Patent Publication Number: US-8986982-B2

Title: Apparatus and method for a continuous rapid thermal cycle system

Description:
CROSS-REFERENCES TO RELATED APPLICATIONS 
     This application is a continuation of U.S. patent application Ser. No. 11/045,526 filed Jan. 28, 2005, which claims the benefit of U.S. Provisional Application No. 60/540,225 filed Jan. 28, 2004. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Award No. 0314742 awarded by the National Science Foundation. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to systems for maintaining multiple temperature regions, and in particular, to a device and associated method for the automated, bulk thermal cycling of fluids, solutions, and/or reactants. 
     2. Description of the Related Art 
     The polymerase chain reaction (PCR) is widely used by research professionals around the world as a means to amplify small strands of DNA. Typically, PCR is performed using automated thermal cyclers that alternately heat and cool numerous small tubes containing the PCR reaction mixture. Such a process uses a static reactor having discrete, confined spaces in which the reaction occurs when exposed to different temperatures in a repetitive sequence. This process is time intensive, labor intensive, and inefficient, as the tubes must be individually filled with the reactants, closed, processed through the automatic cycler, opened, and finally drained of the reaction product that contains the desired amplified DNA. 
     Accordingly, continuous thermal cyclers were developed to eliminate the need for using a multitude of small tubes to amplify DNA via PCR by using a dynamic reactor. Rather than using small tubes, continuous thermal cyclers use a constant or continuous stream of fluid repetitively passed through different temperature zones to amplify DNA. One example of a continuous thermal cycler is disclosed in U.S. Pat. No. 5,270,183 issued on Dec. 14, 1993, to Corbett et al. Corbett et al. disclose a device and method for DNA amplification in which a PCR reaction mixture is injected into a carrier fluid with which the PCR reaction mixture is immiscible, and the carrier fluid then passes through a plurality of temperature zones to facilitate DNA amplification within the PCR reaction mixture. The function of this device is to accelerate the processing of a multitude of different DNA strands contained in discrete pockets or plugs, hence the need for a carrier fluid that is immiscible with the PCR reaction mixture that acts to separate the different DNA strands. This device is not designed to produce mass quantities of DNA. 
     Moreover, the Corbett et al. device is not designed to be easily and quickly adaptable to different PCR reaction requirements. For example, the preferred arrangement for passing the carrier fluid through the temperature zones is to wrap tubing conveying the carrier fluid around separate cylinders maintained at different temperatures. Modifying the device for different reaction conditions therefore requires re-wrapping the tubing around one or more of the cylinders a different number of times, unwrapping the tubing around one or more of the cylinders to replace one or more of the cylinders with different cylinders, re-routing the tubing around the cylinders in different orders, or another such labor-intensive procedure. Additionally, efficiency and fine temperature control is reduced as the reaction mixture pockets pass from one cylinder to the next and thermal energy is unintentionally lost or gained at such “gaps.” 
     Another example of a continuous thermal cycler is disclosed in Curcio, M. and Roeraade, J. (2003, published on web 2002) Continuous Segmented Flow Polymerase Chain Reaction for High-Throughput Miniaturized DNA Amplification, Anal. Chem. 75, 1-7. This device similarly is designed for numerous small sample mixtures separated by an immiscible fluid. Rather than using separate cylinders as different temperature zones as in the Corbett et al. device, however, this device uses separate thermally controlled water baths as temperature zones. This device is not designed for easy modification for providing a number of different reaction conditions, as additional water baths would have to be prepared and added for such modification. Use of this device also entails adding, checking, and draining water from the baths on a periodic basis, as well as cleaning of the water bath containers. 
     For the foregoing reasons, there is a need for a continuous thermal cycler that is designed to mass produce DNA strands, that is easily adaptable to different PCR reaction requirements, and that is efficient in operation. 
     SUMMARY OF THE INVENTION 
     The present invention comprises an apparatus and method for a continuous thermal cycle system capable of the bulk production of DNA strands that is efficient, scalable, easily adaptable to different PCR reaction requirements, and is relatively inexpensive to produce. An embodiment of the present invention has a plurality of temperature-controlled sectors within a temperature control body, thereby resulting in a plurality of temperature zones. A fluid preferably flows continuously through or along the apparatus via a path, and thereby through or along the different temperature zones. 
     A preferred embodiment of the present invention is particularly suited for amplification of DNA fragments quickly, easily, and in large quantities. Mass production of DNA at rates much greater than conventional DNA production rates is thereby effectively achieved using the present invention. Low manufacturing costs and enhanced scalability of the present invention permit relatively inexpensive, continuous amplification of DNA in bulk quantities. In particular, a preferred embodiment of the present invention comprises a single cylindrical temperature control body having twelve pie-shaped or wedge-shaped sectors, each sector having a means for obtaining a desired temperature, and each sector separated from other sectors by a thermal barrier. A grooved channel circles or spirals around the exterior surface of the temperature control body, and a length of tubing placed in or on the channel conveys DNA amplification reactants cyclically from one sector to subsequent sectors. The reactants are thereby exposed to different temperature zones in a cyclical fashion, ultimately resulting in the amplification of the DNA. A means for moving the reactants establishes the flow rate of the reactants through the length of tubing to optimize the amplification via PCR based upon the characteristics of the specific reactants. Any number of sectors may be incorporated into the temperature control body by simply dividing it into additional sectors or reducing the number of sectors. Also, further adaptability can be incorporated into the temperature control body by adding layered sectors and/or using a temperature control body having a shape other than a cylinder. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
       The present invention is described with reference to the accompanying drawings. In the drawings, like reference numbers indicate identical or functionally similar elements. 
         FIG. 1  is an elevation view of an embodiment of a thermal cycle system of the present invention. 
         FIG. 2  is a plan view of the thermal cycle system of  FIG. 1 . 
         FIG. 3A  is an elevation view of an alternate embodiment of the thermal cycle system of the present invention. 
         FIG. 3B  is a expanded view of a portion of an exterior surface of the thermal cycle system of  FIG. 3A . 
         FIG. 3C  is an expanded view of a portion of a channel of the thermal cycle system of  FIG. 3A . 
         FIG. 4  is an elevation view of the thermal cycle system of  FIG. 1  showing an insulating layer substantially surrounding the temperature control body. 
         FIG. 5  is a top plan view of the thermal cycle system of  FIG. 1 . 
         FIG. 6  is a perspective view of a temperature control body of the thermal cycle system of  FIG. 1  showing a portion of an insulating layer. 
         FIG. 7  is a top plan view of a temperature control body of the thermal cycle system of  FIG. 1 . 
         FIG. 8  is a bottom plan view of a temperature control body of the thermal cycle system of  FIG. 1 . 
         FIG. 9  is an elevation view of an alternate embodiment of the thermal cycle system of the present invention. 
         FIG. 10  is a top plan view of the thermal cycle system of  FIG. 9 . 
         FIG. 11  is a bottom plan view of the thermal cycle system of  FIG. 9 . 
         FIG. 12  is a plan view of a top cap of the thermal cycle system of  FIG. 9 . 
         FIG. 13  is a plan view of a bottom cap of the thermal cycle system of  FIG. 9 . 
         FIG. 14  is a photograph of an electrophoresis gel demonstrating the efficiency of an embodiment of the thermal cycle system of the present invention as compared with the efficiency of a conventional system. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The present invention is directed to an apparatus and method for simultaneously maintaining multiple temperature regions within a single physical structure. The present invention is therefore particularly suited for use in the automated thermal cycling of substances, such as those used in the amplification of nucleic acid sequences. With reference to the drawings, and in particular to  FIGS. 1-13 , a thermal cycle system  100  of the present invention preferably comprises a temperature control body  102  having at least two sectors  118  and a path  104  that cyclically passes from one initial sector  118  to each successive sector  118  in turn, thereafter returning to the initial sector  118  and cyclically repeating passes from one sector  118  to the next sector  118  as many times as is desired. The path  104  traverses the sectors  118  by passing along an exterior surface  132  of the temperature control body  102  from one sector  118  to each successive sector  118 , by boring through the sectors  118  internally from one sector  118  to each successive sector  118 , or by a combination of such external or internal travel. 
     Each sector  118  comprises at least one means for changing or obtaining a temperature  120 . The means for changing temperature  120  is capable of achieving and maintaining a specific desired temperature. The means for changing temperature  120  is therefore preferably a heater, cooler, Peltier device, heat pump, oven, firebox, thermal reaction chamber, or similar means. Each sector  118  is preferably substantially made of aluminum, aluminum alloy, metal, metal alloy, a thermal conductor, an asymmetric thermal conductor, or combinations thereof. The means for changing temperature  120  thereby heats, cools, or maintains the temperature of the sector  118  such that the section of the path  104  located in or on each sector  118  is similarly heated, cooled, or maintained at the particular temperature of that sector  118 . 
     Each sector  118  is also preferably separated from other sectors  118  by a thermal barrier  122  located between the sectors  118 . The thermal barrier  122  may be passive, and may comprise a thermal insulator, air, gas, liquid, solid, and/or a combination thereof. The thermal barrier  122  may alternatively or additionally be an active device or material, such as a Peltier device, which can maintain a significant temperature differential. Each sector  118  therefore acts as an independent temperature sink wherein the means for changing temperature  120  for that sector  118  achieves and maintains a desired temperature throughout that sector  118 , and a thermal barrier  122  thermally isolates each sector  118  from the other sectors  118 . Multiple temperature regions are thereby efficiently achieved and maintained in a single body. An insulating layer  124  may optionally substantially surround the temperature control body  102  to minimize thermal transfer between the sectors  118  and the surrounding environment. 
     The temperature control body  102  may have any desired shape, such as a cylinder, cone, triangle, rectangle, pyramid, polygon, block, or cube. The sectors  118  may also have any desired shape conforming to sections, parts, or pieces of the temperature control body  102 . For example, the sectors  118  may be wedge shaped, arc shaped, or pie-slice shaped, or may have the shape of sliced portions of a cylinder, cone, triangle, rectangle, pyramid, polygon, block, or cube. The sectors  118  may also be layered, one atop another. There may be any number of desired sectors  118 . All the sectors  118  may be the same size, or one or more of the sectors  118  may be a different size. 
     The thermal cycle system  100  also preferably comprises a plurality of temperature sensors  130 . Each sector  118  preferably has one or more temperature sensors  130  located within or adjacent to that sector  118  to measure the temperature within that sector  118  or portion of sector  118 . Each temperature sensor  130  produces temperature values output that directly or indirectly represents the temperature of that sector  118 . Such temperature sensors  130  may be any conventional instrument for determining temperature. Such temperature sensors  130  may optionally be placed in or on the insulating layer  124 . 
     The thermal cycle system  100  also preferably comprises a means for regulating temperature  134 . The means for regulating temperature  134  regulates each means for changing temperature  120 , such that desired temperatures within each sector  118  are achieved. Any number of means for regulating temperature  134  may be used to regulate the means for changing temperature  120 . The means for regulating temperature  134  preferably comprises a thermostat. In one embodiment, a computer system executing a software program is in communication with the means for changing temperature  120  and the temperature sensors  130 , wherein the software uses a predefined set of target temperatures for each sector  118  for control and regulation of the means for changing temperature  120 . The target temperatures are dictated by the desired application and use of the thermal cycle system  100 , which in a preferred embodiment is PCR. The software receives the temperature values output from the temperature sensors  130 . Each such temperature value represents directly or indirectly the temperature of a sector  118 . The software compares the temperature value output of each sector  118  with its predefined target temperature for that sector  118 . Then, if the temperature value output received from a temperature sensor  130  falls above or below a minimum predefined value, the software engages one or more of the means for changing temperature  120  in that sector  118  to increase or decrease the heat in that sector  118  or in an appropriate portion of that sector  118 . That is, according to a temperature sensor&#39;s  130  value and position, the system may engage all or a subset of the means for changing temperature in the sector  118 . Alternative means for regulating temperature  134  can be used such as any conventional thermostat system. 
     The thermal cycle system  100  also preferably comprises a means for moving  106  a fluid  128  along the path  104 . The fluid  128  thereby cyclically passes from one sector  118  to another sector  118 , and the temperature of the fluid  128  equilibrates with the temperature of the sector  118  through which or on which the fluid  128  is passing. The temperature of the fluid  128  thereby cyclically changes as it flows along the path  104 . The fluid  128  preferably comprises any thermally dependent reaction mixture, reactants, or reagents. The fluid moving means  106  preferably comprises a pump, such as a peristaltic pump, a pressurized gas system, or similar means. For example, a pressurized helium system can be used to pump the fluid  128  along the path  104 . 
     In a preferred embodiment of the thermal cycle system  100 , the temperature control body  102  is a single substantially cylindrical body having a plurality of substantially pie-slice shaped or wedge-shaped sectors  118 . The path  104  comprises a grooved channel circling or spiraling around the exterior surface  132  of the temperature control body  102 . A length of tubing  126  is placed within or along the grooved channel. The desired temperature for each sector  118  is determined based upon the characteristics and requirements of a particular thermal-dependent reaction. The means for regulating temperature  134  and the means for changing temperature  120  are activated such that the desired temperature for each sector  118  is attained. The temperature sensors  130  measure the actual temperatures of each sector  118 , and each means for changing temperature  120  is activated or inactivated as appropriate to attain and maintain the desired temperature for each sector  118 . The fluid moving means  106  moves or pumps the fluid  128  through the length of tubing  126 . The fluid  128  is thereby subjected to a series of different temperature regions on a cyclical basis that ultimately results in a transformation or reaction of the fluid  128  into a product or products. The temperature control body  102  may optionally be attached to a base for support. A means for rotating the temperature control body  102  may also optionally be used to facilitate placing the length of tubing  126  within or along the grooved channel. Such means for rotating may comprise an electric motor with wheel and gear assemblies or similar alternative. 
     The thermal cycle system  100  is particularly suited for large scale amplification of DNA via PCR. Thus, a preferred embodiment of the thermal cycle system  100  has grooved channel path  104  circling around the exterior surface  132  of a single cylindrical temperature control body  102 . Thus, the channel has a first end  114  near the top edge  110  of the temperature control body  102  and a second end  116  near the bottom edge  112  of the temperature control body  102 . The depth of the groove is discretionary and may depend on the diameter of the length of tubing  126  that can be placed within or along the groove and/or may depend on the particular application of the thermal cycle system  100 . The cylindrical temperature control body has twelve equally sized arc-shaped sectors  118 , and each sector  118  has one means for changing temperature  120 . Each sector  118  has one temperature sensor  130 , specifically a type K thermocouple, internally placed within the sector  118 . A fluid moving means  106 , preferably a pressurized helium system, moves a fluid  128  through the length of tubing  126 . The fluid  128  preferably comprises a DNA strand to be amplified, two primers, and a heat stable Taq polymerase. Additional substances may be included in the fluid  128  to facilitate DNA amplification via PCR. A single means for regulating temperature  134  preferably regulates every means for changing temperature  120 . The fluid moving means  106  moves the fluid  128  from sector  118  to sector  118  such that DNA amplification via PCR is optimized. 
     In one embodiment of the thermal cycle system  100 , the cylindrical temperature control body  102  is divided into 3 equal pie-slice shaped sectors  118 , and there are about 30 to about 40 “turns” of the channel around the cylinder with the preferred number being about 33 turns. Each “turn” of the channel is a “cycle” of the fluid  128  traveling around the circumference of the exterior surface  132  of the cylinder. Also, tubing  126 , e.g., polytetrafluoroethylene (PTFE) tubing or TEFLON tubing or synthetic resinous fluorine-containing polymer tubing, within the channels is surrounded by 3 insulating layers  124  (one per sector  118 ), wherein each insulating layer  124  has eight temperature sensors  130 . A peristaltic pump  106  is positioned about six to about seven inches from the point at which the tubing  126  extends away from the bottom  112  of the cylinder. Using this arrangement of the apparatus, the preferred method for using the present apparatus pumps the fluid  128  through the tubing  126  at a rate of about 45 seconds per sector  118  (temperature zone), resulting in a flow rate of about 135 seconds per cycle (1 “turn” of the tubing  126  around the cylinder). 
     The temperatures and cycle times imposed on the reagents by the sectors/temperature zones  118  are preferably consistent with the well-known and current process of PCR. The preferred use of the present apparatus and method for a continuous thermal cycle system is amplifying DNA, but this use of the present invention is for convenience purposes only. It would be readily apparent to one of ordinary skill in the relevant art to use the apparatus and method of the present invention in a different application requiring the continuous heating or cooling of a fluid  128  through multiple temperature zones. 
     The fluid  128  may be mixed or created in a large batch prior to its introduction into the length of tubing  126 , or the fluid  128  may be created just-in-time or on-the-fly right before it is introduced into the length of tubing  126 . The fluid  128  is preferably a substantially homogeneous temperature-dependent reaction mixture, and there is preferably a continuous supply of such fluid  128  through the length of tubing  126 . A means for controlling the introduction of the fluid  128  may be used, such as a computer system and software program. The software program preferably uses a predefined protocol for determining the proper mix (by proportions), sequential order, and timing for inputting the fluid  128 , and/or the fluid components, into the length of tubing  126 . In one embodiment, the protocol for introducing the fluid  128  components is determined by particular PCR requirements. Any means for introduction of the fluid  128  may be used, such as a pump and valve manifold or network known to those skilled in the art. 
     The resulting fluid  128  output from an end of the tubing is collected by conventional means. In a preferred embodiment, the resulting fluid contains amplified DNA. In addition, it is readily apparent that the apparatus and method of the present invention will provide a continual supply of amplified DNA so long as the pump is feeding the fluid components through the apparatus as described herein. 
     A method of the present invention for the facilitation of a chemical reaction requiring cyclical temperature changes therefore comprises activating a means for changing temperature  120  on a thermal cycle system  100  having a means for conveying a fluid such as a length of tubing  126  extending along a path  104 , introducing a substantially homogeneous temperature-dependent reaction mixture into the means for conveying, activating a means for moving  106  such that the reaction mixture moves through the means for conveying and such that the reaction mixture reacts to form a product, and collecting the product at an end of the means for conveying. The chemical reaction is preferably a polymerase chain reaction. The method optionally further comprises continuously replenishing the fluid at one end of the means for conveying. 
     EXAMPLE 
     A sample was prepared containing: 12% MgCl2 (25 mM), 0.33% Taq DNA polymerase (5 units/μl), 2.0% dNTP&#39;s (deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP) and deoxythymidine triphosphate (dTTP)), 8.0% template (2 μg/ml), 61.66% PLURONIC® F108 solution (1.5% solution; PLURONIC® is a registered trademark of BASF Corporation of Mount Olive, N.J.), 4% forward primer, 4% reverse primer, 8% reaction buffer (10× concentration). The solution can be scaled up to the correct volume using these figures. The twelve vertical sectors  118  of the cylindrical temperature control body  102  were heated to three different temperatures, four adjacent sectors  118  were heated to 95° C., another four adjacent sectors were heated to 59° C., and the final four adjacent sectors  118  were heated to 72° C. 1/32″ ID, 1/16″ OD TEFLON® PTFE tubing (TEFLON® is a registered trademark of E.I. Dupont De Nemours Company of Wilmington, Del.) was wrapped around the temperature control body  102  thirty times to subject the length of tubing  126  and reaction mixture to the three different temperatures thirty different times in succession. The reaction mixture was then pumped through this tubing  126  using a pressurized vessel at 20 PSI. After the reaction mixture was fed to the temperature control body  102 , mineral oil was used to push the sample through the entire length of tubing  126 . The flow rate of the reaction mixture was controlled with a flow valve to 0.25 ml/min. The specific DNA sequence (whose limits are defined by the oligonucleotide primers) present in the sample was amplified as it passed cyclically through the temperature zones. After the thirtieth cycle, the tubing  126  exited the cylinder  102 , and the contents were collected. The sample was then analyzed on a Cambrex Reliant Precast 2% Agarose Gel and stained with ethidium bromide. 
     An image of the gel was acquired using a BIORad Geldoc EQ system and is shown in  FIG. 14 . The lane contents were as follows: lane  1  empty; lane  2  ladder; lane  3  no template negative control (sample A); lane  4  empty; lane  5  sample amplified in an embodiment of the thermal cycle system  100  (sample B); lane  6  empty; lane  7  sample amplified in an embodiment of the thermal cycle system  100  followed by amplification in a conventional Perkin Elmer 480 machine (sample C); lane  8  empty; lane  9  positive control sample run with the conventional Perkin Elmer 480 machine (sample D); lane  10  ladder; lane  11  empty; and lane  12  empty. 
     The image was analyzed using ImageJ version 1.33u software wherein intensity data was extracted to obtain integrated intensities and calculations included a background subtraction, and no other normalization. The band intensity for sample A was 0.07, the band intensity for sample B was 3.62, the band intensity for sample C was 3.77, and the band intensity for sample D was 3.19. 
     This data indicates that the system and method of this invention is as efficient, if not more efficient, than an example of a standard commercial system; a Perkin Elmer 480 machine. Three identical reaction mixtures were prepared and one sample was examined in its unamplified form without template (sample A), one sample was run with the system of this invention (sample B), one sample was first run with the system of this invention and then run through a conventional commercial system (sample C), and one sample was run on a conventional commercial system (sample D). The intensity of the band on a gel at the targeted mass (300 bp) is an indicator of the quantity of DNA product produced. 
     Sample C produced the most intense band, but it is not very much more intense than the sample produced by this invention alone. Since sample C was subjected to thirty cycles with an embodiment of the thermal cycle system  100 , then with thirty cycles of a commercial system, it is reasonable to expect some additional amplification if active reagents remain after exiting the machine of the present invention. 
     Sample B, the DNA produced using the machine of this invention, produced the second most intense band. Sample D is included to demonstrate the relative quantity of DNA to be expected from a conventional commercial system, the Perkin Elmer system. The band from the commercial system, sample D, is less intense than the band from the system and method of this invention, sample B. This means that the system and method of this invention is equal or better in efficiency than the commercial system. Sample A is used to indicate that no DNA (or a negligible amount of signal) is observed in a system subjected to amplification conditions (in the Perkin Elmer commercial system) but lacking template DNA, that there is not a contaminant in the reaction solution which could be misinterpreted as amplification. The important feature of this data is the fact that the sample B band is more intense (indicating a better reaction) than the same reaction carried out on the conventional system. 
     CONCLUSION 
     While various embodiments 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. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined in the appended claims. Thus, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.