Abstract:
The invention relates to systems and methods including a combination of thermal generating device technologies to achieve more efficiency and accuracy in PCR temperature cycling of nucleic samples undergoing amplification.

Description:
[0001]    This application claims the benefit of Provisional Patent Application No. 60/806,440, filed on Jun. 30, 2006, which is incorporated herein by this reference. 
     
    
     BACKGROUND 
       [0002]    1. Field of the Invention 
         [0003]    The present invention relates to systems and methods for efficient thermal cycling in DNA amplification using a combination of energy sources, including electrical and/or magnetic (hereafter electromagnetic) radiation as an energy source. 
         [0004]    2. Discussion of the Background 
         [0005]    The detection of nucleic acids is central to medicine, forensic science, industrial processing, crop and animal breeding, and many other fields. The ability to detect disease conditions (e.g., cancer), infectious organisms (e.g., HIV), genetic lineage, genetic markers, and the like, is ubiquitous technology for disease diagnosis and prognosis, marker assisted selection, identification of crime scene features, the ability to propagate industrial organisms and many other techniques. Determination of the integrity of a nucleic acid of interest can be relevant to the pathology of an infection or cancer. 
         [0006]    One of the most powerful and basic technologies to detect small quantities of nucleic acids is to replicate some or all of a nucleic acid sequence many times, and then analyze the amplification products. Polymerase chain reaction (PCR) is a well-known technique for amplifying DNA. With PCR, one can produce millions of copies of DNA starting from a single template DNA molecule. PCR includes phases of “denaturation,” “annealing,” and “extension.” These phases are part of a cycle which is repeated a number of times so that at the end of the process there are enough copies to be detected and analyzed. For general details concerning PCR, see Sambrook and Russell,  Molecular Cloning—A Laboratory Manual  (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (2000);  Current Protocols in Molecular Biology , F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley &amp; Sons, Inc., (supplemented through 2005) and  PCR Protocols A Guide to Methods and Applications , M. A. Innis et al., eds., Academic Press Inc. San Diego, Calif. (1990). 
         [0007]    The PCR process phases of denaturing, annealing, and extension occur at different temperatures and cause target DNA molecule samples to replicate themselves. Temperature cycling (thermocyling) requirements vary with particular nucleic acid samples and assays. In the denaturing phase, a double stranded DNA (dsDNA) is thermally separated into single stranded DNA (ssDNA). During the annealing phase, primers are attached to the single stand DNA molecules. Single strand DNA molecules grow to double stranded DNA again in the extension phase through specific bindings between nucleotides in the PCR solution and the single strand DNA. Typical temperatures are 95° C. for denaturing, 55° C. for annealing, and 72° C. for extension. The temperature is held at each phase for a certain amount of time which may be a fraction of a second up to a few tens of seconds. The DNA is doubled at each cycle; it generally takes 20 to 40 cycles to produce enough DNA for the applications. To have good yield of target product, one has to accurately control the sample temperatures at the different phases to a specified degree. 
         [0008]    More recently, a number of high throughput approaches to performing PCR and other amplification reactions have been developed, e.g., involving amplification reactions in microfluidic devices, as well as methods for detecting and analyzing amplified nucleic acids in or on the devices. Thermal cycling of the sample for amplification is usually accomplished in one of two methods. In the first method, the sample solution is loaded into the device and the temperature is cycled in time, much like a conventional PCR instrument. In the second method, the sample solution is pumped continuously through spatially varying temperature zones. See, for example, Lagally et al. ( Analytical Chemistry  73:565-570 (2001)), Kopp et al. ( Science  280:1046-1048 (1998)), Park et al. ( Analytical Chemistry  75:6029-6033 (2003)), Hahn et al. (WO 2005/075683), Enzelberger et al. (U.S. Pat. No. 6,960,437) and Knapp et al. (U.S. Patent Application Publication No. 2005/0042639). 
         [0009]    Many detection methods require a determined large number of copies (millions, for example) of the original DNA molecule, in order for the DNA to be characterized. Because the total number of cycles is fixed with respect to the number of desired copies, the only way to reduce the process time is to reduce the length of a cycle. Thus, the total process time may be significantly reduced by rapidly heating and cooling samples to process phase temperatures while accurately maintaining those temperatures for the process phase duration. 
         [0010]    Accordingly, what is desired is a system and method for rapidly and accurately changing process temperatures in PCR processes. 
       SUMMARY OF THE INVENTION 
       [0011]    The present invention relates to systems and methods for rapid temperature change in microfluidic thermal cycling. 
         [0012]    In one aspect, the present invention provides a method for cycling the temperature of a nucleic acid sample. In one embodiment, the method includes: (a) controlling a heating device to cause a temperature of the sample to be at or about a first desired temperature for at least a first time period; (b) after expiration of the first time period, increasing the output of an electromagnetic heating source to cause the temperature of the sample to be at or about a second desired temperature for at least a second time period; (c) during said second time period, lowering the amount of heat the heating device provides to the sample; and (d) immediately after expiration of the second time period, lowering the output of the electromagnetic heating source and controlling the heating device to cause the temperature of the sample to be at or about a third desired temperature for a third time period, wherein the first temperature is less than the second temperature and the third temperature is less than the first temperature. In some embodiments, steps (a) through (d) occur while the sample is flowing through a channel (e.g., a microfluidic channel). 
         [0013]    In another embodiment, the method includes: heating the sample to a first temperature for a first time period using a thermoelectric device; heating the sample to a second temperature for a second time period using primarily an electromagnetic heat source; cooling the sample to a third temperature; and maintaining the third temperature for a third time period using the thermoelectric device, wherein the second temperature is higher than the first and the first temperature is higher than the third. 
         [0014]    In another embodiment, the method includes: (a) heating the nucleic acid sample to about a first temperature; (b) after heating the sample to about the first temperature, maintaining the temperature of the sample at about the first temperature for a first period of time; (c) after expiration of the first period of time, heating the sample to about a second temperature; (d) after heating the sample to the second temperature, maintaining the temperature of the sample at about the second temperature for a second period of time; (e) after expiration of the second period of time, cooling the sample to about a third temperature; and (f) after cooling the sample to the third temperature, maintaining the temperature of the sample at about the third temperature for a third period of time, wherein the first temperature is less than the second temperature and greater than the third temperature, and the step of heating the sample to the second temperature consists primarily of using one or more non-contact heating elements to heat the sample to the second temperature. 
         [0015]    In another aspect, the present invention provides a system for cycling the temperature of a nucleic acid sample. In one embodiment, the system includes: a nucleic acid sample container operable to receive a nucleic acid sample; a first heating device; and a second heating device, wherein the first heating device is configured to heat the nucleic acid sample to at least about a first temperature, the second heating device is configured to heat the nucleic acid sample to a second and third temperature, the first temperature is associated with a denaturing phase of a PCR process, the first heating device is a non-contact heating device, and the second heating device is a contact heating device. 
         [0016]    The above and other embodiments of the present invention are described below with reference to the accompanying drawings. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0017]    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. Additionally, the left-most digit(s) of a reference number identifies the drawing in which the reference number first appears. 
           [0018]      FIG. 1  depicts an apparatus in accordance with an exemplary embodiment of the invention. 
           [0019]      FIG. 2  depicts an exemplary desired PCR temperature cycle. 
           [0020]      FIG. 3  depicts a temperature characteristic of a heating device and of an electromagnetic heating device. 
           [0021]      FIGS. 4 and 5  depict a temperature cycle. 
           [0022]      FIG. 6  depicts steps in an exemplary method in accordance with an embodiment of the present invention. 
       
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
       [0023]    With reference to  FIG. 1 , an exemplary embodiment of an apparatus  100  relating to the present invention may include: a microfluidic device  101  or other device for containing a sample containing a nucleic acid and PCR reagents (which PCR reagents may include PCR primers, dNTPs, polymerase enzymes, salts, buffers, surface-passivating agents, and the like), a heat spreader  102 , a heating device  103 , a heat sink  104 , an electromagnetic heat source  105 , a contact temperature sensing device  107 , a non-contact temperature sensing device  108 , a controller  109  and a fan  110 . 
         [0024]    In some embodiments, device  101  includes a microfluidic channel configured to receive the sample. The sample may flow through the channel as its temperature is cycled, as described herein. Moving the sample through the microfluidic channel can be accomplished by a variety of methods, for example, via conventional methods of pressure-driven flow (e.g., using a pump to create a pressure differential) and the flow rates can vary, for example between 10 nanoliters per minute to 1 ml per minute. 
         [0025]    In the embodiment illustrated, heating device  103  is thermally in contact with the microfluidic device  101  through the metal heater spreader  102 . Device  103  may be implemented using a thermoelectric cooler (TEC) (also referred to as a Peltier device), a resistive heater, a chemical heater, or other heating device. A suitable TEC may be available from Melcor Corporation of Trenton, N.J. (see part number HT6-6-21X43). In some embodiments, heating device  103  may have a different temperature resolution than heat source  105 . More specifically, in some embodiments, heating device  103  may have a finer temperature resolution than heat source  105 . 
         [0026]    As mentioned above, device  103  may be implemented using a Peltier device, a widely used component in laboratory instrumentation and equipment, well known among those familiar with such equipment, and readily available from commercial suppliers such as Melcor. A Peltier device is a solid-state device that can function as a heat pump, such that when an electric current flows through two dissimilar conductors, the junction of the two conductors will either absorb or release heat depending on the direction of current flow. A typical Peltier device consists of two ceramic or metallic plates separated by a semiconductor material, such as bismuth telluride. The direction of heat flow is a function of the direction of the electric current and the nature of the charge carrier in the semiconductor (i.e., n-type or p-type). Peltier devices can be arranged and/or electrically connected in an apparatus of the present invention to heat or to cool a PCR process taking place in microfluidic device  101 . 
         [0027]    The size of heat spreader  102  is related to the sizes of the heating device  103  and microfluidic device  101 . In an exemplary embodiment, a heat spreader of 20 mm×40 mm is used. Heat sink  104  removes waste heat from heating device  103 . The assembly comprising the heat sink  104 , heating device  103 , and heater spreader  102  may be screwed together, bonded together, or clamped in place. Thermal coupling may be enhanced by the use of thermally conductive adhesives, greases, pastes, thermoconducting pads (e.g., a SIL-PAD product available from the Berquist Company of Chanhassen, Minn.). 
         [0028]    A switchable fan  110  may be used to increase airflow towards the assembly and/or heat sink  104  in particular. The fan  110  and heat sink  104  can function together to quickly remove heat from the assembly. In some embodiments, fan  110  may be left on continuously during for an entire PCR process. 
         [0029]    Electromagnetic heat source  105  may radiate energy  106  directed toward the microfluidic device  101  surface. A suitable electromagnetic heat source is any device which generates an electric and/or magnetic field which may be used to heat microfluidic device  101 . An exemplary electromagnetic heat source  105  may be an infrared source including a tungsten filament bulb, such as one from the GE XR series, or a laser. Heat source  105  may be located ½ of an inch or less to twelve inches or more from the surface of microfluidic device  101 . Preferably, heat source  105  is located at an angle so as to facilitate real-time PCR monitoring of the microfluidic zone and is located between about 2-6 inches from the surface of device  101 . 
         [0030]    Contact temperature sensing device  107  may be located inside or on the microfluidic device  101  surface. Suitable temperature sensors include a thin film wire, embedded wire, thermocouple, RTD, resistor, or solid state device. In some embodiments, a temperature measuring sensor available from Analog Devices is used to implement device  107  (e.g., the Analog Devices AD590 temperature transducer may be used). In some embodiments, a non-contact temperature sensing device  108 , such as a pyrometer manufactured by Mikron, may be used in addition to or instead of contact temperature sensing device  107 . 
         [0031]    Controller  109  may be used to energize and deenergize heating device  103  and electromagnetic heat source  105  in a thermostatic fashion such that the temperature sensed by sensor  107  and/or  108  (e.g., the temperature of a region of microfluidic device  101 ) is at, or approximately at, a desired temperature for a desired period of time. Controller  109  include one or more computers or other programmable devices which may be programmed to control heating device  103 , electromagnetic heat source  105 , and/or fan  111  in response to the expiration of a timer and temperature measurements from contact temperature sensor  107  and/or non-contact temperature sensor  108 . 
         [0032]      FIG. 2  depicts an exemplary desired PCR cycle. In this exemplary cycle, phase  211  is the extension phase, phase  212  is the denaturation phase, and phase  213  is the annealing phase. Typically, the denaturation phase  212  requires less precision of temperature and time control, provided that a minimum temperature point is achieved homogeneously in the microfluidic device  101 . 
         [0033]    In a preferred embodiment, controller  109  is programmed to use primarily heating device  103  to heat and/or cool microfluidic device  101  during extension phase  211  and annealing phase  213 . That is, in some embodiments, source  105  may be turned “off” during the extension phase and annealing phase or may output a lower level of radiation  106  during these phases than it outputs during denaturation phase  212 . Controller  109  is operable to energize the heating and/or cooling ability of heating device  103  such that the desired temperatures are quickly reached and maintained for the desired times. For example, an extension phase  211  may have a duration of about 5 seconds and a desired temperature of about 72° C. An exemplary annealing phase  213  may have a desired duration of 2 seconds and a desired temperature of about 55° C. 
         [0034]    In the preferred embodiment, electromagnetic heat source  105  provides to microfluidic device  101  a greater amount of heat during the denaturation phase  212  than during the other two phases of the PCR cycle. Additionally, during denaturation phase  212 , device  103  may be operated to provide less heat to device  101  than device  103  is configured to provide to device  101  during the other two phases of the PCR cycle. Accordingly, in some embodiments, device  103  may actually draw heat from device  101  during denaturation phase  212 . An exemplary desired denaturation phase  212  may last about 500 ms at a desired temperature of about 95° C. 
         [0035]      FIG. 3  depicts exemplary plots of a heating device  103  temperature characteristic  301  and source  105  temperature characteristic  303 . Characteristic  301  illustrates that the heating device  103  is controlled by the controller  109  such that the desired temperature control of the sample for the extension phase  211  and annealing phase  213  is substantially provided through the functioning of the heating device  103 . Characteristic  303  illustrates that the electromagnetic heat source  105  is controlled by the controller  109  such that the desired temperature control of the sample for denaturation phase  212  is substantially provided through the functioning of the electromagnetic heat source  105 . 
         [0036]      FIGS. 4 and 5  depict time and temperature graphs for a sample contained in device  101  when heat sources  103  and  105  are operated according to the diagram shown in  FIG. 3 . Shaded area  441  represents the part of the cycle for which device  103  provides thermal input. Period A, which occurs in area  440 , represents the duration of an energy input from source  105 . 
         [0037]    Accordingly, and with reference to  FIG. 5 , fast thermal ramp/rise rates may be achieved as indicated by slope  530 . Furthermore, high cooling rates may be achieved as indicated by slope  531 . 
         [0038]    Referring now to  FIG. 6 ,  FIG. 6  is a flow chart illustrating a process, according to some embodiments of the invention, for cycling the temperature of a sample that is present in device  101 . 
         [0039]    The process may begin in step S 601 , where the controller controls thermostatically heating device  103  such that the extension phase temperature is reached and maintained for a desired duration of the extension phase  211 . While step S 601  is being performed, source  105  may be in an “off” state. 
         [0040]    In step S 603 , which preferably does not occur until about immediately after the expiration of the desired extension phase duration, controller  109  increases the output of electromagnetic heat source  105 . Preferably, the output of source  105  is raised to a level that causes the temperature of the sample to rapidly increase to the denaturation phase temperature. 
         [0041]    In step  605 , controller  109  may control thermostatically one or more of the heating and cooling devices of apparatus  100  so that the sample is kept at the denaturation phase temperature for the desired duration of the denaturation phase  212 . 
         [0042]    At or about the same time controller  109  causes heat source  105  to heat the sample to the denature temperature, controller  109  may control device  103  such that the heat provided by device  103  to device  101  is less than the heat device  103  provided to device  101  during step S 601  (i.e., during extension phase  211 ). 
         [0043]    In step S 607 , which preferably does not occur until about immediately after the expiration of the desired denature phase duration, controller  109  lowers the energy output (e.g., turns off) electromagnetic heat source  105  and controls heating device  103  such that the annealing phase temperature is reached. In some embodiments, step S 607  includes using the cooling capability of the heating device to quickly drive the temperature of the sample down from the denaturation phase temperature to the annealing phase temperature as shown in ramp  531 . In some embodiments, step S 607  also includes activating and directing fan  110  at the microfluidic device  101  and/or heat sink  104  to drive the temperature down rapidly as shown in ramp  631 . 
         [0044]    In step S 609 , controller  109  may control thermostatically one or more of the heating and cooling devices of apparatus  100  so that the sample is kept at the annealing phase temperature for the desired duration of the annealing phase  213 . For example, if a temperature sensor (e.g., sensor  107  or  108 ) indicates that the temperature of the sample is too low, then controller  109  may control a heat source (e.g., device  103  or source  105 ) to add more heat to the sample, and if a temperature sensor  108  indicates that the temperature of the sample is too low, then controller  109  may control device  103  so that it draws heat from the sample. 
         [0045]    Embodiments of the present invention have been fully described above with reference to the drawing figures. Although the invention has been described based upon these preferred embodiments, it would be apparent to those of skill in the art that certain modifications, variations, and alternative constructions could be made to the described embodiments within the spirit and scope of the invention. 
         [0046]    Furthermore, one of skill in the art will recognize that temperatures enumerated in the following claims should be interpreted to mean “at or about” the enumerated temperature. 
         [0047]    For the claims below the words “a” and “an” should be construed as “one or more.”