zip code primers can be complementary to the universal tail sequences. The use of zip code tails sequences and zip code primers can reduce the need for target specific primers, resulting in significant cost savings as well as greater assay flexibility.
According to various embodiments, pre-processing a sample can comprise adding to the sample reactants to facilitate a nucleic acid amplification reaction. For example, the four dNTP's (dATP, dTTP, dGTP, and dCTP), a polymerase, oligonucleotide primers, and/or chelating agents can be added to the sample. Oligonucleotide primers can be chosen as determined by one of skill in the art to accomplish the desired objective, for example, universal primers can be used.
According to various embodiments, pre-processing a sample can comprise diluting the sample with a miscible solvent, vehicle, or carrier. The sample can be diluted at a ratio of 1:1, 1:10, 1:100, 1:1000, or 1:10,000, for example. Exemplary ranges of dilution can be from about 1:1 to about 1:100, or from about 1:10 to about 1:50. For example, the sample can be diluted such that only a single fragment of nucleic acid is present per 500 nanoliters of diluted sample, or per 200 nanoliters of diluted sample. In some embodiments, the concentration of target fragments can be based on the size of the immiscible-fluid-discrete-volumes generated that carry the target fragments, such that an average of about 1 target fragment is present per 1.4 immiscible-fluid-discrete-volumes generated. According to various embodiments, the sample can be diluted such that at least 50% immiscible-fluid-discrete-volumes produced from a sample in the process described below can each comprise a single target nucleic acid sequence. In various other embodiments, less than about 50% of the immiscible-fluid-discrete-volumes produced can each comprise a single target nucleic acid sequence. In other embodiments, at least 1% or more, 5% or more, 10% or more, or 20% or more can comprise a single target nucleic acid sequence, for example, from about 10% to about 50% or from about 20% to about 40%.
After optional preprocessing, the sample fluid is introduced to system 10 to form one or more discrete volumes of the sample fluid in a spacing fluid with which it is immiscible. A plurality of immiscible-fluid, discrete volumes can be associated together as a set of immiscible-fluid, discrete volumes. Each set of immiscible-fluid, discrete volumes can comprise immiscible-fluid, discrete volumes separated from one another by a spacing fluid, for example, an oil. Each immiscible-fluid-discrete-volume of a set can be equally spaced from one or more adjacent immiscible-fluid, discrete volumes of the set. Multiple sets of immiscible-fluid, discrete volumes can be present at the same time in main conduit 50. Each set of immiscible-fluid, discrete volumes can be separated from one or more other sets of immiscible-fluid, discrete volumes by larger volumes of spacing fluid, which in a tube of constant cross-sectional area size and shape is visible by greater length between the trailing end of the last discrete volume of the upstream set and the leading end of the first discrete volume in the downstream set. In some embodiments, two or more sets of immiscible-fluid, discrete volumes are spaced from one another a distance that is greater than the average distance between adjacent immiscible-fluid, discrete volumes with the same set.
In the embodiment depicted in
According to some embodiments, the method can comprise moving a set of immiscible-fluid, discrete volumes, from T-junction 52, to cross-junction 70, by way of conduit system 50. If a set of immiscible-fluid, discrete volumes does not contain nucleic acid amplification reactants, the reactants can be added to each immiscible-fluid-discrete-volume of the set of immiscible-fluid, discrete volumes at cross-junction 70. More details about exemplary methods of adding additional miscible liquid to immiscible-fluid, discrete volumes is provided herein, at least in FIGS. 2, 21-28, and 29A-C. As illustrated in
According to some embodiments, the method can comprise moving a set of immiscible-fluid, discrete volumes from cross-junction 70, through main conduit system 50, to thermal-spiral 74. Detector D-8 can be used to detect the arrival of a set of immiscible-fluid, discrete volumes at thermal-spiral 74. Detector D-8 can be used to detect the end of a set of immiscible-fluid, discrete volumes, and thereby detect that a set of immiscible-fluid, discrete volumes is disposed in thermal-spiral 74. A set of immiscible-fluid, discrete volumes can be thermally cycled, for one or more cycles, for example, for from about 5 to about 50 temperature cycles or from about 20 to about 30 temperature cycles.
According to various embodiments, the method can comprise introducing polymerase chain reaction inactivating reagents into main tube 50 after amplifying the at least one target nucleic acid sequence and before subjecting the nucleic acid sequence to a sequencing reaction. The reagents can be used to inactivate or remove or eliminate excess primers and/or dNTP's. The inactivating reagents can be introduced at an junction in the capillary channel, for example, after an immiscible-fluid-discrete-volume to be inactivated is aligned with the junction. The junction can comprise, for example, a T-junction.
According to some embodiments the method can comprise moving a set of immiscible-fluid, discrete volumes from thermal-spiral 74, through cross-junction 76. As the set of immiscible-fluid, discrete volumes moves through cross-junction 76, the method can comprise adding exonuclease and shrimp alkaline phosphatase to each immiscible-fluid-discrete-volume of the set of immiscible-fluid, discrete volumes. For example, the exonuclease and shrimp alkaline phosphatase “SAP” can be metered out in discrete volumes which merge respectively with the immiscible-fluid, discrete volumes of a set of immiscible-fluid, discrete volumes at an junction in rotary valve 77. More details about metering “addition” liquid, such as, for example, exonuclease and SAP is provided herein, at least in FIGS. 11A-B, 21-24, and 25-27 and their corresponding descriptions. For example, exonuclease and shrimp alkaline phosphatase can be added to each immiscible-fluid-discrete-volume of the set of immiscible-fluid, discrete volumes in cross-junction 76.
In the exemplary system shown, detector D-6 can detect the arrival of the beginning and/or the end of a set of sample discrete volumes at cross-junction 76. Detector D-18 can detect the arrival of the beginning and/or the end of one or more immiscible-fluid, discrete volumes of exonuclease and shrimp alkaline phosphatase at cross-junction 76. Valve V-8 can control the movement of a set of immiscible-fluid, discrete volumes out of cross-junction 76. In some embodiments, V8 can be used to isolate one or more thermal spirals from each other. More details about an exemplary method of isolating a thermal spiral is provided herein, at least in
In the exemplary embodiment shown, a set of immiscible-fluid, discrete volumes containing exonuclease and shrimp alkaline phosphatase can be moved into thermal-spiral 80, via main conduit system 50. Detector D-9 can detect the arrival of the beginning and/or the end of a set of immiscible-fluid, discrete volumes at thermal-spiral 80. The set of immiscible-fluid, discrete volumes can be incubated at from about 25° C. to about 35° C. for a time period of from about one minute, to about 60 minutes or from about two minutes to about 10 minutes. The incubation step can function to facilitate the activities of the exonuclease and shrimp alkaline phosphatase. A set of immiscible-fluid, discrete volumes can be further incubated at a temperature of from about 75° C. to about 85° C., for a time period of from about 10 seconds to about 10 minutes, or from about one minute to about five minutes. The incubation at from about 75° C. to about 85° C. can function to heat-kill any enzymes that might still be present in the set of immiscible-fluid, discrete volumes.
According to some embodiments, the method can comprise moving a set of immiscible-fluid-discrete-volumes to T-junction 84. Valve V-9 can control the movement of a set of immiscible-fluid-discrete-volumes from thermal spiral 80, to T-junction 84. Detector D-10 can detect the arrival of the beginning and/or the end of a set of immiscible-fluid-discrete-volumes at T-junction 84. The method can comprise dividing one or more immiscible-fluid, discrete volumes of a set of immiscible-fluid discrete volumes into two or more smaller immiscible-fluid-discrete volumes to form two newly formed sets of equal number of immiscible-fluid discrete volumes, but containing immiscible-fluid discrete volumes of smaller volume. More details about an exemplary method of splitting immiscible-fluid, discrete volumes into smaller immiscible-fluid, discrete volumes is provided herein, at least in FIGS. 11A-B, and 12-16 and the corresponding description. The method can comprise moving one newly created set of immiscible-fluid, discrete volumes along main conduit system 50, to cross-intersection 86. Forward primers and chain terminating dyes can be moved from reservoirs 91 and 93, to rotary valve 77. The forward primers and chain terminating dyes can be metered out by rotary valve 77. The forward primers and chain terminating dyes can be moved to cross-intersection 86 and be added to each immiscible-fluid-discrete-volume of the newly-created set of immiscible-fluid, discrete volumes, thereby creating a forward set of immiscible-fluid, discrete volumes. According to various embodiments, the method can comprise moving the second newly created set of immiscible-fluid, discrete volumes along main conduit system 50, to cross-intersection 88. Reverse primers and chain terminating dyes can be moved from reservoirs 95 and 97, to rotary valve 79. The reverse primers and chain terminating dyes can be metered out by rotary valve 79. The reverse primers and chain terminating dyes reagent can be moved to cross-intersection 86 and be joined with each immiscible-fluid-discrete-volume of the second newly-created set of immiscible-fluid, discrete volumes, thereby creating a reverse set of immiscible-fluid, discrete volumes.
In some embodiments, the method can comprise moving the forward set of immiscible-fluid-discrete-volumes from cross-junction 86, along main conduit system 50, to thermal spiral 90. The forward set of immiscible-fluid-discrete-volumes can be thermally cycled for from about 5 to about 50, temperature cycles, for example, from about 20 to about 40 thermal cycles.
In some embodiments, the method can comprise moving the reverse set of immiscible-fluid-discrete-volumes from cross-junction 88, along main conduit system 50, to thermal spiral 92. The reverse set of immiscible-fluid-discrete-volumes can be thermally cycled for from about 5 to about 50 thermal cycles, for example, from about 20 to about 40 cycles, temperature cycles.
In some embodiments, the method can comprise thermal cycling at least four different sets of immiscible-fluid-discrete-volumes, one in each of thermal spirals 74, 80, 90, and 92. In some embodiments, valves, such as, for example, can be used to isolate each thermal spiral from all other thermal spirals. In some embodiments, the different thermal spirals operate at the same time, but with different thermal profiles. In some embodiments, isolating the thermal expansion and contraction of the fluid in the different spirals can be desirable. In some embodiments, the total time for more than one temperature cycling can be the same. In embodiments where different length thermal cycles are desired, an additional step at non-process inducing temperature can be added for the remainder of the longest thermal cycles.
According to various embodiments, the method can comprise moving the forward and the reverse sets of immiscible-fluid-discrete-volumes from their respective thermal spiral to output conduit 54. Movement can be caused by syringe pumps 82A and 82B that can be controlled independently, or together, by a motor 88A operatively connected thereto. Syringe pumps 82A and 82B can push and pull fluids through respective T-junctions 84A and 84B. This arrangement is useful as syringe pumps 82A and 82B can initially pull immiscible-fluid-discrete-volumes into place in the respective thermal spirals 90 and 92, in conjunction with the positive pressure from the pumps on the upstream side of tee 84. Valves V-10 and V-11 can be switched so that immiscible-fluid-discrete-volumes can be pushed out of system 10. In some embodiments, the pushing can be done with one of pumps 82A and 82B at a time; therefore, there is no need to merge two separate sets of immiscible-fluid-discrete-volumes back together into a single set, but rather the separate sets can be individually dispensed. Output conduit 54 can deposit both sets of immiscible-fluid-discrete-volumes on, for example, a multi-well plate.
According to some embodiments, a dye can be added to one or more immiscible-fluid, discrete volumes of a set of immiscible-fluid, discrete volumes. The dye can comprise a double-strand (ds), nucleic acid intercalating dye, for example, SYBR green, SYBR gold, EVA green, LC green, or the like. The dye can be added to an aqueous immiscible-fluid-discrete-volume-forming fluid, such as an aqueous sample, before it is added to system 10. The dye can be added to a set of immiscible-fluid, discrete volumes at any cross-junction of system 10. The dye can be used to discriminate between immiscible-fluid, discrete volumes that contain ds nucleic acids and immiscible-fluid, discrete volumes that do not contain ds nucleic acids. The immiscible-fluid, discrete volumes that do not contain ds nucleic acids can be removed from output tube 54 before the immiscible-fluid, discrete volumes are deposited on a multi-well plate 47. The immiscible-fluid, discrete volumes that do not contain ds nucleic acids can be moved through second waste tube system 48, to waste reservoir 85. In some embodiments, a dye can be detected by detector 98 to determine whether a discrete volume should be sent to second waste reservoir 85 or be collected. Pump 87 can apply a negative pressure to waste tube system 48, which can cause the movement of immiscible-fluid, discrete volumes into waste reservoir.
Immiscible-fluid, discrete volumes deposited on multi-well plate 47 can be subjected to a sequencing reaction to form a detectable product, and the method of the present teachings can comprise detecting the detectable product. In various embodiments, the detectable product can be detected using, for example, a flow cell or a capillary electrophoresis sequencer. In various other embodiments, an off-capillary detector can be used as deemed appropriate.
Shown below is a table showing a state diagram of various settings that can be implemented for the various valves and detectors of the system shown in
Footnotes:
1 Pull SMAF into T-intersection (67);
2 Pull oil through T-intersection (67);
3 Pull MM through T-intersection (67);
4 Pull SMAF + MM through D-17;
5 Push SMAF + MM towards T-intersection (66) until D-5 detects AF;
6 Pull, Push oil towards T-intersection (66) until D-4 detects oil;
7 Push oil + SMAF + MM through thermal cycler until D-6 detects zebras or, more likely, D-2 sees only oil;
8 Pull SMAF + MM through D-17;
9 Push oil + SMAF + MM through thermal cycler until D-6 detects zebras or, more likely, D-2 sees only oil;
10 Pull SMAF + MM towards D-17. After total volume of SMAF has entered T-intersection (67), close V-18. After total volume of MM has left Rotarty Valve (71), switch Rotary Valve (71) to “oil” position. Continue pulling SMAF + MM towards D-17 until D-2 sees a;
11 Push oil + SMAF + MM through thermal cycler until D-6 detects zebras or, more likely, D-5 sees only oil;
12 Push oil until D-16 detects oil;
13 Push ES until D-18 detects ES, then push further distance calculated to advance ES to Zebra path.;
14 Push until D-6 detects end of batch, then push further distance calculated to advance batch just past ES adder;
15 Push until D-9 detects end of batch, then push farther distance calculate to advance batch completely into cleanup thermal cycler;
16 Push SP (78) until D-19 sees oil. Push SP (82) until D-20 sees oil.;
17 Pull portion of FP into SP (78). Pull portion of RP into SP (82);
18 Pull portion of BD into SP (78). Pull portion of BD into SP (82);
19 Pull alternating sub-portions of primers and big dyes until complete portion has been loaded;
20 Pull small amount of oil so all aqueous fluids advance into syringe;
21 Push SP (78) until D-19 sees FP + BD. Push SP (82) until D-20 sees RP + BD. Push farther distance calculated to advance FP + BD and RP + BD to Zebra path;
22 Push with pumps until D-11 and D-7 see oil, then push further distance calculated to advance batch just past RP + BD and FP + BD adders;
23 Push with pumps further distance calculated to advance batch into cycle sequencing thermal cycler;
24 Push until FSD-1 detects sample-laden FP slug, then push further distance calculated to move downstream boundary of sample-laden slug just inside dispense tip;
25Push distance calculated to bead sample-laden slug on dispense tip. Touch bead to bottom of sample well.
According to various embodiments, the present teachings can encompass a resequencing method using system 10. In general, the resequencing method is similar to the de-novo method described herein with modifications as discussed herein.
In some embodiments, the pre-processing of a sample for resequencing comprises shearing a robust sample of nucleic acid having a plurality of copies of one or more nucleic acids of interest, herein also referred to as target sequences. The nucleic acids in the sample can be sheared. The method can comprise adding a set of gene specific primers, for example, at cross-junction 10, to a set of immiscible-fluid, discrete volumes generated from the sample. Immiscible-fluid, discrete volumes made from the sample can contain a single copy of a nucleic acid fragment or can contain a plurality of copies of one or more different nucleic acid fragments. Each immiscible-fluid-discrete-volume can contain, for example, from about 50 to about 150 or more; 384 or a thousand, or several thousands, or fewer.
In some embodiments, the method can comprise moving a set of immiscible-fluid, discrete volumes comprising the concentrations of primers discussed above, to thermal-spiral 74. The set of immiscible-fluid, discrete volumes can be thermally cycled and thereafter processed in any of the many manners disclosed herein for the de novo sequencing method.
Various sequencing and re-sequencing methods that can be carried out according to various embodiments can include, for example, those depicted in
Shown below are Tables 2A and 2B which are the first and second halves of another state diagram of various settings that can be implemented for the various valves and detectors of the system shown in
A simplified system 200 is illustrated in
System 200, as illustrated in
Next in line, as illustrated in
As illustrated in
Reference will now be made to various embodiments of devices, apparatus, systems, and methods for controlling the fluid flow to manipulate immiscible-fluid, discrete volumes of a first fluid separated from one another by an immiscible spacing fluid, examples of which are illustrated in the accompanying drawings. Various embodiments of these can be used in the system described above with reference to
Referring now to
In some embodiments, annular stator 302 and cylindrical rotor 304 can be formed from PTFE. In some embodiments, annular stator 302 or cylindrical rotor 304 can be formed poly oxy-methylene, Nylon 66, Tefzel (a modified ETFE (ethylene-tetrafluoroethylene) fluoropolymer (manufactured by Du Pont)), Polytetrafluoroethylene (PTFE), Noryl classico family of modified PPE resins consists of amorphous blends of PPO* polyphenylene ether resin and polystyrene. (manufactured by General Electric), ultra high density polyethylene, polyphenol sulfide, and red Turcite TX or Turcite X ((Ethylene-chlorotrifluoroethlyene [ECTFE]) (manufactured by Busak+Shamban). In some embodiments, annular stator 302, cylindrical rotor 304, tube 312 and tube 314 comprise the same material. In some embodiments, at least one of annular stator 302, cylindrical rotor 304, tube 312, and tube 314 comprises a different material than at least the remaining listed components.
In some embodiments, conduits 312 and 314 are connected to annular stator 302 via fittings. In some embodiments, conduits 312 and 314 do not touch the cylindrical surface of cylindrical rotor 304, but fit in bores coaxially aligned with through-hole 306 or 308.
Use of rotary valve 300 does not displace a volume of fluid relative to its longitudinal location in tube 312 or 314. If a slug is located partially in one of conduits 312 and 314 and partially in through-hole 310, rotation of annular rotor 304 will split the slug into at least two parts. However, upon rotating annular rotor 304 back such that through-hole 310 aligns with conduits 312 and 314, the two or more volumes of the “split” slug will coalesce and the original slug can be flowed out of rotary valve 300.
Rotary valves as depicted in
In some embodiments of a system such as that depicted in
When solenoid 1020 actuates, actuator 1022 extends under force, rotating lever 1006 a pre-determined number of degrees, thereby collapsing or “pinching” tube 1002 between lever 1006 and radiused valve body 1004.
The ratio of volumes of the two smaller discrete volumes formed when a slug is split in a T-junction depends on the differences in the flow rate, diameter, and length of each of the two conduits flowing away from the T-junction. If the diameter and length are the same, then the ratio of the flow rates determines the ratio of the volumes of the two smaller, immiscible-fluid, discrete volumes.
The other side of T-junction 1502 can be plumbed in a separate, but similar way. Thus, one end of tube 1506 connects to and is in fluid communication with T-junction 1502 and the other end is connected to and is in fluid communication with port 1520 of three-way valve 1522. One end of tube 1523 is connected to and is in fluid communication with port 1524 of three-way valve 1522. Port 1526 of three-way valve 1522 is connected to and is in fluid communication with one end of tube 1528. The other end of tube 1528 is connected to and is in fluid communication with an outlet of displacement pump 1530.
Displacement mechanism 1531 and 1519 can be moved at the desired speed, in the same direction, and starting at the same time to create a desired ratio of flow rates through the fluid pathway between an outlet of T-junction 1502. If the movement does not start at the same time, in the same direction, or at the desired speed, the ratio of flow rates in each divergent pathway may not be as desired, resulting in an undesired volumetric split of a slug in T-junction 1502.
To reduce the likelihood of an undesired volumetric split, the displacement mechanisms of displacement pumps 1530 and 1518 can both be moved by a single motion. As illustrated in
After motor 1534 has moved link 1532 the desired distance in the positive Y direction, it stops. Three-way valves 1510 and 1522 change position to close internal pathways between ports 1526 and 1520 and 1514 and 1508, respectively, and open internal pathways between ports 1526 and 1524 and 1514 and 1512, respectively.
The configuration of displacement pumps, three-way valves and connected conduits described above can also be used to control the volumes of miscible fluid combined in a T, if the flow directions are reversed, along with the steps. In other words, the three-way valves start in the position shown in
The method of operation of the configuration described immediately above can also generate immiscible-fluid discrete volumes of a first fluid in a consistent ratio with a second fluid with which it is immiscible. Thus, referring to
Another method of use can combine slugs as in the zipper approach to merging samples and primer sets. After creation of slugs sets of primers in tubes 1523 and 1513 they can be pulled into 1528 and 1516, and then pushed so that they merge in tube 1502. A pair of detectors can monitor the position of the slugs, oil adding devices can be used to add oil into tubes 15061504 so that the slugs will meet precisely.
Another system for splitting aqueous immiscible-fluid, discrete volumes spaced apart by spacing fluid, in a conduit, is depicted in
Housing 1206 is provided with an upper wall 1210 and a lower wall 1208. Lower wall 1208 is provided with through holes 1226 and 1216 to accommodate and/or provide a fluid communication with aqueous immiscible-fluid-discrete-volume supply tube 1202 and spacing-fluid supply tube 1204, respectively. Upper wall 1210 is provided with through holes 1224 and 1218 to accommodate and provide fluid communication with a right-split-slug tube 1222 and a left-split-slug tube 1220, respectively. In the position shown in
In the next step of a method using system 1200, slider 1212 is shifted to the right-side position shown in
Next, spacing fluid is flowed upwardly through the spacing-fluid supply tube 1204 to move a slug 1230 out of through-hole 1214 and into right-split-slug tube 1222, as shown in
Subsequently, slider 1212 is moved back to the left-side position as shown in
Thereafter, as shown in
Thereafter the series of steps described in conjunction with
In some embodiments, the initial immiscible-fluid-discrete-volume can be flowed into through hole 1214 such that part of it remains in through-hole 1214, and part of it extends into left-split-part conduit 1220. Then, when slider 1212 is moved back to align with conduit 1220 after “splitting” slug 1228, slug 1232 can be moved further into conduit 1220 with less, if any, spacing fluid flowing through through-hole 1214.
Another embodiment of a method of splitting slugs using a two part slider mechanism described in U.S. patent application Ser. No. ______, entitled “Device and Method for Making Immiscible-Fluid-Discrete-Volumes,” to Cox et al. (attorney docket number 5010-363) comprises splitting a slug into at least two parts and moving the first and second sliders to align with distinct sets of through-holes and conduits to deliver their respective split portion 1230 or 1232 of initial slug 1228, wherein each of those conduits is not in alignment with the immiscible-fluid-discrete-volume supply conduit. An advantage of this embodiment is it requires less time to flow both smaller split slugs in their respective conduits.
In some embodiments, conduit 1702 and 1704 can form an angle of about 90 degrees and conduit 1702 and conduit 1706 can form an angle of about 180 degrees. In this embodiment, slugs bend approximately ninety degrees as the move from the junction of conduits 1702 and 1704. Gas bubbles 1710 flow straight up from conduit 1702 into conduit 1706.
Application of additional pressure to the system can cause the gas phase to be dissolved into solution, which is in effect, another embodiment of a bubble remover. Such pressure is sometimes called “back pressure.” The use of back pressure can be useful during batch thermal cycling of the fluids can apply to all thermal cycling processing sections, whether in thermal spirals, for example, in a system as illustrated in
In some embodiments, the pressure of pressurized fluid 1916 is supplied by a pressure pump 1922. In some embodiments, the pressure of pressurized fluid 1916 is regulated to a set pressure by regulator 1920 connected between pressure pump 1922 and enclosure 1914. In some embodiments, and as illustrated in
Detectors 2116 and 2118 are coupled to sense circuitry 2120 and 2122, respectively, which senses the detected signal and provides it in a useable format to a controller 2124. Controller 2124 can include for example, a set of instructions, memory, and a processing unit. Controller 2124 can communicate with one or more valves 2126, which permit or prevent flow upstream or downstream of a detector, one or more pumps 2128 and 2130, which pressurize (negatively or positively), displace, or otherwise move fluid upstream and/or downstream of a detector, motors 2132 to move the relative locations of, for example, containers of liquids, and conduits for removing liquid from or adding liquid to those containers, and one or more monitors for viewing and further interface with the presented information. In communicating, controller may direct a component to maintain or change its state based on the signal received from sense circuitry 2120 and/or 2122.
In some embodiments, a method is provided that can comprise using the system described herein to process an aqueous immiscible-fluid-discrete-volume. Additional compounds may be needed in the aqueous discrete volume during the processing and, as discussed above, the spacing of adjacent aqueous discrete volumes may need to change to accommodate the addition of liquids comprising the desired compounds involved in the reaction or processing within an aqueous discrete volume.
According to various embodiments of the present teachings illustrated, for example, in
According to the various embodiments, manifold 720 can comprise a fluorocarbon material, for example, a perfluorocarbon material such as polytetrafluoroethylene. In some embodiments, manifold 720 can comprise the same material as is used for the tubes 710, 712, 714, 716, and/or 718, or other materials known to those skilled in the art. Materials can be selected that are non-reactive or minimally reactive with the liquids passing through manifold 720.
According to various embodiments, tube 710 can be connected to manifold 720 by any appropriate connection, for example, using a fitting or connector that extends from manifold 720, by frictionally fitting tube 710 into a bore formed in manifold 720 wherein the bore has an inner diameter that is about equal to the outer diameter of tube 710, or by using an adhesive, or the like. Similarly, tubes 712, 714, 716, and 718, can be connected to manifold 720.
In the embodiment shown, tube 712 is connected at a first end to manifold 720 and at an opposite, second end to a first liquid supply unit 736. First liquid supply unit 736 can comprise, for example, a supply of a first liquid 738 and a pump for moving first liquid 738 into and through tube 712. First liquid supply unit 736 can comprise a pump of the same type, or of a different type, as the type used for supply unit 734. Controller 2124 (shown in
A tube 714 can be connected at a first end to manifold 720 and at a second, opposite end, to a second liquid supply unit 740. Second liquid supply unit 740 can comprise, for example, a supply of a second liquid and a pump for moving the second liquid 742 into and through tube 714. Second liquid supply unit 740 can comprise a pump that can be the same type as, or different than, the type of pump used in supply unit 734. Controller 2124 (shown in
A tube 716 can be connected at a first end to manifold 720 and at a second, opposite end to a third liquid supply unit 744. Third liquid supply unit 744 can comprise, for example, a supply of a third liquid 746 and a pump for moving third liquid 746 into and through tube 716. The pump can be the same type as, or different than, the type of pump used for supply unit 734. Controller 2124 can control the flow rate of third liquid 746 in tube 716, whether a result of differential pressure or displacement/time or other motive force.
According to various embodiments, first liquid 738, second liquid 742, and third liquid 746, can each comprise an aqueous medium, for example, an aqueous solution, and each can be miscible with the other two reagents. In some embodiments, each of first liquid 738, second liquid 742, and third liquid 746, can be immiscible with spacing fluid 766 in contact with aqueous discrete volume 732. In some embodiments, the first liquid 738, second liquid 742, or third liquid 746 can comprise spacing fluid 766 to increase or decrease the spacing between immiscible-fluid discrete volumes prior to receiving an additional volume of aqueous liquid at a subsequent junction. Controller 2124 (see
In an exemplary embodiment, a first aqueous immiscible-fluid-discrete-volume 732 flowing through immiscible-fluid-discrete-volume channel 756 can be supplemented with second liquid 742 flowed out at a constant rate or injected by second liquid supply unit 740 as aqueous immiscible-fluid-discrete-volume 732 lines-up with the junction of passageway 752 and immiscible-fluid-discrete-volume-forming channel 756. As a result, a supplemented immiscible-fluid-discrete-volume 760 can be formed that comprises a mixture of the aqueous liquid of aqueous immiscible-fluid, discrete volume 732 and second liquid 742. As supplemented immiscible-fluid-discrete-volume 760 proceeds through immiscible-fluid-discrete-volume-forming channel 756 and reaches the junction of immiscible-fluid-discrete-volume-forming channel 756 with passageway 754, third liquid 746 can be flowed out at a constant rate or injected from third liquid supply unit 744, when properly timed, to form a further supplemented immiscible-fluid-discrete-volume 762 comprising a mixture of the aqueous liquid of aqueous immiscible-fluid, discrete volume 732, second liquid 742, and third liquid 746. It is to be understood that additional tubes can be connected to additional respective passageways (not shown) if it is desired to provide such additional features in a system. It is to be understood that system 2300 can also comprise a separate manifold for each junction of a liquid adding or removal conduit and an immiscible-fluid-discrete-volume supply conduit, adjacent manifold being connected by tubes. While the manifold is not illustrated, such a concept as just mentioned is illustrated in
Such an embodiment that permits the addition of one or more different miscible liquids to be added to an immiscible-fluid-discrete-volume, can assist in adding a first individual primer to a selected slug at a first junction, and a second individual primer to a selected slug at a second junction. After receiving both primers, the selected slug will contain a desired primer set for amplification in a later section. In this way, the number of different primer liquids needed to provide a predetermined number of primer pairs for requencing methods, such as those described in conjunction with SYSTEM APPLICATION, can be reduced. Table 3 illustrates the combination of eight different primer liquids to provide 16 different primer pairs for use with a set of slugs, or other discrete volumes.
Referring again to
According to various embodiments, system 2300 shown in
System 2400 illustrated in
As illustrated, tube 716 and passageway 754 contain a third liquid 746 (which number is not in the figure) in three separate discrete volumes 2406, 2408, and 2410 separated by spacing fluid 766. As illustrated, the volumes of third liquid 746 in volumes 2406, 2408, and 2410 is roughly equal to the volume of the passing immiscible-fluid, discrete volumes 732 in through-hole 756. By controlling the flow rates in each conduit, discrete volumes, 2406, 2408, and 2410 can each coalesce with a discrete volume 732 to form a supplemented (not illustrated) discrete volume of combined liquids 732 and 746. In some embodiments, a particular liquid is desired to be added to only certain slugs, and having preformed volumes of the addition liquid can accomplish this task with minimal contamination of the slugs to which no addition liquid is to be added with that addition liquid, as may be the case when the addition liquid is present up to the junction of the addition conduit and the main conduit, even if its flow rate is zero.
While the methods described in conjunction with
The various reagents, mixtures, samples, oils, and other fluids and liquids that can be used with or moved through the systems described herein include those fluids and liquids described in detail in U.S. Provisional Patent Application No. 60/710,167 entitled “Sample Preparation for Sequencing,” to Lee et al., filed Aug. 22, 2005 (Attorney Docket No. 5841P), and in U.S. Provisional Patent Application No. 60/731,133 entitled “Method and System for Spot Loading a Sample,” to Schroeder et al., filed Oct. 28, 2005 (Attorney Docket No. 5010-288), each of which is incorporated herein in its entirety by reference.
According to various embodiments, different approaches or mechanisms can be used to pump or drive liquid into a manifold. According to various embodiments, and as illustrated in
A consideration in this regard can be the number of liquids to be introduced, in the case when a large number of samples are to be introduced into a tube. According to various embodiments, and as illustrated in
One method of operating the system in
Another method of operating the system illustrated in
According to various embodiments, and as illustrated in
Another method to pump or transport liquids through the manifold can involve displacement of liquids localized near the junction of the addition conduit and the immiscible-fluid-discrete-volume supply conduit. Compression of the tube with a solenoid, roller, or other mechanism, may more dispense a more consistent volume of addition liquid each time in comparison to metered flow of pressurized liquid. Displacing the liquid in the addition conduit by compressing or pinching the tubes with rollers to press the tubes down is one example. As a roller is moved along the tube the pinching action of the tube can be used to push liquid into or out of the manifold. In some embodiments, a roller can be positioned in a range from about 2 inches to 10 inches away from the junction. Another is actuating a solenoid against the tube. In some embodiments, a solenoid can be positioned in a range from about 1 inch to about 1 foot away from the junction. Positioning may depend on conduit thickness and the actuation force of a selected solenoid.
Pumping and routing techniques, according to various embodiments of the present teachings, can eliminate the need for re-dipping a tube tip into different supply or other wells. This in one regard can minimize contamination. In various embodiments, the disclosed techniques can be performed in a totally enclosed, vacuum-sealed or otherwise isolated system, such that the introduction of air bubbles can be avoided.
In
As shown in
Using the system shown in
In order to have great control over very small fluid volumes, a conventional syringe pump can be used as syringe pump 3016, and in some embodiments, gearing can be implemented to gear down the otherwise conventional syringe pump to accommodate small movements of finite volumes of fluid. In some embodiments, a reciprocating pump can be used with appropriate gearing to provide both negative pressure and positive pressure, alternately.
According to various embodiments, an immiscible-fluid-discrete-volume can be generated in an immiscible-fluid-discrete-volume-forming conduit, and spaced apart by spacing fluid, according to any of the various methods described herein. To minimize and/or eliminate the formation of air bubbles in an immiscible-fluid-discrete-volume-forming conduit, and to minimize or eliminate merging of adjacent spaced-apart immiscible-fluid-discrete-volumes, methods of pushing a pattern of immiscible-fluid-discrete-volumes and spacing fluid through a conduit can be used after the immiscible-fluid-discrete-volumes are generated. In so doing, a pattern of immiscible-fluid-discrete-volumes can be moved through a processing conduit without the use of negative pressure. An exemplary system for pushing a pattern of immiscible-fluid-discrete-volumes through a conduit, after the pattern is formed, is depicted in
As shown in
As shown in
After a first set of aqueous immiscible-fluid-discrete-volumes is formed in conduit 3101, for example as illustrated, 15 spaced-apart aqueous immiscible-fluid-discrete-volumes, tip 3103 is then held in spacing fluid vessel 3112 for a period of time sufficient to enable the uptake of a large spacing fluid spacer following the first set of 15 aqueous immiscible-fluid-discrete-volumes. The large spacer can be used to separate the first set of aqueous immiscible-fluid-discrete-volumes from a subsequent set of aqueous immiscible-fluid-discrete-volumes, as shown in the third and fourth steps depicted in
After two complete sets of aqueous immiscible-fluid-discrete-volumes are generated in immiscible-fluid-discrete-volume-forming conduit 3101, intake tip 3103 is held in spacing fluid 3112 and valve 3116 is closed such that the first set of aqueous immiscible-fluid-discrete-volumes, but not the second set of aqueous immiscible-fluid-discrete-volumes, is located along conduit 3101 between port 3106 and 3108, as shown in the fifth step of the process identified in
An alternative method to that shown in
As can be understood with reference to
According to various embodiments of the present teachings, a method is provided that comprises: alternately introducing a first fluid and a second fluid, that is immiscible with the first fluid, into a conduit, to form a set of immiscible discrete volumes of the second fluid, each immiscible discrete volume of the set being separated from one or more other immiscible discrete volumes of the set by the first fluid, the set comprising a first end and a second end; moving the set of immiscible discrete volumes in a first direction by withdrawing from the conduit, some of the first fluid from the first end of the set; and moving the set in the first direction by adding to the conduit, more first fluid at the second end of the set. In some embodiments, the method can involve processing a first fluid that comprises an oil and a second fluid that comprises an aqueous liquid, for example, an aqueous sample that is immiscible in the oil. In some embodiments, the method can further comprise moving the set past a valve in the conduit and closing the valve before moving the set in the first direction by adding to the conduit more first fluid at the second end of the set. In some embodiments, closing a valve can comprise rotating a rotary valve as described herein, for example, in connection with
Other embodiments of the present teachings will be apparent to those skilled in the art from consideration of the present specification and practice of the present teachings disclosed herein. It is intended that the specification and examples be considered as exemplary only and not be limiting. All cited references, patents, and patent applications are incorporated in their entireties herein by reference.
The present application claims the benefit of priority under 35 U.S.C. 119 of earlier filed U.S. Provisional Patent Application No. 60/710,167, filed Aug. 22, 2005, U.S. Provisional Patent Application No. 60/731,133, filed Oct. 28, 2005, and U.S. Provisional Patent Application No. 60/818,197, filed Jun. 30, 2006, which are incorporated herein in their entireties by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60710167 | Aug 2005 | US | |
| 60731133 | Oct 2005 | US | |
| 60818197 | Jun 2006 | US |
