WO2014113663A1

WO2014113663A1 - In-line polymerase chain reaction

Info

Publication number: WO2014113663A1
Application number: PCT/US2014/012039
Authority: WO
Inventors: David A. Hill
Original assignee: Hill David A
Priority date: 2013-01-18
Filing date: 2014-01-17
Publication date: 2014-07-24

Abstract

A method and apparatus for In-Line Polymerase Chain Reaction (PCR) is disclosed. In In-Line PCR an emulsified reaction mixture moves through a tube or channel, which remain in contact with thermal elements that maintain a specified temperature. This departs from the prior art PCR methods and apparatuses where the reaction mixture remains static in a reaction well, tube, vessel, or chamber and the environment surrounding it changes. The emulsified reaction mixture flows through a tube propagating it along or through thermal elements of various temperatures. This permits the stages of PCR to progress as the reactants flow by each thermal element. The emulsified reaction mixture then enters into fluorescent reader for sorting or analysis of the DNA target product generated by the PCR or can be analyzed by conventional analysis.

Description

[0001] IN-LINE POLYMERASE CHAIN REACTION

[0002] CROSS REFERENCE TO RELATED APPLICATION

[0003] This application claims the benefit U.S. provisional application No.

61/754,111, which was filed January 18, 2013 and is incorporated herein by reference as if fully set forth.

[0004] FIELD OF INVENTION

[0005] The disclosure relates to Polymerase Chain Reactions (PCR). More specifically it is related to a method and apparatus for In- Line PCR.

[0006] BACKGROUND

[0007] The Polymerase Chain Reaction (PCR) is used to amplify DNA and has become one of the most commonly used techniques in the medical and molecular biological fields. PCR is used to amplify single or multiple copies of a piece of DNA across several orders of magnitude in order to produce millions of copies of a particular DNA sequence.

[0008] PCR is an indispensable technique used in medical and biological research labs for a variety of applications including but not limited to: DNA cloning for sequencing, DNA-based phylogeny, functional analysis of genes, the diagnosis of hereditary diseases, the identification of genetic fingerprints for use for example in forensic sciences and paternity testing, and the detection and diagnosis of infectious diseases.

[0009] In current PCR protocols, reaction mixtures are prepared for thermal cycling, put into reaction chambers or vessels, and placed inside a thermocycler. In typical applications the thermocycler is programmed to perform a series of cycles that precisely heat and cool the reaction. The heating and cooling is performed by changing the temperature of a conductive block or water bath, in which the PCR reaction vessels had been placed for defined periods of time. This series of heating and cooling steps melts, anneals, and elongates the DNA template.

[0010] Typically, a PCR is a three-step reaction. The sample containing a dilute concentration of DNA is mixed with a heat- stable DNA polymerase, such as Taq polymerase, which is an enzyme originally isolated from the bacterium Thermus Aquaticus . The DNA is also typically mixed with DNA oligonucleotides or primers, Deoxynucleoside triphosphates (dNTPs), and magnesium. The DNA primers are required for initiation of the DNA synthesis.

[0011] In the first step of PCR, the reaction mixture is heated. The heating causes DNA melting of the DNA template, which denatures the double-stranded DNA. During this step the hydrogen bonds between the complementary bases are disrupted yielding a single-stranded DNA.

[0012] In the second step, the temperature is decreased allowing the primers to anneal to specific complementary sequences of the single-stranded DNA template. Stable hydrogen bonds are formed during this step when the primer sequence closely matches the DNA template sequence. During this step the polymerase binds to the primer-DNA template hybrid and begins to synthesize DNA.

[0013] In the third step, the temperature is altered depending on the type of polymerase used. This allows the DNA polymerase to extend the primers to create a new strand of DNA, complementary to the template strand, thus doubling the quantity of double-stranded DNA in the reaction.

[0014] This sequence of denaturation, annealing, and extension is repeated for several cycles, resulting in the exponential amplification of the input DNA. As the DNA polymerase loses activity or the dNTPs and primers are consumed, the reaction levels off and plateaus.

[0015] After nearly three decades of use and innovation, there have been many improvements to PCR instruments and protocol. For examples, thermocycling reaction vessels have been constructed with thinner walls to increase efficiency of heat transfer between the thermal element of the thermocycler and the reaction, different types of thermo stable polymerases have been engineered to be heat activated, have faster elongation and/or better fidelity, and reaction components have been adjusted to achieve more reliable reactions. Additionally, the reaction volume has been decreased to the extent possible to minimize cost per reaction as demonstrated by the production and use of 384-well and 1536-well reaction plates. Even though there have been many improvements to increase the efficiency of PCR, there has been very little innovation in the manner in which the reactions are conducted; PCR reactions are placed in a reaction vessel which is placed statically into a thermocycler instrument that is programmed to vary the temperature of the thermal elements in order to conduct PCR cycling.

[0016] Methods for conducting PCR amplifications fall into two general classes. The approach typically utilized is a time domain approach in which the amplification reaction mixture is kept stationary and the temperature is cycled (see, e.g., Cheng, et al. (1996) Nucleic Acids Res 24:380-385; Shoffer, et al. (1996) Nucleic Acids Res. 24:375-379; and Hong, et al. (2001) Electrophoresis 22:328- 333, which are all incorporated herein by reference as if fully set forth). While methods utilizing this approach can be conducted with relatively small sample volumes, the methods require complex regulation of heater elements and relatively long reaction times. Another approach that has been discussed is limited to a space domain approach in which three temperature zones are constantly kept at the different temperatures and the reaction mixture runs in a serpentine flow channel above it (see, e.g., Kopp et al. (1998) Science 280:1046- 1048, which is incorporated herein by reference as if fully set forth). A method such as this requires the use of relatively large sample volumes.

[0017] In recent developments, PCR reactions have been miniaturized to unprecedented low volumes using emulsion technology, generating individual reactions on the order of 20 nanoliters and less. The potential efficiencies to PCR by using micro-scale emulsified reactions have not yet been fully realized since emulsified PCR are still processed in conventional PCR reaction vessels and conducted using conventional thermocyclers. Development of In-Line PCR instrumentation and methods will advance PCR by improvements by capturing more of the associated benefits of micro-scale PCR.

[0018] SUMMARY

[0019] In an aspect, the invention relates to an apparatus for In-Line

Polymerase Chain Reaction. The apparatus includes a machine configured for receiving a tube for passing a PCR reaction mixture across at least one thermal element. The thermal element is configurable to be equilibrated at a specified temperature.

[0020] In an aspect, the invention relates to an apparatus for In-Line

Polymerase Chain Reactions. The apparatus includes a first thermal element configurable to be equilibrated at a first temperature, a second thermal element configurable to be equilibrated at a second temperature, and a third thermal element configurable to be equilibrated at a third temperature. The first thermal element, the second thermal element, and the third thermal element are configured to receive a tube for passing an emulsion of Polymerase Chain Reaction reactants and oil across the first thermal element, the second thermal element, and the third thermal element.

[0021] In an aspect, the invention relates to configuring the In-Line PCR apparatus to receive PCR reactants as an emulsion.

[0022] In an aspect, the invention relates to a method of conducting an In-

Line Polymerase Chain Reaction. The method includes moving an emulsion of In- Line Polymerase Chain Reaction reactants and oil through a tube extending successively across a first thermal element equilibrated at a first specified temperature, a second thermal element equilibrated at a second specified temperature, and a third thermal element equilibrated at a third specified temperature. The method includes repeating the moving step 0— n times across at least one thermal element.

[0023] In an aspect, the invention relates to a method of conducting an In-

Line Polymerase Chain Reaction. The method includes creating an emulsion of reactants and oil; moving said emulsion through a tube; and heating and cooling in alternative steps said tube through the use of at least one thermal element equilibrated at specified temperatures through which the tube passes.

[0024] In an aspect, the invention relates to the In-Line PCR apparatus being central to emulsion generation and product analysis. The flow of the PCR reactants of In-Line PCR may be a continuous flow from droplet generation to product analysis.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The following detailed description of the preferred embodiments will be better understood when read in conjunction with the appended drawings. For the purpose of illustration, there are shown in the drawings embodiments which are presently preferred. It is understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. In the drawings:

[0027] FIG. 1 is a diagram of the flow of the In-Line PCR in the preferred embodiment.

[0028] FIG. 2 is a diagram of the top side view of the flow of the In-Line

PCR in the preferred embodiment.

[0029] FIG. 3 is a diagram of the PCR emulsion according to the various embodiments.

[0030] FIG. 4 is a diagram of the PCR emulsion flowing through the tube according to the various embodiments.

[0031] FIG. 5 is a diagram of the flow of the In-Line PCR in the embodiment including a emulsification generator.

[0032] FIG. 6 is a diagram of the flow of the In-Line PCR in the embodiment including a fluorescent reader.

[0033] FIG. 7 is a diagram of an In-Line PCR machine.

[0034] DETAILED DESCRIPTION

[0035] This description of the embodiments is intended to be read in connection with the accompanying drawings, which are to be considered part of the entire written description. In the description, relative terms such as "top," bottom," "right," and "left" as well as derivatives thereof should be construed to refer to the orientation as then described or as shown in the drawing under discussion. These relative terms are for convenience of description and do not require that the apparatus be constructed or operated in a particular orientation. The words "a" and "one," as used in the claims and in the corresponding portions of the specification, are defined as including one or more of the referenced item unless specifically stated otherwise. This terminology includes the words above specifically mentioned, derivatives thereof, and words of similar import. The phrase "at least one" followed by a list of two or more items, such as "A, B, or C," means any individual one of A, B or C as well as any combination thereof.

[0036] A method and apparatus for In-Line Polymerase Chain Reaction

(PCR) is provided. In In-Line PCR, the reaction mixture moves through a tube, which remain in contact with multiple thermal elements that maintain a fixed temperature. This departs from the prior art PCR methods and apparatuses where the reaction mixture remains static in a reaction tube, vessel, or chamber and the environment surrounding it changes.

[0037] During In-Line PCR, the reaction is moved through a tube. While moving through the tube, the reaction is equilibrated to various temperatures for a specific duration of time. The duration of time for each temperature is dependent upon the flow rate of the sample and the length of the thermal element. The result is that the sample is alternatively heated and cooled in discrete steps as it proceeds through the tube.

[0038] The materials of the tubing can be of any formulation, including but not limited to stainless steel, polyethylene, poly propylene, nylon, vinyl, latex, and the like.

[0039] The flow of a liquid through tubing is not equal across the cross section of the tubing, as described by Hagan-Poiseuille's law. If the tube were frictionless, all liquid throughout the tube's cross section would flow at the same rate. However, since friction near the tube walls slows the liquid in the tube, the liquid near the edges of a cylindrical tube will flow more slowly than the liquid near the center of the tube. In an embodiment, In-Line PCR reactants are isolated from the frictional forces at the tube walls though the use of an emulsion, thus, the reaction droplets are exposed to each of the alternating thermal elements for a consistent duration of time. Separating the reactants from the frictional forces at the tube walls is critical to establish a constant flow of droplet reactions to be constant reactants throughout the entire tube cross section.

[0040] This separation of the In- Line PCR reactants from the tube walls may be accomplished by creating an emulsion of PCR and oil. Oil is used as a medium in which the aqueous droplet PCR reactions are formed. In one aspect of In-Line PCR, the reactants are surrounded by oil to create a droplet of emulsified PCR reaction mixture. The emulsion is then propagated through the tube. The PCR reactants within the droplet may be propagated through the tubing at a set rate. Different types of oils may help form different size droplets or other physical attributes of the droplet like integrity. Tubing size may be chosen to provide a particular velocity of the droplets or orient the droplets with respect to one another. For example, the capillary may be small enough to physically restrict the droplets into a single file orientation - one droplet may not pass by another. Or, it may be larger allowing multiple droplets to occupy the same cross section

[0041] Creating an emulsion has the additional benefit that the reactants in each droplet of emulsified PCR reaction mixture do not contact the tube walls. As a result the PCR reaction mixture remains pristine within the reaction droplet. The emulsion prevents retention of the reactants in the tubing and cross mixing of reactants from reaction droplet to reaction droplet. Following propagation through the reaction cycles, if the In-Line PCR generated product is needed for further characterization or experimentation, it can be released from the emulsion by disrupting the emulsion with physical agitation, alcohol, a non- ionic detergent or similar amphipathic agent or other methods. The formulation and type of disruption used depends on the type of emulsion created and would be known to a skilled artisan. For example emulsions used in next generation sequencing (NGS) are typically broken with alcohol so the reaction product can be collected and further processed. Droplet digital emulsions droplets are "hardened" using a proprietary formulation within the oil and PCR reaction mix. The "hardening" is done by heating the emulsion to 95 degree C for 5 minutes. This type of emulsion can be disrupted by agitation. Droplet digital PCR methods are not designed to produce a recoverable product, so no protocol is available. However, a skilled artisan would readily know a method to collect the PCR product by disrupting the emulsion created.

[0042] In-Line PCR has several advantages over typical PCR methods and apparatuses. The tubes for propagating the droplets in In-Line PCR are smaller than typical PCR reaction vessels used in conventional PCR thermocyclers. The smaller volume within the tubes will reach equilibrium with the environment or thermal elements much more rapidly than the conventional, larger reaction vessels. Reaching equilibrium more rapidly using In-Line PCR may lead to much faster cycling times. Faster cycling times result in completion of the reaction more quickly. Additionally, smaller reaction volumes may be used, typically emulsion droplets are 20 nL or less, providing micro-scale PCR advantages when compared to convention reactions that are orders of magnitude larger.

[0043] Flowing the PCR emulsion through the In-Line PCR apparatus overcomes the limitation in the current art of thermocycling emulsified PCR reactions. Currently, when thermocycling a PCR emulsion (created for NGS or droplet digital PCR) in a conventional PCR tube or plate, the ramp time between temperature steps needs to be more gradual than those used for non-emulsified reactions of the same volume. This is because the emulsified reaction droplets inhibit the natural convection that occurs when heating or cooling the reaction in a typical PCR reaction vessel. Thus, the emulsified droplets in the center of the standard PCR reaction vessel come to equilibrium with the block much later than the droplets near the edge of the reaction standard reaction. Hence, thermocycling emulsified reactions in standard reaction vessels, like those used in droplet digital PCR and next generation sequencing, have protocols that use a slower ramp time in the thermocyclers. Using the In-Line PCR apparatus would overcome this limitation by treating each droplet individually as it passes by or through each thermal element.

[0044] The extremely small size of the reaction droplets in the emulsified

PCR reaction mixture may reduce temperature gradients within the reaction. As a result, all components within each reaction droplet achieves equilibrium with the thermal element at essentially the same time and remain at the equilibrium temperature for the same duration of time. Thus, In-Line PCR will be able to cycle faster and may produce far fewer non-desired default sequences.

[0045] During In-Line PCR, the thermal elements of an In-Line PCR apparatus may remain at a defined fixed temperature. Having each thermal element at a defined fixed temperature would essentially eliminate the ramp time between each temperature step because In-Line PCR reactants would flow from one thermal element to another without the need to heat or chill the mass of the thermal element to a new temperature. This is in contrast to current PCR thermocyclers that have a ramp time due to physical limitations; the ramp time is defined as the time it takes the thermocycler to heat and/or chill the mass of the thermal element. Additionally, the fixed temperature elements may be easier to manufacture and may be longer lived than the Peltier driven reaction current temperature control blocks found in conventional thermocyclers.

[0046] Furthermore, development of apparatus to support In-Line PCR may not be limited to current designs of thermal elements used in conventional thermocyclers. The miniature scale of the tubing in the In-Line PCR path may allow for other technologies to establish a thermal element, such as using micro electronics or a focused laser. These advancements will further distinguish In- Line PCR as a method of choice for rapid PCR.

[0047] Embodiments will now be described with reference to the figures showing how the In-Line Polymerase Chain Reaction (PCR) can be made and used. Like reference numerals are used throughout the description and several views indicate like or corresponding parts.

[0048] Referring to FIG. 1 and FIG. 3, an In-Line Polymerase Chain

Reaction (PCR) machine 10 is shown in the preferred embodiment. The In-Line PCR machine 10 is configured to receive a tube 12, which is adapted for In-Line PCR. The tube 12 can be a micro-capillary tube or tube of greater cross section depending on the desired flow rate for the In-Line PCR.

[0049] The tube 12 adapted for use in In-Line PCR allows the emulsified

PCR mixture 16 to pass through the machine 10, which is configured for receiving the tube 12. The machine 10 provides a path for the tube 12 to be propagated across a first thermal element 13 equilibrated to a first temperature, a second thermal element 14 equilibrated to a second temperature, and a third element 15 equilibrated to a third temperature. A tube "propagated across" a thermal element may be within a channel along the surface of the thermal element or through the thermal element, or otherwise associated with the thermal element such that the contents of the tube are equilibrated with exposed to the temperature of the thermal element. The emulsified PCR mixture may be passed through the tube by any suitable mechanism including but not limited to gravity, pumping or suction. The tube 12 may be etched into slide, or disk or be otherwise fabricated to be a one-use consumable that is placed in contact with the thermal element(s) of the In-Line PCR machine.

[0050] The reaction droplets in the PCR mixture 16 are part of an emulsion of PCR reactants and oil. This emulsion results in the aqueous phase (the PCR reaction) being surrounded by oil creating droplets of PCR reactants. The use of an emulsion results in the frictional forces of the tube 12 walls being separated from the reaction droplets in the emulsified PCR mixture 16. The oil surrounding each reaction droplet in the emulsified PCR mixture 16 absorbs the friction of the tube 12 wall. Because these frictional forces are isolated from each reaction droplet, the flow or propagation of each reaction droplet is constant throughout the cross section of the tube 12. This allows each individual reaction droplet to be exposed to the various temperatures of the separate thermal elements for the same duration of time. The length of the tube and/or the rate of flow across the elements can be varied in order to establish the duration of time that the reaction spheres in the emulsified PCR mixture 16 are exposed to the temperature of that particular thermal element. The velocity at which the emulsion of PCR reactants 16 pass through the tube can be varied in order to establish the duration of time that the reaction droplets in the emulsified PCR mixture 16 are exposed to the temperature of that particular thermal element.

[0051] In an embodiment, In-Line PCR is set up by installing a tube 12 that is adapted for the In-Line PCR in the In-Line Polymerase Chain Reaction (PCR) machine 10. The emulsified PCR mixture 16 can be placed in the installed tube 12. During a first step of In-Line PCR, the emulsified PCR mixture 16 is heated as it flows through the portion of the machine 10 that propagates the installed tube 12 across the first thermal element 13 equilibrated to a first temperature. The heating causes DNA melting of the DNA template in the emulsified PCR mixture 16, which denatures the double-stranded DNA. During this step the hydrogen bonds between the complementary bases are disrupted yielding a single- stranded DNA. In the second step In-Line PCR, the temperature is decreased as the emulsified PCR mixture 16 flows through the portion of the machine 10 that propagates the installed tube 12 across the second thermal element 14 equilibrated to a second temperature. The decreased temperature of the second thermal element 14 allows the primers to anneal to specific complementary sequences of the single- stranded DNA template created in the first step. Stable hydrogen bonds are formed during this step when the primer sequence is closely complementary matches the DNA template sequence. During this step the polymerase binds to the primer-DNA template hybrid and begins to synthesize a strand of DNA complementary to the template strand. In the third step of In-Line PCR, the temperature is altered depending on the type of polymerase used as the emulsified PCR mixture 16 flows through the portion of the machine 10 that propagates the installed tube 12 through the third thermal element 15 equilibrated to a third temperature. This allows the DNA polymerase to extend the primers to create a new strand of DNA, complementary to the template strand of DNA, thus doubling the quantity of DNA in the reaction.

[0052] The temperature of the first thermal element, the second thermal element, or the third thermal element may be selected based on the particular nucleic acids subject to the reaction, the polymerase utilized, the magnesium concentration, and other components etc. as known to the skilled artisan.

[0053] Referring to FIG. 2, a top-side view of an In- Line Polymerase Chain

Reaction (PCR) machine 10. The tube 12 is adapted to allow the reaction spheres in the emulsified PCR mixture 16 to pass through the machine 10, which is configured to receive a tube 12. In this embodiment the tube 12 is propagated in a circular path through a first thermal element 13 equilibrated to a first temperature, a second thermal element 14 equilibrated to a second temperature, and a third element 15 equilibrated to a third temperature.

[0054] The path of the tube 12 can also be altered in other embodiments of the machine 10 such that the path of the tube 12 is not circular. Regardless of the shape of the path of the tube 12, each sphere in the emulsified PCR mixture 16 is exposed to the alternate temperature of each thermal element for a consistent duration of time because of the constant flow rate caused by the absorption of the frictional forces of the walls of the tube 12 by the oil in the emulsified PCR mixture 16. The duration of time for each thermal element may vary from the other thermal elements as defined by the length of the passage or the speed of the droplets passing through that thermal element.

[0055] Referring to FIG. 3, the emulsified PCR mixture 16 passes through the tube 12. The frictional forces of the walls of the tube 12 are absorbed by the oil in the emulsified mixture 16 such that the reaction droplets flow at a constant rate and are therefore exposed to the various temperatures of each thermal element for a consistent duration of time. The duration of time for each temperature is dependent upon the flow rate of the emulsified PCR mixture 16 and the length of the tubes path within the thermal element. The result is that the reaction droplets in the emulsified PCR mixture 16 are alternatively heated and cooled in discrete steps as it proceeds through the tube 12.

[0056] Referring to FIG. 4, several of the reaction droplets spheres in the emulsified PCR mixture 16 pass through the tube 12 adapted for use in the Inline Polymerase Chain Reaction machine 10. A single droplet may be an isolated reaction. The reaction spheres in the emulsified PCR mixture 16 are designed not to coalesce through the process but may remain separated from each other. As a result the PCR reaction mixture remains pristine within each sphere in the emulsified PCR mixture 16. The emulsion also prevents retention of the reactants in the tubing and cross mixing of reactants from sphere to sphere. Following propagation through the tube 12, the emulsion can be disrupted if the PCR generated product needs to be collected for further investigation or analysis.

[0057] In an embodiment, the tube 12 is configured to remain on the machine 10 between uses. In this embodiment the tube 12 does not need to be installed before conducting the In- Line PCR because it is part of the machine 10 configuration. The tube 12 may, however, be removable for cleaning or maintenance purposes.

[0058] In an embodiment, the tube 12 is configured to be removed from the machine 10 between uses as a one-use consumable. In this embodiment the tube 12 needs to be installed before conducting the In- Line PCR because together they form the In-Line PCR apparatus 10. The tube 12 may, however, be reused if desired.

[0059] Referring to FIG. 5, in an embodiment the emulsion generator is part of the In-Line PCR apparatus. The emulsion generator 17 forms the droplets of reactants dispersed in oil. The emulsion is propagated from the emulsion generator 17 through the tubel2 passing thermal elements 13, 14, and 15 of the In-Line PCR machine 10.

[0060] Referring to FIG. 6, in an embodiment the In-Line PCR concludes with analysis of the PCR products. In this embodiment, following the flow of the reaction through the thermal elements 13, 14 and 15 of the In-Line PCR machine, the spheres of emulsified PCR mixture 16 flow out of the tube 12 and are analyzed.

[0061] Analysis may be performed on an isolated reaction, which may correspond to one reaction sphere. The analysis of the droplets maybe performed using a flow through cell analyzing device 18. Since the droplets remain intact through the analysis process, they may be analyzed qualitatively and/or quantitatively. This includes counting or sorting droplets containing product when analyzed using technology similar to Fluorescence Activated Cell Sorting

(FACS). Analytical devices include instrumentation that manipulate, sort, quantify signal based on the type of reaction readout designed into the reaction.

[0062] Following the product analysis by sorting, counting, and/or quantification, the contents from one or more isolated reaction droplets may be collected and analyzed. The reaction products from collected by disrupting the droplet. The collected isolated reaction(s) may be combined with a substance to disrupt the droplets. The collected isolated reaction(s) may be separated from oil by a physical method. Collected isolated reaction(s) may be manually or robotically collected and processed, or a combination thereof.

[0063] Still referring to FIG. 6, an embodiment of In-Line PCR concludes with the use of fluorescent reporting where fluorescent analyzer 18 is a type of analyzer. In preparing the In-Line PCR for this embodiment, the spheres of emulsified PCR mixture 16 may be further combined with a dsDNA-binding fluorescent dye or marker. The dsDNA-binding-fluorescent dye may be SYBR

(Green Mackay IM, Arden KE, Nitsche A (March 2002). "Real-time PCR in virology". Nucleic Acids Res. 30 (6): 1292-305). Other reaction components can be probes labeled with any one of many available fluorophores such as TaqMan or similar probes. Holland, P. M.; Abramson, R. D.; Watson, R.; Gelfand, D. H.

(1991). "Detection of specific polymerase chain reaction product by utilizing the

5'— 3' exonuclease activity of Thermus aquaticus DNA polymerase". Proceedings of the National Academy of Sciences of the United States of America 88 (16):

7276-7280. The use of dyes or probes is to detect which droplets went to completion; i.e., had target, primers and other necessary reactants.

[0064] In the application of qPCR, the product would be analyzed after each elongation step. The qPCR reaction design would work in In-Line PCR if the instrument includes a detector to detect product after passing through the elongation thermal element in the instrument. And since each droplet is a unique reaction vessel, each droplet would have to be uniquely labeled or propagated in single file in order to establish a quantification curve for that droplet. In an embodiment, following the third step of the In-Line PCR, the reaction(s) may be processed as discussed in reference to FIG. 5, and then provided to fluorescence reader 18, which analyzes collected isolated reaction(s) a fluorescent signal. Software controlling on the reader 18 may read and calculate the concentration of the DNA target in the isolated reaction(s).

[0065] Still referring to FIG. 6, in an alternative, the primers may include a fluorescent label and a quencher. The workflow may be described as above, but where the fluorescence reader 18 is adapted to detect and analyze the increase in fluorescence due to release of the fluorescent dye from the proximity of the quencher. Reaction components may be adapted for In- Line PCR but similar to those for Droplet Digital PCR as described in Hindson, et al., High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number (2011) Anal. Chem. 83: 8604 - 8610, which is incorporated herein by reference as if fully set forth.

[0066] Referring to FIG. 7, in an embodiment the In-Line PCR machine includes the droplet generator 17, the tube to propagate the reaction 12 and the analytical device 18 as a continuous path through the In-Line PCR machine. In this embodiment the droplets are generated, flow through the thermal elements 13, 14 and 15 of the In-Line PCR machine, and through an analytical device 18 in a continuous path 12. The design of this embodiment for an instrument for an In- Line PCR connects the steps from droplet generation to the droplet detection by a continuous flow through a machine with thermal elements eliminating the need for external manual or robotic manipulation. The process used with this instrument is superior to the process described in Hindson et.al. in which the droplets are generated in one machine, manually removed from the droplet generation vessel, placed in a 96- well plate for thermocycling and then, following thermocycling they are removed from the thermocycler and placed in another instrument that separates each droplet and detects if the reaction within the droplet went to completion by the detection of fluorescent signal.

[0067] Conditions for PCR known in the art may be adapted for In-Line

PCR. See VanGuilder HD et al. Twenty-five years of quantitative PCR for gene expression analysis (2008) Biotechniques 44 (5): 619-626, which is incorporated herein by reference as if fully set forth. See also Udvardi MK, et al. Eleven Golden Rules of Quantitative RT-PCR(2008) Plant Cell 20 (7): 1736-1737; Spackman E et al. Type A influenza virus detection and quantitation by real-time RT-PCR (2008) Methods Mol Biol 436: 19-26; Gertsch J et al. Relative quantification of mRNA levels in Jurkat T cells with RT-real time-PCR (RT-rt- PCR): new possibilities for the screening of anti-inflammatory and cytotoxic compounds (2002) Pharm Res 19 (8): 1236-1243; Julie Logan, Kirstin Edwards, and Nick Saunders (Eds.) Real-Time PCR: Current Technology and Applications (2009) Caister Academic Press; S. Dhanasekaran et al. and TB Trials Study Group Comparison of different standards for real-time PCR-based absolute quantification (2010 Mar) Immunol Methods. 354 (1-2): 34-9; Scheie JH et al. Quantitative real-time RT-PCR data analysis: current concepts and the novel "gene expression's CT difference" formula (2006) J Mol Med 84: 901-10; Nailis H et al. Development and evaluation of different normalization strategies for gene expression studies in Candida albicans biofilms by real-time PCR (2006) BMC Mol Biol. 7: 25; Nolan T et al. Quantification of mRNA using real-time RT-PCR (2006) Nat. Protoc. 1 (3): 1559-1582; Boggy G et al. A Mechanistic Model of PCR for Accurate Quantification of Quantitative PCR Data (2010) PLOS One 5 (8): el2355; and Sails AD Applications in Clinical Microbiology. Real-Time PCR: Current Technology and Applications (2009) Caister Academic Press; Filion, M (editor) Quantitative Real-time PCR in Applied Microbiology (2012) Caister Academic Press. ISBN 978-1-908230-01-0, which are all incorporated herein by reference as if fully set forth. Other PCR applications not explicitly described here could potentially be adapted and used with In- Line PCR. The skilled artisan would be able to recognize and adapt their method to this instrument.

[0068] Embodiments can make use of more or fewer thermal elements depending on the output of the reaction to be generated and the number of alternate temperatures necessary for the reaction. In embodiments comprising additional thermal elements, each droplet resulting from the emulsified PCR mixture 16 passes through the additional thermal elements at the same rate and the time spent with an additional thermal element will depend on the path length across of the additional thermal elements and the rate of flow.

[0069] EMBODIMENTS

[0070] The following list includes particular embodiments of the present invention. The list, however, is not limiting and does not exclude embodiments otherwise described herein or alternate embodiments, as would be appreciated by one of ordinary skill in the art.

1. An apparatus for In-Line Polymerase Chain Reaction comprising: a machine configured for receiving a tube for passing an emulsified reaction mixture across at least one thermal element; and said thermal element configurable to be equilibrated at a specified temperature.

2. The apparatus of embodiment 1 wherein said emulsified reaction mixture is an emulsion of Polymerase Chain Reaction reactants and oil.

3. The apparatus of any one or more of the preceding embodiments, wherein the apparatus includes a fluorescent reader for detecting the DNA product based on the level of fluorescence in a sample droplet produced from In Line Polymerase Chain Reaction on said emulsified reaction mixture.

4. The apparatus of any one or more of the preceding embodiments, wherein said emulsified reaction mixture is combined with a dsDNA-binding fluorescent dye.

5. The apparatus of any one or more of the preceding embodiments, wherein said emulsified reaction mixture is combined with primers having attached fluorescent dye and quencher.

6. The apparatus of any one or more of embodiments 3— 5, wherein the fluorescent reader is for detecting the concentration of target DNA produced based on the level of fluorescence in a sample droplet produced from In Line Polymerase Chain Reaction on said emulsified reaction mixture.

7. The apparatus of any one or more of the preceding embodiments further comprising a flow-through product analyzing device.

8. The apparatus of any of the preceding embodiments further comprising an emulsion generator.

9. An apparatus for In-Line Polymerase Chain Reactions comprising: a first thermal element configurable to be equilibrated at a first temperature, a second thermal element configurable to be equilibrated at a second temperature, and a third thermal element configurable to be equilibrated at a third temperature; the first thermal element, the second thermal element, and the third thermal element configured to receive a tube for passing an emulsion of Polymerase Chain Reaction reactants and oil across the first thermal element, the second thermal element, and the third thermal element.

10. The apparatus of embodiment 9 including the features of any one or more of embodiments 1-8.

11. The apparatus of any one or more of embodiments 9 or 10 further comprising an emulsion generator, and wherein the emulsion of Polymerase Chain Reaction reactants and oil are mixed to generate the emulsification of PCR reactants droplets in oil.

12. The apparatus of any or more of embodiments 9-11 further comprising a fluorescent reader, and wherein the emulsion of Polymerase Chain

Reaction reactants and oil includes a dsDNA-binding fluorescent dye and the fluorescent reader is adapted for detecting the presence concentration of target

DNA produced based on the level of fluorescence in a sample produced from In Line Polymerase Chain Reaction on said emulsified reaction mixture, wherein the fluorescent reader is optionally part of a FACS apparatus.

13. The apparatus of any of one or more of embodiments 9-11 further comprising a fluorescent reader, and wherein the emulsion of Polymerase Chain Reaction reactants and oil includes primers having attached fluorescent dye and quencher and the fluorescent reader is adapted for determining the presence of target DNA produced based on the level of fluorescence in a sample produced from In Line Polymerase Chain Reaction on said emulsified reaction mixture.

14. The apparatus of any one or more of embodiments 9-13 further comprising of a fluorescent detector used to sort the emulsion droplets of Polymerase Chain Reaction based on fluorescent signal of each droplet.

15. The apparatus of any one or more of embodiments 9-14 further comprising a flow-through product analyzing device.

16. The apparatus of any one or more of embodiments 9-15 further comprising an emulsion generator.

17. A method of conducting an In- Line Polymerase Chain Reaction, the steps comprising:

moving an emulsion of In-Line Polymerase Chain Reaction reactants and oil through a tube received and extending across the thermal element(s) in the apparatus of any one more of embodiments 1-16.

18. The method of embodiment 17 further comprising repeating the moving step 0-N times.

19. A method of conducting an In-Line Polymerase Chain Reaction, the steps comprising: moving an emulsion of In-Line Polymerase Chain Reaction reactants and oil through a tube extending successively across a first thermal element equilibrated at a first specified temperature, a second thermal element equilibrated at a second specified temperature, and a third thermal element equilibrated at a third specified temperature; and

repeating the moving step 0-n times;

20. The method of any one or more of embodiments 17-19 further comprising after the repeating step isolating the product from the emulsion by disrupting the emulsion with physical agitation, alcohol, a non-ionic detergent or similar amphipathic product to release In-Line PCR generated product.

21. The method of any one or more of embodiments 17-20 further comprising steps of combining said emulsion with a dsDNA-binding fluorescent dye, collecting isolated reactions, and flowing the collected isolated reactions into a fluorescent reader.

22. The method of any one or more of embodiments 17-21 further comprising steps of combining said emulsion with primers having attached fluorescent dye and quencher, collecting isolated reactions, flowing the collected isolated reactions into a fluorescent reader for counting the number of droplets containing the target DNA produced based on the level of fluorescence in the collected isolated reactions.

23. The method of any one or more of embodiments 17-22 further comprising a step of collecting isolated reactions, and providing the collected isolated reactions to an electrophoresis apparatus.

24. A method of conducting In-Line Polymerase Chain Reactions, the steps comprising:

creating an emulsion of reactants and oil; moving said emulsion through a tube; and

heating and cooling in alternative steps said tube through the use of at least one thermal element equilibrated at specified temperature through which the tube passes.

25. The method of embodiment 24, wherein the at least one thermal element is within the apparatus of at least one or more of embodiments 1-16 and the method includes implementing the apparatus of at least one or more of embodiments 1-16.

26. The method of embodiment 24 or 25, wherein the method includes the method of any one or more of embodiments 17-23.

27. The method of any one or more of embodiments 24-26 further comprising collecting isolated reactions and disrupting the emulsion in an isolated reaction with physical agitation, alcohol, a non-ionic detergent or similar amphipathic product to release the generated PCR product.

28. The method of any one or more of embodiments 24-27 further comprising steps of combining said emulsion with a dsDNA-binding fluorescent dye, collecting isolated reactions, and flowing the collected isolated reactions into a fluorescent reader for determining the presence of the target DNA produced based on the level of fluorescence in the collected isolated reactions.

29. The method of any one or more of embodiments 24-27 further comprising steps of combining said emulsion with primers having attached fluorescent dye and quencher, collecting isolated reactions, and flowing the collected isolated reactions into a fluorescent reader for determining the presence of the target DNA produced based on the level of fluorescence in the collected isolated reactions. [0071] The methods and features described above may be performed, mutatis mutandis, using any appropriate architecture and/or computing environment. The apparatuses above may be controlled using any appropriate architecture and/or computing environment to perform a method herein. Although features and elements are described above in particular combinations, each feature or element can be used alone or in any combination with or without the other features and elements. The methods provided herein may be implemented in a computer program, software, or firmware incorporated in a non-transitory computer-readable storage medium for execution by a general purpose computer or a processor. Examples of non-transitory computer-readable storage mediums include a read only memory (ROM), a random access memory (RAM), a register, cache memory, semiconductor memory devices, magnetic media such as internal hard disks and removable disks, magneto-optical media, and optical media such as CD-ROM disks, and digital versatile disks (DVDs).

[0072] Further embodiments herein may be formed by supplementing an embodiment with one or more element from any one or more other embodiment herein, and/or substituting one or more element from one embodiment with one or more element from one or more other embodiment herein.

[0073] U.S. Patent No. 8,535,889, which issued from U.S. Appln. No.

13/026,107 (filed Feb. 11, 2011) and is incorporated herein by reference as if fully set forth, describes droplet formation, PCR of emulsified droplets and analysis. The invention generally relates to droplet based digital PCR and methods for analyzing a target nucleic acid using the same. In certain embodiments, U.S. Patent No. 8,535,889 described methods involving forming sample droplets containing, on average, a single target nucleic acid, amplifying the target in the droplets, excluding droplets containing amplicon from the target and amplicon from a variant of the target, and analyzing target amplicons. Any device, method, or reagent from U.S. Patent No. 8,535,889 may be included in an embodiment herein.

[0074] U.S. Pat. No. 7,833,708, which issued from U.S. Appln. No.

11/133,805 (filed May 19, 2005) and is incorporated herein by reference as if fully set forth, describes flowing PCR through stationary chambers on a microfluidic level. Devices include a rotary microfluidic channel and a plurality of temperature regions at different locations along the rotary microfluidic channel at which temperature is regulated. Solution can be repeatedly passed through the temperature regions such that the solution is exposed to different temperatures. Any device, method, or reagent from U.S. Pat. No. 7,833,708 may be included in an embodiment herein.

[0075] Temperatures in in-line PCR could be the same as those used in standard PCR reactions. Without limiting the embodiments, protocols could reference ranges from 4 degree C to 100 degree C, although typically melting is 90 degree C to 100 degree C, annealing is from 22 degree C to 72 degree C, and elongation is performed from 60 degree C to 72 degree C. The temperatures may be optimized for the polymerase being used as well as the template being amplified. These are non-limiting examples of temperatures that maybe utilized in methods herein.

[0076] Non-limiting examples of resident times at a particular temperature may be adapted from standard PCR protocols for melt, annealing and elongation times. Shorter times may be adapted for microfluidics embodiments, since microfluidics minimizes the time needed to equilibrate the oil/droplet solution with the surrounding environment. Ranges of resident times may be 0 to 30 minutes for each step. However, longer times may be needed for enzyme activation or high GC melting in the first step. Typical times for standard PCR is melt 30 seconds to 2 minutes, anneal 15 seconds to 5 minutes, and elongation from 10 seconds to 2 minutes (sometimes hours for long-range PCR). These times, or ranges around these times framed by 5 second increments from the stated time out to +/- 10 times the stated time, may be resident times in embodiments herein.

[0077] Flow rates are coupled with the size of the fluid channel/tube as stated. The flow rates may be set to obtain optimal reaction sensitivity in the apparatus with the reaction components and polymerase. Flow rates could be optimized with size of flow tube, or length of heated element. A flow range that could be 0 to 1 liter per minute, or a value in a range between and including any two microliter per minute increments selected from 0 to 1 liter per minute. Flow could be stopped and started to accommodate resident times. Droplets may be produced with a flow of about 100 uL/min, or a flow in a range between and including any two values selected from microliter increments of uL/min from 0 to 100 uL/min. Reading the droplets may be accomplished at 16 uL/min, or at any value in a range between any two microliter values selected from increments of uL/min from 0 to 20 uL/min.

[0078] The references cited throughout this application are incorporated for all purposes apparent herein and in the references themselves as if each reference was fully set forth. For the sake of presentation, specific ones of these references are cited at particular locations herein. A citation of a reference at a particular location indicates a manner(s) in which the teachings of the reference are incorporated. However, a citation of a reference at a particular location does not limit the manner in which all of the teachings of the cited reference are incorporated for all purposes.

[0079] It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but is intended to cover all modifications which are within the spirit and scope of the invention as defined by the appended claims; the above description; and/or shown in the attached drawings.

Claims

CLAIMS What is claimed is:

1. An apparatus for In-Line Polymerase Chain Reaction comprising: a machine configured for receiving a tube for passing an emulsified reaction mixture across at least one thermal element; and

said thermal element configurable to be equilibrated at a specified temperature.

2. The apparatus of claim 1 wherein said emulsified reaction mixture is an emulsion of Polymerase Chain Reaction reactants and oil.

3. The apparatus of any of the preceding claims wherein said emulsified reaction mixture is combined with a dsDNA-binding fluorescent dye the apparatus includes a fluorescent reader for detecting the DNA product based on the level of fluorescence in a sample droplet produced from In Line Polymerase Chain Reaction on said emulsified reaction mixture.

4. The apparatus of any of claims 1 or 2 wherein said emulsified reaction mixture is combined with primers having attached fluorescent dye and quencher, and the apparatus includes a fluorescent reader for detecting the concentration of target DNA produced based on the level of fluorescence in a sample droplet produced from In Line Polymerase Chain Reaction on said emulsified reaction mixture.

5. The apparatus of any of the preceding claims further comprising a flow-through product analyzing device.

6. The apparatus of any of the preceding claims further comprising an emulsion generator.

7. An apparatus for In-Line Polymerase Chain Reactions comprising: a first thermal element configurable to be equilibrated at a first temperature, a second thermal element configurable to be equilibrated at a second temperature, and a third thermal element configurable to be equilibrated at a third temperature; the first thermal element, the second thermal element, and the third thermal element configured to receive a tube for passing an emulsion of Polymerase Chain Reaction reactants and oil across the first thermal element, the second thermal element, and the third thermal element.

8. The apparatus of claim 7 further comprising an emulsion generator, and wherein the emulsion of Polymerase Chain Reaction reactants and oil are mixed to generate the emulsification of PCR reactants droplets in oil.

9. The apparatus of any of claims 7 or 8 further comprising a fluorescent reader, and wherein the emulsion of Polymerase Chain Reaction reactants and oil includes a dsDNA-binding fluorescent dye and the fluorescent reader is adapted for detecting the presence concentration of target DNA produced based on the level of fluorescence in a sample produced from In Line Polymerase Chain Reaction on said emulsified reaction mixture, wherein the fluorescent reader is optionally part of a FACS apparatus.

10. The apparatus of any of claims 7 or 8 further comprising a fluorescent reader, and wherein the emulsion of Polymerase Chain Reaction reactants and oil includes primers having attached fluorescent dye and quencher and the fluorescent reader is adapted for determining the presence of target DNA produced based on the level of fluorescence in a sample produced from In Line Polymerase Chain Reaction on said emulsified reaction mixture.

11. The apparatus of any of claims 7— 10 further comprising of a fluorescent detector used to sort the emulsion droplets of Polymerase Chain Reaction based on fluorescent signal of each droplet.

12. The apparatus of any of claims 7—10 further comprising a flow- through product analyzing device.

13. The apparatus of any of claims 7— 12 further comprising an emulsion generator.

14. A method of conducting an In- Line Polymerase Chain Reaction, the steps comprising:

moving an emulsion of In-Line Polymerase Chain Reaction reactants and oil through a tube extending successively across a first thermal element equilibrated at a first specified temperature, a second thermal element equilibrated at a second specified temperature, and a third thermal element equilibrated at a third specified temperature; and

repeating the moving step 0 - n times;

15. The method of claim 14 further comprising after the repeating step isolating the product from the emulsion by disrupting the emulsion with physical agitation, alcohol, a non-ionic detergent or similar amphipathic product to release In-Line PCR generated product.

16. The method of claim 14 further comprising steps of combining said emulsion with a dsDNA-binding fluorescent dye, collecting isolated reactions, and flowing the collected isolated reactions into a fluorescent reader.

17. The method of claim 14 further comprising steps of combining said emulsion with primers having attached fluorescent dye and quencher, collecting isolated reactions, flowing the collected isolated reactions into a fluorescent reader for counting the number of droplets containing the target DNA produced based on the level of fluorescence in the collected isolated reactions.

18. The method of claim 14 further comprising a step of collecting isolated reactions, and providing the collected isolated reactions to an electrophoresis apparatus.

19. A method of conducting In-Line Polymerase Chain Reactions, the steps comprising:

creating an emulsion of reactants and oil;

moving said emulsion through a tube; and

20. The method of claim 19 further comprising collecting isolated reactions and disrupting the emulsion in an isolated reaction with physical agitation, alcohol, a non-ionic detergent or similar amphipathic product to release the generated PCR product.

21. The method of claim 19 further comprising steps of combining said emulsion with a dsDNA-binding fluorescent dye, collecting isolated reactions, and flowing the collected isolated reactions into a fluorescent reader for determining the presence of the target DNA produced based on the level of fluorescence in the collected isolated reactions.

22. The method of claim 17 further comprising steps of combining said emulsion with primers having attached fluorescent dye and quencher, collecting isolated reactions, and flowing the collected isolated reactions into a fluorescent reader for determining the presence of the target DNA produced based on the level of fluorescence in the collected isolated reactions.