WO2020210420A1 - Réaction accélérée en chaîne par polymérase basée sur le thermocyclage activé par le chauffage par ondes millimétriques - Google Patents

Réaction accélérée en chaîne par polymérase basée sur le thermocyclage activé par le chauffage par ondes millimétriques Download PDF

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WO2020210420A1
WO2020210420A1 PCT/US2020/027360 US2020027360W WO2020210420A1 WO 2020210420 A1 WO2020210420 A1 WO 2020210420A1 US 2020027360 W US2020027360 W US 2020027360W WO 2020210420 A1 WO2020210420 A1 WO 2020210420A1
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pcr
nucleic acid
mmw
temperature
reaction mixture
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PCT/US2020/027360
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English (en)
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Andrey SAMSONOV
Andrea KLARICH
Dmitry Malkov
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Sigma-Aldrich Co. Llc
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • H—ELECTRICITY
    • H05—ELECTRIC TECHNIQUES NOT OTHERWISE PROVIDED FOR
    • H05B—ELECTRIC HEATING; ELECTRIC LIGHT SOURCES NOT OTHERWISE PROVIDED FOR; CIRCUIT ARRANGEMENTS FOR ELECTRIC LIGHT SOURCES, IN GENERAL
    • H05B6/00—Heating by electric, magnetic or electromagnetic fields
    • H05B6/64—Heating using microwaves
    • H05B6/80—Apparatus for specific applications
    • H05B6/806—Apparatus for specific applications for laboratory use
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/14—Process control and prevention of errors
    • B01L2200/143—Quality control, feedback systems
    • B01L2200/147—Employing temperature sensors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1822—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using Peltier elements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1861—Means for temperature control using radiation
    • B01L2300/1866—Microwaves

Definitions

  • PCR cycler models use conventional heating to increase the temperature of the PCR mixture solution inside a PCR tube: the tube is placed in a metal heat block that is heated by electric resistance heating. Conventional heating raises the temperature of the PCR tube content by transferring heat energy from the PCR tube walls (that contact the heat block) to the liquid inside the tube (see, FIG. 3A). There are several steps in this process: first, a massive (relative to the liquid volume in the PCR tube to be heated) metal block is heated by an electrical resistive heater; second, plastic PCR tube walls contacting the metal block are heated and; third, the PCR tube content is heated. The block is cooled via Peltier cooler elements. Average PCR reaction volume is about 20 pi.
  • the mass of water solution that needs to be heated and cooled for PCR steps (such as denaturation, annealing, and extension) is negligible compared to the mass of the metal heat block that needs to be heated.
  • the time it takes to heat the PCR sample is referred to as“ramp rate.”
  • Collins developed a microwave-assisted PCR amplification procedure.
  • the method involved the use of pulses of microwave radiation to assist nucleic acid amplification by subjecting the PCR reaction mix to microwave radiation at the denaturalization and annealing steps.
  • any increased speed in PCR cycles resulting from this procedure was, at least in part, accomplished by maintaining the DNA at a maximum temperature of less than 60 °C and not by the use of microwave radiation.
  • This limitation may be caused by an inherent limitation with regard to the use of microwave radiation in heating PCR samples, as discussed below. Further, this limitation does not allow the use of Taq polymerase at its optimal temperature (75 - 80 °C), the polymerase enzyme of choice in most PCR reactions.
  • Giordano, et al. (Analytical Biochemistry 2001 , 201 :124- 132) also explored the use of IR radiation in PCR.
  • the researchers used a tungsten lamp to specifically heat the PCR solution.
  • the reaction was restricted to use on microchips with volumes of 1.7 mI. Typical PCR volumes are about 20 pi. It is unknown if the procedure would work effectively or efficiently on these volumes.
  • Ouyan, et al. (Analytica Chima Acta 2015, 901 :59-67) disclose a laser print-cut-laminate microchip for multiplexed PCR via infrared-mediated thermal control.
  • the present invention provides a new method for rapid and convenient heating of PCR reaction mixtures via millimeter wave-mediated (MMW-based) internal heating.
  • MMW-based millimeter wave-mediated
  • the present invention provides a new device for performing the new method.
  • the new device can be produced without undue cost.
  • the new device comprises a waveguide and a vessel for holding polymerase chain reaction (PCR) reaction tubes, e.g., a conical horn antenna or vessel for holding a PCR reaction tube.
  • PCR polymerase chain reaction
  • the MMW radiation exiting the waveguide via the conical horn antenna is completely absorbed by the PCR reaction mixture volume. This results in internal heating of the PCR solution with a heating rate of about 10°C/s or faster, as compared to minutes for conventional PCR devices, thereby greatly reducing the ramp rate.
  • MMW heating can be performed in any standard PCR tubes, which are manufactured from materials that are completely transparent to MMW radiation.
  • the instrument of the present invention is inexpensive, simple and, in combination with the method of the present invention, decreases PCR reaction time for 25-30 cycles to 20 minutes or less compared to 90-120 minutes in PCR cyclers that use conventional external heating.
  • amplification simultaneously increases the amplification rate by reducing the apparent activation energy for a DNA polymerase. Both advantages, faster temperature ramp rates and increased amplification rates, lead to a reduction in the total reaction time for 25 - 30 PCR cycles from 1.5 - 2.0 hours to 15— 20 minutes or less and can create a paradigm shift in PCR techniques and devices.
  • the present invention relates to a method and an apparatus for optimization and acceleration of nucleic acid amplification reaction by means of MMW heating of the PCR reaction mix.
  • the invention provides a method for amplification of a nucleic acid fragment where MMW energy is used to control the heating (/. e., temperature increase) at all PCR steps: DNA double strand denaturation at about 95°C, annealing at an average temperature of 63°C and polymerization ⁇ elongation) at an average temperature of 73°C (see, FIG. 2).
  • MMW radiation applied to a small PCR volume (about 20 mI or less) allows heating it with ramp rate of about 10 °C/s or faster and provides stable temperature levels by the use of regulated MMW power.
  • Rapid cooling of PCR tubes may be done via Peltier cooler elements connected to brass (or other suitable material having rapid heat transfer capabilities) PCR conical horn antennas that serve as PCR tube holders (see, FIG. 4).
  • the background temperature of the horn antennas (about 57°C) can be maintained by conventional resistive heating or other suitable means as is known to one of ordinary skill in the art.
  • the invention utilizes on MMW energy to achieve two goals with regard to increasing the overall rate of PCR amplification reactions: 1) rapid heating of PCR reaction volume; 2) significant acceleration of PCR reaction rate. Combination of both processes allows performing 30 - 35 cycles of PCR amplification of double stranded DNA in 15 - 20 minutes rather than in 1.5 - 2.0 hours.
  • the present invention proposes a new method for fast and convenient heating of small PCR reaction volumes via MMW-based internal heating (FIG. 3). Since MMW radiation energy is absorbed only by water, there is no need to heat any other parts of the PCR cycler. The MMW radiation is absorbed by PCR volume only, without heating even the PCR tube walls. The MMW heating could be performed in any standard PCR tubes. Standard PCR tubes are manufactured from materials transparent to MMW. The proposed instrument is inexpensive, simple and safe for the users.
  • the present invention relates to a device for the amplification of nucleic acid, the device comprising: a millimeter wave source, a rotary vane attenuator, a power amplifier, a power divider, one or more vessels (e.g., conical horns) for holding polymerase chain reaction tubes, wherein the millimeter wave (MMW) source, rotary vane attenuator, power amplifier, power divider and one or more vessels for holding polymerase chain reaction (PCR) tubes are connected with waveguides.
  • a millimeter wave source millimeter wave
  • rotary vane attenuator e.g., conical horns
  • PCR polymerase chain reaction
  • the MMW source is a Gunn oscillator.
  • the device additionally comprises Peltier cooling.
  • the rotary vane attenuator is programmable and is remote controlled.
  • the power amplifier is selected from CMOS-based power amplifier and solid-state power amplifier.
  • the vessels for holding polymerase chain reaction tubes are conical horns.
  • thermocoupler inside at least one the PCR reaction tube.
  • the present invention relates to a method for the amplification of nucleic acids, the method comprising: a) providing: i) nucleic acid to be amplified, a reaction mixture comprising reagents suitable for the synthesis of nucleic acid and primers, wherein the nucleic acid to be amplified and the reaction mixture are combined to create an amplification reaction mixture (ii) a device for raising the temperature of reaction mixture, wherein said device raises the temperature of the amplification reaction mixture by, at least in part, exposure of the amplification reaction to millimeter wave (MMW) radiation; b) contacting the nucleic acid to be amplified with the reaction mixture to create an amplification reaction mixture; cycling the amplification reaction mixture through temperature changes to induce denaturization of the nucleic acid to be amplified, annealing of primers to the denatured nucleic acid and synthesis of new copies of the nucleic acid to be amplified, wherein the temperature of the amplification reaction mixture
  • the MMW radiation is from 12 to 110 GHz.
  • the MMW radiation is from 25 to 42 GHz.
  • the device lowers said temperature of the amplification reaction with Peltier coolers.
  • step b) is performed from 5 to 40 times (or more) in sequence, step b) is performed from 10 to 30 times in sequence or step b) is performed from 15 to 25 times in sequence.
  • the reagents suitable for the synthesis of new nucleic acid comprise nucleotide bases, nucleic acid polymerase and buffers.
  • the nucleic acid polymerase is Taq polymerase.
  • the nucleic acid to be amplified is selected from deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and peptide nucleic acid (PNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • PNA peptide nucleic acid
  • FIG. 1 shows a schematic diagram of nucleic acid (DNA) amplification via PCR.
  • FIG. 2 shows an illustration of 3 PCR steps within 1 cycle performed in commercial PCR cyclers that use conventional heating.
  • FIG. 3 show (A) an illustration of conventional heating and internal heating approaches.
  • the object's temperature rises by transferring the heat from the surface to the inside the object (external heating).
  • the object In the case of the microwave heating, the object is heated from the inside due to the microwave absorption in its volume resulting in much faster heating for small volume objects. Note: in contrast to millimeter waves, microwave radiation could not be used for heating the PCR samples as its water penetration depth (about a few centimeters) is much longer than the characteristic size of a PCR sample (about a few millimeters).
  • FIG. 4 show (A) a schematic illustration of an embodiment of the MMW-based PCR apparatus of the present invention, as is discussed in detail, below; and, (B) a schematic illustration of the intensity of the MMW used in heating the PCR reaction mixture as different stages of the PCR cycle.
  • FIG. 5 is real block and sample temperature curves from six zones during up ramp on the ProFlexTM 96-well PCR system (Kim, YH, et ai, Performance evaluation of thermal cyclers for PCR in a rapid cycling condition. Biotechniques, 2008, 44(4): 495-506).
  • the dotted lines indicate the temperature range sampled for the maximum ramp rate and the average ramp rate.
  • FIG. 6 shows temperature profiles of PCR volumes inside PCR tubes (Kim, YH, et al., Performance evaluation of thermal cyclers for PCR in a rapid cycling condition. Biotechniques, 2008, 44(4): 495-506).
  • A Normal temperature profile (cycler I).
  • B Curving temperature profile (cycler F).
  • C Overshooting temperature profile (cycler P).
  • D Undershooting temperature profile (cycler J). See Table 1 for corresponding cycler types.
  • FIG. 7 is an illustration showing the contribution of the intracycle "waste time” within one PCR cycle to the net duration of the cycle.
  • A Real temperature of a sample in a PCR cycler with conventional heating showing that the intracycle "waste time” (heating and cooling ramps) is comparable in duration to the net step time for fast cycles.
  • B Ideal PCR cycle where temperature drops happen instantaneously.
  • FIG. 8 shows an output waveguide of an embodiment of the MMW- based thermal cycler pf the present invention.
  • FIG. 9 shows the cycle temperature profile of a PCR reaction mixture (Q5 polymerase, sample volume 12 mI) heated by an embodiment of the MMW-based thermal cycler pf the present invention.
  • FIG. 10 shows an electrophoresis gel showing the performance comparison between an embodiment of the MMW-based thermal cycler pf the present invention to a commercial PCR machine.
  • PCR Polymerase Chain Reaction
  • US Patent Nos. 4,683,195 and 4,683,202 to Mullis both of which are incorporated herein in their entirety.
  • PCR is a method to generate thousands to millions of copies of specific segments of nucleic acids, especially DNA.
  • PCR methods rely on thermocycling. Thermocycling repeats cycles of heating and cooling to permit different temperature-dependent reactions in the synthesis of new strands of nucleic acid to occur. Specifically, temperatures are cycled to cause DNA denaturization (often referred to as “melting”), annealing of primers to the melted DNA, and enzyme-driven DNA replication.
  • Primers are short sequences of DNA specific for the sequences of DNA that is to be replicated, i.e., the“target DNA.”
  • the PCR process doubles the number of copies of the target DNA with each cycle. Thus, in only a limited number of cycles the number of copies of the target DNA replicated can reach the millions. For example, after 20 cycles the number of copies equals 2 20 or over 1 million copies. After 30 cycles (2 30 ) the number of copies equals over a billion copies.
  • the number of copies are ultimately limited by depletion of reagents (e.g., nucleotide bases) available in the reaction mixture, however the process is usually terminated prior to that point by the technician.
  • Thermocycling originally was performed by physically moving reaction tubes from one temperature-controlled water bath to another. Flowever, PCR automated thermal cyclers (PCR cyclers) are now typically used.
  • All commercially available PCR cycler models use conventional heating to increase the temperature of the PCR mixture solution inside a PCR tube: the tube is placed in a metal heat block that is heated by electric resistance heating. Conventional heating raises the temperature of the PCR tube content by transferring heat energy from the PCR tube walls (that contact the heat block) to the liquid inside the tube (see, for example, FIG. 3). There are several steps in this process: first, a massive (relative to the liquid volume to be heated) metal block is heated by an electrical resistive heater; second, plastic PCR tube walls contacting the metal block are heated and; three, finally the PCR tube content (the“PCR reaction mixture” or“PCR volume”) is heated. The block is typically cooled via Peltier cooler elements
  • the average PCR reaction volume is about 20 mI, i.e., the mass of reaction solution than needs to be heated and cooled for each of the PCR steps (such as denaturation, annealing and extension) is negligible compared to the mass of the metal heat block that needs to be heated and cooled.
  • the initial step is a denaturing step where the target DNA is denatured (melted) by heating it to 94°C or higher for 20 seconds to 2 minutes.
  • the two intertwined strands of DNA separate from one another, producing the necessary single-stranded DNA template for replication by a thermostable DNA polymerase such as, for example, Taq polymerase.
  • a thermostable DNA polymerase such as, for example, Taq polymerase.
  • Other suitable polymerases are known to those of ordinary skill in the art.
  • the temperature is reduced to approximately 40 - 60°C. At this temperature, the oligonucleotide primers can form stable associations (anneal) with the denatured target DNA and serve as primers for the DNA polymerase.
  • step (elongation) the synthesis of new DNA begins as the reaction temperature is raised to the optimum temperature for the DNA polymerase. For most thermostable DNA polymerases, this temperature is in the range of 70 - 74°C.
  • the extension step lasts approximately 1 - 2 minutes.
  • the next cycle begins with a return to 94°C for denaturation.
  • Each set of denaturation, annealing and elongation steps comprises a“cycle.” Typically, PCR reactions are run from 20 - 30 cycles.
  • FIG. 1 shows a schematic diagram illustrating one cycle.
  • FIG. 2 is an illustration of the timing required to perform the 3 PCR steps within 1 cycle of PCR performed in commercial PCR cyclers that use a conventional heating system. Heating and cooling lag time is shown in dashed lines.
  • FIG. 3A is a schematic diagram illustrating the differences between MMW heating and conventional heating of PCR reaction tubes.
  • the object's temperature rises by transferring the heat from the surface of a metal heating block, through the PCR tube wall to the PCR reaction mix inside the PCR tube (external heating).
  • the PCR reaction mix is heated from the inside due to the millimeter wave absorption by the reaction mix volume thus resulting in much faster heating.
  • FIG 3B shows a graphical representation of the difference in heating between conventional and MMW heating of a PCR reaction mix.
  • temperature cycling is fundamental to all PCR reactions.
  • Commercial thermal cyclers use conventional external heating to heat PCR reaction volumes in PCR tubes inserted in a metal heat block that is heated by the electric resistance heating and cooled by the Peltier cooler elements.
  • it takes significant time for the PCR machine to transition between each set of temperatures see,
  • FIG. 5 termed the intracycle “waste time” (WT). It takes about 25 - 30 seconds for a modern PCR machine to change the temperature between the steps of the cycle (see, FIG. 6). How fast an instrument can "ramp” between temperatures dictates the overall speed of the PCR run. Because of this, thermal cycler manufacturers publish their “ramp rates” to indicate the change in temperature over time. Ramp rates are typically expressed in °C/second. Looking at this concept graphically in a plot of temperature versus time, the ramp rate is simply expressed by the slope of the curve. A steeper curve represents a higher ramp rate and means that a specific temperature range can be covered in a shorter time. The terms “up ramp” and “down ramp” refer to the ramp rate when heating and cooling, respectively. A faster ramping block will result in a faster PCR run, so many thermocycler manufacturers seek to show the highest possible ramp rate.
  • FIG. 6 shows temperature profiles of PCR volumes inside PCR tubes (data from: Kim, YH, et al., Performance evaluation of thermal cyclers for PCR in a rapid cycling condition. Biotechniques, 2008, 44(4): 495-506).
  • A Shows a typical temperature profile (cycler I).
  • B Shows a curving temperature profile (cycler F).
  • C shows an overshooting temperature profile (cycler P).
  • (D) shows an undershooting temperature profile (cycler J). See Table 1 , below, for corresponding PCR cycler types. Additionally, with a“curving temperature profile” time intervals of PCR reaction steps can be much shorter than required because WT periods between the steps are much longer than the step time. Even in a "normal temperature profile” (/.e., an ideal temperature profile) the total intracycle "waste time” is comparable to the net step duration of the cycle (see, FIG. 7 A).
  • thermocouple probe
  • the present invention proposes a new method for rapid and convenient heating of PCR volumes via MMW- based internal heating. That is, only the PCR reaction mix is heated thereby eliminating the time and energy needed to heat the heating block and PCR tube walls.
  • the MMW radiation exits a waveguide by way of a conical horn antenna and is completely absorbed by the PCR reaction mixture. This results in internal heating of the PCR reaction mixture with a heating rate of about 10°C/s or faster.
  • the MMW heating can be performed in any standard PCR tube.
  • Standard PCR tubes are manufactured from materials that are completely transparent to MMW radiation.
  • the instrument of the present invention is inexpensive, simple and can decrease PCR reaction time for 25 - 30 cycles to 20 minutes or less compared to 90 - 120 minutes in PCR cyclers that use conventional external heating.
  • the present method is based on water absorption of a MMW electromagnetic field that occurs in water-based solutions.
  • MMWs enter solutions with polar molecules such as water, these molecules attempt to align themselves to the applied electric field by rotation but do not have enough time to follow the electric field. This failure to keep in phase results in energy loss via friction and collision with other molecules giving rise to the dielectric (internal) heating.
  • Millimeter wave generators are known to one of ordinary skill in the art. For example, see, Remley, et al., (A Precision Millimeter-wave Modulated- signal Source; June 06, 2013 in IEEE International Microwave Symposium Digest) as well as commonly available from vendors.
  • An example of a suitable MMW generator is, e.g., a Gunn oscillator made by ZAX Millimeter Wave Corporation (Upland, CA).
  • Other suitable MMW generators are known to those of ordinary skill in the art and can be selected based on the teachings of this specification.
  • the method employs millimeter waves in Ku, K, Ka, Q, U, V and W-bands with frequencies ranges from 12 GHz to 1 10 GHz.
  • the penetration depth (Do) is the depth at which approximately 63% of the radiation energy is absorbed (the electric field amplitude falls to e-1 of its value).
  • Do The penetration depth
  • the Do should be in 1 - 1.5 mm range
  • the millimeter wave frequencies useful in the present invention are from 12 - 110, GHz, 15 - 100 GHz, 18 - 75 GHZ, 20 - 60 GHz, 24 - 50 GHz and 26 - 40 GHz.
  • the MMW are guided to the bottom of PCR tubes via a special waveguides that provide localized irradiation of a controllable power.
  • the waveguides characteristic impedance has a different value than the impedance of the free space (or air). This is a mismatch of the impedance and in this case a significant part of the power will be reflected.
  • the conical horn (the“horn”) is essentially a flared waveguide (see, FIG. 8).
  • the flaring structure of the waveguide acts as an impedance transformer and matches the waveguide impedance to the free space impedance and therefore, enables the flared waveguide structure to radiate guided wave into free space.
  • the horn is, for example, a hollow pipe that has been tapered to a larger opening.
  • Horns may be made of any suitable material. Material that is conductive of heat (for faster cooling of the PCR reaction mixture) is preferred. Metal, particularly brass, is most preferred. [0055] Horn antennas are typically fed by a section of a waveguide. As is known to one of ordinary skill in the art, a waveguide refers to any linear structure that conveys electromagnetic waves between its endpoints.
  • the rectangular waveguide transitions into a short circular horn antenna with internal dimensions that fit the conical bottom of a standard 0.2 ml PCR tube (see, FIG. 8).
  • This horn antenna has two functions: 1) it provides more effective irradiation of the PCR volume inside the PCR tube reducing the power reflection; 2) it serves as a PCR tube holder and as an effective heat sink providing more uniform heating of the reaction volume and accelerating cooling of the PCR tube and its content when needed.
  • One of ordinary skill in the art can design other variations of the present invention with the guidance and teachings provided herein.
  • MMW can penetrate diamagnetic materials, such as glass and plastic, without power attenuation.
  • Plastic PCR tubes are completely transparent for MMW radiation and therefore will not be heated, and therefore, will not absorb heat or act as heat sinks (and, therefore, will cool faster).
  • the MMW radiation heats only the solution contained inside the PCR tube but not the PCR tube itself.
  • the temperature of the solution within the PCR volume depends on the energy (amplitude and frequency) of MMW.
  • the temperature of the PCR volume depends on the supplied MMW power. To reach the maximal temperature ramp rate in the PCR tube volume, MMW power is applied and, after the desired temperature is reached, the MMW power is decreased to lower level(s) to maintain the desired
  • the equipment can be calibrated to heat different PCR reaction mixture volumes placed in standard PCR tubes.
  • the calibration procedure is performed using a micro-thermocouple inserted in a PCR tube with known reaction volume (see, FIGS. 4, 8).
  • the ideal thermal cycler should have an ultra-rapid rate of sample heating and cooling (see, FIG. 7, right) that essentially eliminates the intracycle "waste time" intervals.
  • MMW internal heating (/. e., when MMW power directly heats the PCR volume) provides heating rate >10 °C/s that allows changing the temperature between the steps of the cycle within 4 seconds or less (see, FIG. 9) as compared to about 25 - 30 seconds for a conventional PCR cycler (see, FIG. 6).
  • the present invention is not limited by theory, the present inventors believe that another surprising advantage of MMW heating is a substantial enhancement in chemical and enzymatic reaction rates due to enhancement of the chemical reactions as a result of the millimeter waves. Since the first use of microwaves for synthesis reactions appeared in 1986, numerous studies have investigated the use of microwave heating or microwave irradiation for organic and materials synthesis, and significant improvements that cannot be obtained by conventional heating methods have been reported. Microwave-enhanced synthesis allows organic chemists to work faster, generate higher yields and increase product purity. In addition, microwave irradiation has been successfully applied in a range of
  • MMW-based PCR tube heating is safe for the operator because the PCR volume completely absorbs all MMW power. Even if the equipment is accidently turned on without PCR tubes inserted in the horns, the MMW irradiation power is too low to harm the operator. Even 100 mW MMW fanning out from the mouth of the conical horn will expose a person to much less than the maximum permissible exposure for uncontrolled environments of 10 Wrrr 2 (IEEE, 2005). All the same, to exclude the possibility of having the power-on without a water layer placed over the waveguide mouth, a simple MMW detector could be placed in the lid of the device. It would power off the MMW oscillator if a high MMW intensity in a vertical direction is detected.
  • FIG. 4A An embodiment of the device on the present invention is schematically illustrated in FIG. 4.
  • MMWs are originated at the source of MMW radiation (1 ) and then delivered via a waveguide (2) to a remotely controlled motorized programmable rotary vane attenuator (3) and then to the power amplifier (4) via another waveguide (2).
  • MMWs enter the power divider (5) to be split into multiple waveguides (2) ending with conical antenna horns (6).
  • PCR plastic tubes (7) are inserted in conical horns antennas.
  • the conical horn antenna has 2 functions: 1) the horn is a flaring structure of the waveguide that acts as an impedance transformer and matches the waveguide impedance to the free space impedance and therefore, enables the flared waveguide structure to radiate guided wave into free space; 2) conical horn serves as a PCR tube holder that tightly contacts the tube walls and serve as an effective heat sink.
  • the horns are connected to the resistive heater and Peltier cooler elements (8).
  • the background temperature of the PCR tubes is maintained by the resistive heater blocks.
  • the Peltier elements provide for faster cooling.
  • FIG 4B illustrates schematically the level of MMW radiation used in each step of the PCR reaction. To reach maximal temperature ramp rate in the PCR tube volume, MMW power is applied (# 1).
  • the tube is cooled both by the conical horn acting as a heat sink and with proactive cooling by the Peltier coolers.
  • a lower level of MMW power is used at (# 2) to increase the temperature of the PCR reaction mix for the elongation step.
  • a further increase in MMW power is used at (#3) to increase the temperature back to the temperature necessary for the denaturization of the duplex DNA for the nest PCR cycle.
  • FIG. 8 shows the embodiment of the output waveguide of the MMW based thermal cycling prototype used in this experiment.
  • the prototype was designed as schematically illustrated in FIG. 4A and shown in FIG. 8.
  • a standard 0.2 ml PCR tube was inserted into the conical antenna horn that terminates the standard WR10 waveguide.
  • a Gunn MMW oscillator (ZAX Millimeter Wave Corporation, Upland, CA) providing 94 GFIz MMW was used to generate the MMW for heating the PCR reaction mixture.
  • a micro thermocoupler was used to monitor the temperature inside the PCR tube.
  • Thin film resistors were used to maintain the horn temperature at ⁇ 57°C and were monitored by the thermistors.
  • the prototype did not have Peltier coolers or an attenuator.
  • the tube was allowed to cool down by release of heat into the surrounding area (convection) and MMW generation was performed manually by turning the MMW source“on” and“off” based on temperature readout from the micro-thermocoupler.
  • FIG. 9 shows the cycle temperature profile of a PCR reaction mixture (Q5 polymerase, sample volume 12 pi) heated by the MMW based device of the present invention.
  • the cool down time for the sample to cool from 96 °C to 61 °C was 25 seconds per cycle.
  • the 25 seconds cool down time can be reduced substantially by the implementation of the Peltier cooler elements that will allow completion of each cycle in 30 seconds or less.
  • FIG. 10 shows the performance comparison of the MMW based thermal cycling prototype to a commercial PCR machine.
  • a fragment of BYSL gene was amplified from a genomic DNA using the same conditions for both instruments (primers, polymerase, reaction mix and volume, cycling protocol).
  • the cycle temperature profile of the reaction mix for the MMW prototype is shown in FIG. 9.
  • the amplified product was analyzed by the agarose gel electrophoresis. While the intensity of the band was lighter in the case of the MMW-based prototype as compared to the commercial device, the time for the PCR reaction was greatly reduced as compared to the commercial device taking less than half of the time for performing the same number (35) of PCR cycles.
  • the lighter intensity band in the case of the MMW based prototype can be explained by the heterogeneity of heat distribution in the reaction volume due to less than ideal conditions of MMW-based thermocycling; that is, the MMW frequency would more effectively heat the PCR reaction mixture if be decreased from the 94 GFIz used to 30-40 GHz. Further, the prototype lacked automated attenuation, feedback and active cooling. Adding these elements to the device should eliminate the difference in the levels of the product produced between the commercial device and the device of the present invention.

Abstract

La présente invention concerne un cycleur de réaction en chaîne par polymérase basé sur des ondes millimétriques et ses procédés d'utilisation.
PCT/US2020/027360 2019-04-09 2020-04-09 Réaction accélérée en chaîne par polymérase basée sur le thermocyclage activé par le chauffage par ondes millimétriques WO2020210420A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201962831440P 2019-04-09 2019-04-09
US62/831,440 2019-04-09

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