SYSTEMS AND METHODS FOR THE AMPLIFICATION OF DNA

Abstract

A system for amplifying nucleic acids is disclosed which, in one embodiment, includes a fluidic device having a sample channel and a heat exchange channel disposed sufficiently close to the sample channel such that a heat exchange fluid in the heat exchange channel can cause a sample in the sample channel to gain or lose heat at desired levels. In one illustrative embodiment, the system further includes three reservoirs coupled to the heat exchange channel and a temperature control system configured to heat fluids stored in the respective reservoirs at different temperatures. One or more pumps and a controller are configured to cause fluid stored in the reservoirs to enter and flow through the heat exchange channel at different times.

Claims

1. A method of amplifying DNA using a system comprising a fluidic device having a sample channel and a heat exchange channel that is used to heat and/or cool a sample in the sample channel, the method comprising: causing a sample of a test solution containing PCR reagents to move through the sample channel of the fluidic device; and while the sample is moving through at least a section of the sample channel: (1) for a first period of time, causing a first heat exchange fluid stored in a first container and regulated at a first temperature while stored in the first container to: (1) exit the first container and (2) move through the heat exchange channel after exiting the first container; (2) for a second period of time, causing a second heat exchange fluid stored in a second container and regulated at a second temperature while stored in the second container to: (1) exit the second container and (2) move through the heat exchange channel after exiting the second container; and (3) for a third period of time, causing a third heat exchange fluid stored in a third container and regulated at a third temperature while stored in the third container to: (1) exit the third container and (2) move through the heat exchange channel after exiting the third container, wherein the first period of time may be different than the second period of time, which may be different than the third period of time, and the first temperature is different than the second temperature, which is different than the third temperature.

2. The method of claim 1, further comprising causing the first heat exchange fluid to enter the third container after exiting the heat exchange channel.

3. The method of claim 2, further comprising causing the second heat exchange fluid to enter the first container after exiting the heat exchange channel.

4. The method of claim 3, further comprising causing the third heat exchange fluid to enter the second container after exiting the heat exchange channel.

5. The method of claim 1, further comprising causing the first heat exchange fluid to enter the first container after exiting the heat exchange channel.

6. The method of claim 5, further comprising causing the second heat exchange fluid to enter the second container after exiting the heat exchange channel.

7. The method of claim 6, further comprising causing the third heat exchange fluid to enter the third container after exiting the heat exchange channel.

8. The method of claim 1, wherein the first temperature is a temperature such that when the first heat exchange fluid moves through the heat exchange channel said fluid heats a sample in the sample channel to a temperature over 80 degrees Celsius, the second temperature is a temperature such that when the second heat exchange fluid moves through the heat exchange channel said fluid cools a sample in the sample channel to a temperature under about 60 degrees Celsius, and the third temperature is a temperature such that when the third heat exchange fluid moves through the heat exchange channel said fluid heats a sample in the sample channel to a temperature between 60 and 80 degrees Celsius.

9. The method of claim 1, wherein at least a portion of the heat exchange channel is beneath the sample channel and parallel with the sample channel.

10. The method of claim 1, wherein at least one dimension of the heat exchange channel and the sample channel is less than about 3000 micrometers.

11. The method of claim 10, wherein the heat exchange channel has a width between about 20 and 2000 micrometers and a depth between about 20 and 2000 micrometers.

12. The method of claim 1, wherein said first heat exchange fluid is identical with the second heat exchange fluid, which is identical with the third heat exchange fluid, and the first heat exchange fluid comprises a gas and/or a liquid.

13. The method of claim 1, wherein said heat exchange fluids comprise water and/or compressed air with pressure from 1 to 200 psia.

14. The method of claim 1, wherein said first heat exchange fluid is different than the second heat exchange fluid, which can be the same or different than the third heat exchange fluid.

15. A method of thermal exchange in a microfluidic chip comprising: directing a first heat exchange fluid at a first temperature through a heat exchange channel for a first period of time, wherein the heat exchange channel is configured to exchange heat with a portion of a sample channel, wherein at least one dimension of the heat exchange channel and the sample channel are less than 1000 micrometers; directing a second heat exchange fluid at a second temperature through the heat exchange channel for a second period of time; and directing a third heat exchange fluid at a third temperature through a heat exchange channel for a third period of time.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings, which are incorporated herein and form part of the specification, illustrate various embodiments of the present invention. In the drawings, like reference numbers indicate identical or functionally similar elements.

[0029] FIG. 1 is a block diagram illustrating a system according to embodiments of the invention.

[0030] FIGS. 2A-2B illustrate a temperature control system according to some embodiments of the invention.

[0031] FIGS. 3A-3C illustrate various configurations of a heat exchange channel according to embodiments of the invention.

[0032] FIG. 4 illustrates a process according to some embodiments of the invention.

[0033] FIGS. 5A-5B illustrate portions of processes according to some embodiments of the invention.

[0034] FIG. 5C illustrates a temperature profile that can result from the use of a temperature control system according to at least one exemplary embodiment.

[0035] FIG. 6 illustrates a temperature control system according to other embodiments of the invention.

[0036] FIG. 7 illustrates a portion of a process according to some embodiments of the invention.

[0037] FIG. 8 illustrates a temperature control system in accordance with other embodiments of the invention.

[0038] FIG. 9 illustrates a temperature control system in accordance with still other embodiments of the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0039] FIG. 1 illustrates a functional block diagram of a system 100 for the amplification of DNA according to some embodiments of the invention. As illustrated in FIG. 1, system 100 may include a microfluidic device 102. Microfluidic device 102 may include one or more microfluidic channels 104. In the example shown, device 102 includes two microfluidic channels, channel 104a and channel 104b. Although only two channels are shown in the exemplary embodiment, it is contemplated that device 102 may have fewer than two or more than two channels. For example, in some embodiments, device 102 includes eight channels 104.

[0040] Device 102 may include two DNA processing zones, a DNA amplification zone 131 (a.k.a., PCR zone 131) and a DNA melting zone 132. A DNA sample traveling through the PCR zone 131 may undergo PCR, and a DNA sample passing through melt zone 132 may undergo high resolution thermal melting. As illustrated in FIG. 1, PCR zone 131 includes a first portion of channels 104 and melt zone 132 includes a second portion of channels 104, which is down stream from the first portion.

[0041] In order to achieve PCR for a DNA sample flowing through the PCR zone 131, the temperature of the sample must be cycled, as is well known in the art. Accordingly, in some embodiments, system 100 includes a temperature control apparatus 120. The temperature control apparatus 120 may include a temperature sensor, a heater/cooler, and a temperature controller. In some embodiments, temperature controller 120 is interfaced with main controller 130 so that main controller 130 can control the temperature of the samples flowing through the PCR zone and the melting zone.

[0042] To monitor the PCR process and the thermal melting process that occur in PCR zone 131 and melt zone 132, respectively, system 100 may include an imaging system 118. Imaging system 118 may include an excitation source, a detector, a controller, and an image storage unit.

[0043] Further features of system 100 are described in U.S. patent application Ser. No. 11/770,869, which is incorporated herein by this reference.

[0044] FIGS. 2A and 2B further illustrate a temperature control apparatus 120 in accordance with some embodiments of the invention. FIG. 2A illustrates a single heat exchange channel 210 and a single sample channel 104 (although, as discussed above, the invention is not limited to a single heat exchange channel 210 and/or sample channel 104). The heat exchange channel 210 is configured to carry a heat exchange fluid and configured to exchange heat with a portion 230 of a sample channel 220. The sample channel 220 can be configured to carry a bolus 240 of genomic sample material, which experiences temperature variation due to the heat exchange through the portion 230. In the one exemplary embodiment, temperature variations are performed temporally by varying the temperature of the heat exchange fluid temporally or by switching to a different heat exchange fluid at a particular time that has a different temperature.

[0045] At least one exemplary embodiment includes multiple reservoirs of various heat exchange fluids at various temperatures. For example, FIG. 2B illustrates a reservoir system for storing and directing heat exchange fluids through the heat exchange channel 210. FIG. 2B illustrates three fluid containers (a.k.a., reservoirs) T1, T2, and T3, each of which stores a fluid (e.g., a gas, a liquid or a gas and liquid mixture) and pump 290 coupled to each container for pumping fluid out of the containers and into a heat exchange channel 210 formed in chip 102. In one embodiment, the first heat exchange fluid is identical with the second heat exchange fluid, which is identical with the third heat exchange fluid, and the first heat exchange fluid comprises a gas and/or a liquid. In other embodiments, the heat exchange fluids comprise water and/or compressed air with pressure from 1 to 200 psia. In still other embodiments, the heat exchanges fluids can be different from one another. In one non-limiting example, the first heat exchange fluid is a gas, the second heat exchange fluid is a liquid and the third heat exchange fluid is gas and liquid mixture..

[0046] Each container T1-T3 includes an output port that is coupled to an input of the heat exchange channel through a forward valve. For example, the output port of T1 is coupled to the heat exchange channel through forward valve V1F, the output port of T2 is coupled to the heat exchange channel through forward valve V2F, and the output port of T3 is coupled to the heat exchange channel through forward valve V3F.

[0047] Each container T1-T3 also includes an input port that is coupled to an output of the heat exchange channel through a return valve. For example, the input port of T1 is coupled to the heat exchange channel through return valve V1R, the input port of T2 is coupled to the heat exchange channel through return valve V2R, and the input port of T3 is coupled to the heat exchange channel through return valve V3R.

[0048] As further illustrated, temperature control apparatus 120 may include a temperature control system that includes one or more temperatures controllers. For example, in the illustrated embodiment of FIG. 2B, temperature control apparatus 120 includes a temperature controller C1 for regulating the temperature of the fluid stored in T1 at a first temperature (e.g., C1 attempts to maintain the temperature of the fluid in T1 at, or close to, a predetermined temperature), a temperature controller C2 for regulating the temperature of the fluid stored in T2 at a second temperature, and a temperature controller C3 for regulating the temperature of the fluid stored in T3 at a third temperature. Each of C1, C2 and C3 may include, a sensor for sensing temperature, heating/cooling elements, and computerized controllers for controlling the heating/cooling elements based on output from a sensor.

[0049] Referring now to FIGS. 3A-C, cross-sectional, end views of chip 102 are shown and serve to illustrate various different embodiments of heat exchange channel 304 and to illustrate the relationship between a sample channel 104, which carries a sample 302, and heat exchange channel 304. Sample 302 may include a solution that contains, among other things, a piece of DNA, DNA polymerase, and a primer.

[0050] As illustrated in FIGS. 3A-C, heat exchange channel 304 may only run along one side of channel 104 (see FIG. 3A), heat exchange channel 304 may be generally L shaped and run along two sides of channel 104 (see FIG. 3B), and heat exchange channel 304 may be generally U shaped and run along three side of channel 104 (see FIG. 3C). In some embodiments, channel 304 may have a width between about 10 and 3000 micrometers (more preferably between about 20 and 2000 micrometers) and a depth between about 10 and 3000 micrometers (more preferably between about 20 and 2000 micrometers).

[0051] Referring now to FIG. 4, a flow chart illustrates a process 400 according to some embodiments of the invention. Process 400 may begin in step 402, where a fluid is stored in a first container (e.g., container T1). In step 404, the temperature of the fluid in the first container is regulated at a first temperature (e.g., at least about 80 degrees Celsius). In step 406, a fluid is stored in a second container (e.g., container T2). In step 408, the temperature of the fluid in the second container is regulated at a second temperature (e.g., a temperature not more than about 60 degrees Celsius). In step 410, a fluid is stored in a third container (e.g., container T3). In step 412, the temperature of the fluid in the third container is regulated at a third temperature (e.g., a temperature between about 60 and 80 degrees Celsius). In step 414, a sample (e.g., sample 302) is caused to flow though sample channel 104. While the sample is flowing through channel 104, steps 416-420 can be performed.

[0052] In step 416, the fluid stored in the first container is caused to flow through heat exchange channel 304 for a first amount of time. Next, in step 418, the fluid stored in the second container is caused to flow through heat exchange channel 304 for a second amount of time. Next, in step 420, the fluid stored in the third container is caused to flow through heat exchange channel 304 for a third amount of time. After step 420, steps 416-420 may be repeated a number of times. The first amount of time may be different than the second amount of time, which may be different than the third amount of time.

[0053] In one exemplary, non-limiting embodiment, the fluid stored in the first container (e.g. water) can be heated to a temperature of approximately 97 degrees Celsius so that the sample material can be heated to a temperature of approximately 95 degrees Celsius. The fluid stored in the second container (e.g. water) can be maintained at a temperature of approximately 53 degrees Celsius so that the sample material can be cooled to a temperature of approximately 55 degrees Celsius. The fluid stored in the third container (e.g. water) can be heated to a temperature of approximately 74 degrees Celsius so that the sample material can be heated to a temperature of approximately 72 degrees Celsius. Also in this exemplary embodiment, the fluid stored in the first container is caused to flow through heat exchange channel 304 for a first amount of time that can be, for example, approximately 0.3 to 2 seconds and preferably approximately 0.5 seconds. The fluid stored in the second container is caused to flow through heat exchange channel 304 for a second amount of time that can be, for example, approximately 1 to 5 seconds and preferably approximately 2 seconds. The fluid stored in the third container is caused to flow through heat exchange channel 304 for a third amount of time that can be, for example, approximately 1 to 10 seconds and preferably approximately 5 seconds. Of course, the fluid stored in the containers can be heated or cooled to different temperatures and the time periods during which the fluid flows through the heat exchange channel can be decreased or increased depending on the requirements for a given amplification reaction.

[0054] Referring now to FIG. 5A, steps 416-420 are further illustrated according to some embodiments where process 400 is implemented using the apparatus shown in FIGS. 2A-B. As shown in FIG. 5A, step 416 may include opening valve V1F, opening valve V3R and closing the other valves (V2F, V3F, V1R, and V2R), step 418 may include opening valve V2F, opening valve V1R and closing the other valves (V1F, V3F, V2R, and V3R), step 420 may include opening valve V3F, opening valve V2R and closing the other valves (V1F, V2F, V1R, and V3R). Preferably, while all the steps 416-420 are being performed, pump 290 is activated, thereby causing the fluids to flow out of a container and back into a container. The container from which the fluid flows and to which the fluid returns, of course, depends on the valves that are open at the time. For example, when step 416 is performed in accordance with the flow shown in FIG. 5A, fluid will flow out of container T1 and into container T3. Directing the fluid flow out of container T1 and into container T3, in this particular embodiment, is one exemplary way to allow more time for the fluid to reach the desired temperature level, which can increase the temperature accuracy and efficiency of the temperature cycling process.

[0055] Referring now to FIG. 5B, steps 416-420 are further illustrated according to another embodiment where process 400 is implemented using the apparatus shown in FIGS. 2A-B. As shown in FIG. 5B, step 416 may include opening valve V1F, opening valve V1R and closing the other valves (V2F, V3F, V2R, and V3R), step 418 may include opening valve V2F, opening valve V2R and closing the other valves (V1F, V3F, V1R, and V3R), step 420 may include opening valve V3F, opening valve V3R and closing the other valves (V1F, V2F, V1R, and V2R). Preferably, while all the steps 416-420 are being performed, pump 290 is activated, thereby causing the fluids to flow out of a container and back into a container. In this example, when step 416 is performed in accordance with the flow shown in FIG. 5B, fluid will flow, for example, out of container T1 and back into container T1.

[0056] In another embodiment, one or more of the containers T1-T3 are constructed to have an internal bladder or baffle that separates the internal portion of the container into a first chamber and a second chamber, and wherein the first and second chambers are in fluid communication with one another by, for example, a controllable valve. In this embodiment, fluid can be controllably released from one chamber of the container (e.g. T1) through a forward valve (e.g. V1F) and can be controllably caused to flow back into the other chamber of the container through the return valve (e.g. V1R). As stated above, fluid also can controllably flow between the first chamber and the second chamber of a container through, for example, a controllable valve in the bladder or baffle separating the chambers. This embodiment may be useful, for example, in an embodiment where fluid flows out one container and back into the same container before fluid flows out of, or into, another container, as discussed in connection with the process illustrated in FIG. 5B. This embodiment also may be useful in connection with other embodiments where different fluids are used in the containers such as, for example, when a gas is used in container T1, a liquid is used in container T2 and a mixture of gas and liquid is used in container T3.

[0057] FIG. 5C illustrates an example of a temperature versus time plot of the temperature experienced by bolus 240 as it traverses through the sample channel 220 as various heat exchange fluids flow through the heat exchange channel 210 at various times, in accordance with at least one exemplary embodiment of the present invention.

[0058] Referring now to FIG. 6, an apparatus 120 is illustrated according to another embodiment. The embodiment shown in FIG. 6 is similar to that shown in FIGS. 2A-B, with the exception that the forward valves V1F, V2F, and V3F are replaced with pumps 602, 604 and 606, respectively, and pump 290 is not present. The operation of the apparatus 120 in accordance with this embodiment is discussed below.

[0059] Referring now to FIG. 7, steps 416-420 are further illustrated according to some embodiments where process 400 is implemented using the apparatus shown in FIG. 6. As shown in FIG. 7, step 416 may include, activating only pump 602, opening valve V3R and closing the other return valves (V1R and V2R), step 418 may include activating only pump 604, opening valve V1R and closing the other return valves (V3R and V2R), step 420 may include activating only pump 606, opening valve V2R and closing the other return valves (V1R and V3R). The container from which the fluid flows and to which the fluid returns, of course, depends on the valves that are open at the time and the pump that is activated. For example, when step 418 is performed in accordance with the flow shown in FIG. 7, fluid will flow out of container T2 and into container T1. The apparatus of FIG. 6 also can be controlled such that fluid will flow out of one container and back into the same container, as discussed above.

[0060] FIG. 8 illustrates a thermal exchange system 800 in accordance with another exemplary embodiment. The thermal exchange system 800 is directed to a thermal exchange system that includes a plurality of heat exchange channels (e.g., 810a-c), each configured to carry a heat exchange fluid, where each heat exchange fluid preferably is at a different temperature. The plurality of heat exchange channels (810a-c) can be configured to lie substantially perpendicular (orthogonal) (although the invention is not limited to an orthogonal orientation) to a sample channel 820 and configured to exchange heat with a portion (e.g., 830a-c) of the sample channel. In this exemplary embodiment, a bolus 840 of genomic material traveling along the sample channel 820 experiences temperature change associated with heat exchanged in from portions of heat exchange channels 830a-c, as discussed above in connection with other embodiments.

[0061] In the embodiment of FIG. 8, heat exchange channel 810a is in fluid communication with at least container T1, heat exchange channel 810b is in fluid communication with at least container T2, and heat exchange channel 810c is in fluid communication with at least container T3. In one aspect of this embodiment, fluid is caused to flow from containers T1-T3 and through heat exchange channels 810a-c through one or more pumps and is caused to return to the containers through one or more return valves, as disclosed herein. The container from which the fluid flows and to which the fluid returns, of course, depends on the valves that are open at the time and the pump that is activated.

[0062] FIG. 9 illustrates a thermal exchange system 900 in accordance with another exemplary embodiment, which includes a curved sample channel 920, that directs a bolus 940 of genomic material back and forth (905A-C) near a plurality of heat exchange channels (910a-c), each configured to carry a heat exchange fluid, where each heat exchange fluid preferably is at a different temperature. In this exemplary embodiment, a bolus 940 of genomic material traveling along the curved sample channel 920 experiences temperature change associated with heat exchanged with portions of heat exchange channels 910a-c, as discussed above in connection with other embodiments. In this embodiment, the fluid flows from the containers T1-T3 through heat exchange channels 910a-c, for example, in the same manner described above in connection with the FIG. 8 embodiments and other embodiments described herein.

[0063] While various embodiments/variations of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. Thus, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments. Further, unless stated, none of the above embodiments are mutually exclusive. Thus, the present invention may include any combinations and/or integrations of the features of the various embodiments.

[0064] Additionally, while the processes described above and illustrated in the drawings are shown as a sequence of steps, this was done solely for the sake of illustration. Accordingly, it is contemplated that some steps may be added, some steps may be omitted, and the order of the steps may be re-arranged.