Classifications

• • 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
• • 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
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
  • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
• • 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
  • B01L2300/1838—Means for temperature control using fluid heat transfer medium

Definitions

• TITLE GAS JET PROCESS AND APPARATUS FOR HIGH-SPEED AMPLIFICATION OF DNA
• the present invention relates to methods and apparatus for perfonning high-speed amplification of a sample. More specifically, the present invention relates to methods and apparatus for performing pressurized jet polymerase chain reaction, wherein each cycle can be completed in as little as a few seconds.
• PCR Polymerase Chain Reaction
• the Polymerase Chain Reaction is one of the most widely used techniques in molecular biology (U.S. Pat. No 4,683,202 to Mullis; Saiki et al, 1985; Erlich, 1989; Mullis et al, 1994).
• PCR-amplified DNA can be used to diagnose mutations responsible for human genetic diseases (Saiki et al, 1985; Kogan et al, 1987), in blood and tissue typing (Saiki et al, 1989a), or to detect pathogens responsible for important infectious diseases (Persing et al, 1993; Nicoll et al, 2001).
• template DNA sequences lying between the ends of two defined oligonucleotide primers can be amplified in 1 to 2 hours (FIG. 4a).
• Three sequential steps are normally employed: (1) double-stranded DNA is denatured (D) to a single-stranded form at a high temperature (90°C to 95°C), (2) the resulting single-stranded DNA strands are annealed (A) to oligonucleotide primers at ⁇ 45°C to 65°C, and (3) primer: template complexes are elongated (E) using a thermostable DNA Polymerase such as Thermus aquaticus (Taq) Polymerase at ⁇ 72°C (Saiki, 1989b).
• a thermostable DNA Polymerase such as Thermus aquaticus (Taq) Polymerase at ⁇ 72°C (Saiki, 1989b).
• Robotic devices such as Stratagene's ROBOCYCLER move tubes containing PCR reaction samples to and from a series of heat baths, which are thermostated at different temperatures. Although these devices maybe useful in certain research applications, they are incapable of high-speed PCR. They require >60 minutes for 30 cycles of amplification; equivalent to >2 minutes per PCR cycle.
• thermocyclers have become familiar devices in many biochemistry laboratories. Most commercially available PCR devices (Perkin-Ehner, MJ Research, Ericomp, Techne, Eppendorf, BioRad, Hybaid) are thermocyclers (Johnson, 1998). In general, two types of thennocyclers are employed: programmable heat blocks and forced hot-air thermocyclers.
• thermocyclers are heat blocks with holes in them where plastic reaction tubes are heated and cooled under electronic control.
• Several such devices have been reviewed by Johnson (1998).
• the problem with programmable heat block designs is that most of the time is spent waiting for a bulky piece of metal to heat up or cool down.
• 14 seconds/cycle is lost in transition between D, A, and E temperatures ( ⁇ 94°C, ⁇ 55°C, and 72°C).
• a single heating/cooling cycle from 94°C to 55°C wastes 28 seconds per cycle; or -14 minutes during 30 PCR cycles.
• thermocycling protocol spends one minute at 94°C (denaturation), one minute at ⁇ 55°C (annealing), and one minute at 72°C (elongation).
• a 536 b.p. human ⁇ -globin DNA fragment was amplified using 30 cycles of (1 min at 94°C, 1 min at 55°C, 1 min at 72°C).
• Sobczak et al. (1995) have shown that faster PCR protocols can be employed in heat block thermocyclers when denaturation, annealing, and elongation steps are reduced to one to two seconds per cycle.
• a 139 base pair long human p53 gene fragment was amplified through 35 cycles of (1 sec at 94°C, 1 sec at 58°C, 1 sec at 72°C).
• the heating/cooling time of the thermocycler was still >25 seconds/cycle. Therefore, the overall thermocycling time was ⁇ 30 seconds/cycle or -15 minutes for 30 PCR cycles.
• 6,200,781 have used gases other than air, pressures other than atmospheric, cooling gases colder than 22°C, or time constants faster than one second, they have not employed flow conditioners to mix hot and cold gases and/or to increase gas pressure and velocity. They have not considered the possibility that different gases may be employed at different stages of the PCR process, and have not considered the role of fluid mechanics, the physics of heat transfer, or flow conditions for optimal heat transfer. For example, that "Air is an ideal heat transfer medium which can change temperature quickly because of its low density.” (Wittwer et al.
• a pressurized gas jet thermocycler operates at gas pressures, velocities, turbulent non- isothermal flow conditions, and geometries which have not previously been considered — let alone implemented in high-speed DNA amplification experiments, as shown in FIG. 5 through 12.
• thermocycler Comparison of Heat Transfer in Hot-air and Pressurized Gas Thermocyclers.
• the differences between the hot-air thermocycler and the pressurized gas thermocycler are best described by a brief analysis of heat transfer around a thin cylinder (capillary).
• the cylinder is chosen because both of these thermocyclers use capillaries for holding the DNA samples; but the analysis would be analogous for any other shape of sample holder.
• d 0 and d i denote the outer and inner diameters of the capillary tube. Natural convection heat transfer and internal thermal gradients in the reacting liquid mixture are negligible due to the dominant role of viscous forces and small inner diameter. The thermal energy balance of the aqueous solution inside the capillary is then given by
• any heating or cooling of biochemical reaction samples can be described. It is evident from equation (2b) that the time t is reduced when the effective heat transfer coefficient h e f is maximized. This coefficient links the performance of the cycler to the heat transfer and the fluid mechanics.
• the coefficient h e ff includes the heat transfer through the capillary wall ( h c ) and through the boundary layer between the gas and outer surface of the capillary,
• the heat transfer coefficient for the capillary depends upon the thermal conductivity
• Equation (4c) hot-air thermocycler
• Equation (5b) pressurized air thermocycler
• Equation (4c) By comparing Equation (4c) to Equations (5b) and (6b), there is an expected fivefold improvement in the pressurized air jet as compared to the forced hot-air thermocycler and over twelve-fold improvement using pressurized helium as an operating gas.
• This example demonstrates that superior heat transfer translates into faster PCR. Improved heat transfer is accomplished by using pressurized gases; preferably helium; however, even suboptimal gases like air perform far better than non-pressurized devices.
• the pressurized gas thermocycler differs from the hot-air thermocycler in six aspects:
• Velocities are much higher (decreased boundary layer thickness): v > 5 m/sec.
• Gases other than air can be used; for example, helium can be used for rapid heating and CO 2 can be used for superior thermal control and efficient cooling.
• Digitally programmable electronic valves are used to deliver hot and cold gas bursts to the reaction chamber via conditioning chambers to regulate and mix the gas flows.
• DNA denaturation, primer.template annealing, and enzymatic elongation are programmable in time intervals of ⁇ 100 milliseconds.
• Convective cooling/cooling rates depend on: (1) the temperature difference between the gas and the object; and (2) the thickness of the boundary layer around the object (the object is sunounded by a thin layer of gas that is stationary with respect to the object, termed the "boundary layer"). The thickness of this layer strongly conelates with the velocity of the gas relative to the object. At higher gas velocities, the boundary layer thins and heat transfer rate (termed “heat flux”) improves.
• High velocities can be achieved by using pressurized gas: molecules will naturally flow from a region of high pressure to lower (atmospheric) pressure, as regulated by opening/closing of electronic and/or mechanical control valves.
• the transfer of thermal energy (heat) across the boundary layer is also strongly influenced by the type of gas that is used.
• Helium gas is particularly efficient for rapid heating of objects.
• Carbon dioxide (CO 2 ) gas cools to -24°C upon isenthalpic expansion and it is therefore an efficient, inexpensive cooling gas. This property depends on the polytropic coefficient of the gas - that is a thermodynamic property intrinsic and unique to each gas or gas mixture. When gas velocities approach values of Mach 0.3 (0.3 times the sound velocity of the gas) the gas becomes compressible.
• the thermal conductivity coefficient of helium was measured by Ubbink (1947) and is listed in tables published by Johnston and Grilly (1946), Bosworth (1952), Azbel (1984), and Chapman (1984). As shown in FIG. 1, helium has a thermal conductivity coefficient seven times that of air. Neon transfers heat about twice as fast as air. Therefore, hot-air thermocyclers have used the wrong gas for rapid heat transfer. Among the non- combustible gases, air is available; helium is optimal; in particular, the isotope of helium, 3 He. U.S. Pat. No. 5,455,175 to Wittwer et al.
• Zurek et al. does not contemplate the possibility of employing gas pressures greater than atmospheric (P > 1 bar) because the gas pressure in their machine was never measured.
• P > 1 bar gas pressure in their machine was never measured.
• 30 cycles of PCR amplification are achieved in as little as 78 seconds — about the same time needed for one cycle using the method of Zurek et al. (U.S. Pat. No. 5,576,218).
• a biochemical reaction chamber can be cooled much more rapidly using gases other than air at temperatures less than 25°C.
• forced hot-air thermocyclers utilize the wrong gases for heating and cooling, the wrong gas pressures, and the wrong configuration of heat chamber to reaction chamber.
• the time constants employed >1 second are too slow for high-speed thermocycling (see FIG. 2), forced hot-air thermocyclers gases lack optimal CFD-modeled gas dynamics and utilize no flow conditioners.
• Much faster thermocyclers can be designed using pressurized gases, particularly helium/air/CO 2 combinations, which are delivered to a thennostated reaction chamber using a flow conditioner (FIG. 3c) and electronically actuated valves.
• the present invention allows for high-speed amplification of viral, bacterial, and single-copy human gene fragments on a time scale which is over an order of magnitude faster than the method described by Chiou et al. (2001).
• the pressurized gas thermocycler routinely amplifies viral and bacterial DNA fragments 85 to 2331 b.p. long through 30 PCR cycles in 1.3 to 10.3 minutes (78 to 617 seconds). Single copy human gene fragments in this size range are amplified through 35 PCR cycles to detectable quantities in 3.5 to 6.5 minutes (FIG. 5a, 9a, and 13).
• the present invention includes a high-speed process for amplifying DNA.
• a reaction chamber containing a biological sample, a DNA polymerase, oligonucleotide primers, and deoxynucleoti.de precursors is provided.
• the reaction chamber accepts the flow of one or more heat transfer gases.
• a first heat transfer gas is heated in a heating chamber which is physically separated from the reaction chamber.
• the heated gas is delivered to the reaction chamber at a pressure greater than or equal to 1.3 bar (P >20 p.s.i.).
• Heat from the heated gas is utilized to denature DNA.
• a second heat transfer gas which may be the same kind as, or a different kind of gas than, the first heat transfer gas is delivered to the reaction chamber.
• the second heat transfer gas ( ⁇ 20°C) cools the reaction chamber to a temperature low enough to allow the denatured DNA to anneal to the oligonucleotide primers.
• the temperature of the reaction chamber is increased to a sufficient temperature (typically 68° to 75°C) to allow for enzymatic elongation of primer:template complexes.
• thermocycling When hot (>95°C) and cold ( ⁇ 20°C) gases are delivered to a biochemical reaction chamber under the control of either two-way, three-way electronic valves, or multiport electronic valves, extremely fast thermocycling is possible.
• a flow conditioner or a set of flow conditioners
• the temperature of an enzyme-catalysed reaction can be controlled on a time scale of tens of milliseconds.
• the gas of choice is helium, but for convenience, other gases can also be employed.
• An embodiment of the present invention also includes an apparatus for high-speed amplification of DNA.
• the apparatus includes a reaction chamber into which compressed hot (>95°C) and cold ( ⁇ 20°C) gas bursts are delivered on a time scale of fractions of a second.
• the overall configuration of the device consists of six parts: (1) a heater to raise the temperature of the heating gas to a pre-set value (> 95" C ), (2) one or more flow conditioning chambers for mixing cold and hot gases and regulate flow and attenuate pressure pulses, (3) an intake manifold with attached mixing chamber to mix hot/cold gases, (4) a gaseous reaction chamber, (5) an exhaust nozzle and (6) heat exchangers to recover heat from reactor outlet and hot gas by-pass.
• the pressurized gas thermocycler therefore resembles jet and/or rocket engines.
• hot and cold non-combustible gases are mixed under the control of electronic valves; and no ignition occurs.
• the essential process described in the present invention is therefore properly termed “gas jet PCR” and the device is aptly named a “PCR jet”.
• PCR jet Fast, precise, reliable thermal control of enzyme-catalysed reactions, are the purpose of the PCR jet.
• a heating chamber which is physically separated from the reaction chamber, is fluidly connected to the reaction chamber.
• a first container having a heating gas at a pressure greater than the reaction chamber pressure, is fluidly connected to the heating chamber. This connection may include a flow conditioning chamber for flow regulation and suppression of pressure pulses.
• a second container having a cooling gas (which may be the same kind or different kind of gas as the heating gas), is fluidly connected to the reaction chamber. This connection may include a flow conditioning chamber for flow regulation and suppression of pressure pulses.
• One or more cooling gas inlet valves are positioned between the second container and the reaction chamber, and one or more heating gas inlet valves are positioned between the first container and the reaction chamber.
• a programmable controller having electrical inputs and outputs, is used to control opening of the inlet valves. Temperature sensors, positioned in the reaction chamber, provide output to the controller. The controller opens and closes the inlet valves to reach or maintain a desired temperature within the reaction chamber. If the temperature of an optimal heat transfer gas, such as helium, is raised to >95°C in a chamber which is physically separated from the reaction chamber, and then delivered to the reaction chamber electronically actuated valves, then extremely fast thermocycling is possible. Even faster heat transfer is possible if cold ( ⁇ 20°C) gas is delivered to the chamber. For automated heating and cooling of the reaction chamber, preferably at least two electronic valves (hot gas valve and cold gas valve) and one or more mechanical relief valves are employed.
• an optimal heat transfer gas such as helium
• Bottled pressurized carbon dioxide (CO 2 ) gas expands and cools upon leaving its storage container (isenthalpic cooling). It is therefore convenient to use pressurized helium gas (or compressed air) to heat the reaction chamber and bottled CO 2 gas to cool the chamber.
• pressurized helium gas or compressed air
• PCR Polymerase Chain Reaction
• pre-heated and/or pre-cooled gases which are delivered to a thermostated reaction chamber using electronic valves.
• hot (>95°C) or cold ( ⁇ 20°C) pressurized gases are delivered to a physically separate reaction chamber containing biological samples, in order to control the temperatures used for DNA denaturation, primer:template annealing, and polymerase-catalysed elongation.
• Yet another object of the of the present invention to use one or more chambers prior to the reaction chamber to regulate the hot and cold gas flows in order to mitigate pressure pulsations. Still a further object of the present invention to use a mixing chamber before the reaction chamber in order to achieve homogeneous temperature in the flow admitted into the reaction chamber.
• FIG. 1 is a table of Thennal Conductivity of Gases, with thermal conductivity units (k-values) expressed in Watts/meter-°C.
• FIG. 2 a is a schematic diagram which compares a hot-air thermocycler to a pressurized gas thennocycler.
• FIG. 2b is a table which compares properties of a hot-air thermocycler to a pressurized gas jet thermocycler.
• FIG. 3a is a perspective view of an embodiment of an apparatus to amplify DNA according to the present invention.
• FIG. 3b is a perspective view of reaction chamber 38, flow conditioners 30 and 34, and the thermocouples 44, 46, 48, and 50.
• FIG. 3c is a view of a flow conditioner used in the reaction chamber 38 of FIG. 3a.
• FIG. 3d shows a computational fluid dynamic mesh of a flow conditioner as shown in FIG. 3c.
• FIG. 3e shows a perspective view of the reaction chamber 38 as shown in FIG. 3a.
• FIG. 4 shows temperature versus time profiles of different types of thermocyclers.
• thermocycler is -10X faster than a hot-air thermocycler
• FIG. 5a is a picture of a gel electropherogram, which shows high-speed gas jet PCR amplification of four different DNA templates, including (a) a 91 b.p. E.coli O157:H7 Stx2 amplicon, (b) a 333 b.p. bacteriophage ⁇ 'D' gene fragment, (c) a 364 b.p. human Platelet Antigen HPA-4 allele gene fragment, and (d) a human ⁇ -globin 536 b.p. gene fragment.
• a gel electropherogram which shows high-speed gas jet PCR amplification of four different DNA templates, including (a) a 91 b.p. E.coli O157:H7 Stx2 amplicon, (b) a 333 b.p. bacteriophage ⁇ 'D' gene fragment, (c) a 364 b.p. human Platelet Antigen HPA-4 allele gene fragment, and (d) a human ⁇ -globin
• FIG. 5b is a temperature versus time profile for gas jet amplification of the four DNA fragments shown in FIG. 5a.
• FIG. 6 is a picture of a gel electropherogram, which shows the reaction products of high-speed gas jet PCR amplification of an 85 b.p. E.coli O157:H7 Stx DNA fragment using 2.8 bar pressurized air with 2.9 bar CO 2 cooling.
• FIG. 7a is a picture of a gel electropherogram, which shows He/CO 2 gas jet PCR amplification of an 85 b.p. E.coli O157:H7 Stx DNA amplicon through 30 high-speed PCR cycles in 85 seconds (lane a), 81 seconds (lane b), or 78 seconds (lane c).
• FIG. 7b is a temperature versus time profile for the experiment shown in FIG. 7a (lane c). This experiment represents the fastest DNA amplification experiment which has been carried out.
• FIG. 8a is a picture of a gel electropherogram, which shows the reaction products of high-speed gas jet PCR amplification of a 368 b.p. DNA fragment from Bacillus anthracis.
• FIG. 8b is a temperature versus time profile for PCR jet amplification of the 368 b.p. B. anthracis DNA fragment shown in FIG. 8a (lane e). Total time for 30 high-speed PCR cycles was 233 seconds.
• FIG. 9a is a picture of a gel electropherogram, wltich shows high-speed gas jet PCR amplification of (a) 191 b.p. human Accute Intermittant Porphyria (AIP) amplicon, (b) 147 b.p. human ABO blood group 'A-transferase' gene fragment, (c) 486 b.p. E.coli uidA gene fragment, (d) 297 b.p. E.coli uidA gene fragment, (e) 145 b.p. E.coli uidA gene fragment.
• AIP Accute Intermittant Porphyria
• FIG. 9b is a temperature versus time profile of the PCR jet reaction used to amplify the 147 b.p. human ABO blood group A-transferase gene fragment shown in FIG. 9a. 35 PCR cycles were carried out in 228 seconds.
• FIG. 10a is a picture of a gel electropherogram, which shows nested PCR amplification of an outer 486 b.p. and nested inner 186 b.p. E.coli uidA DNA fragment.
• FIG. 10b is a diagram showing the DNA sequences and locations of primers used in the nested PCR experiment shown in FIG. 10a.
• FIG. 11a is a picture of a gel electropherogram, which shows gas jet PCR amplification of a 2206 b.p. long DNA fragment from bacteriophage ⁇ .
• FIG. lib is a picture of a gel electropherogram, which shows gas jet PCR amplification of a 2331 b.p. long DNA fragment from bacteriophage ⁇ .
• FIG. 12a is a picture of gel electropherogram, which shows gas jet PCR amplification of a 297 b.p. long DNA fragment from the E.coli uidA ( ⁇ -glucuronidase) gene.
• FIG. 12b is a temperature versus time profile of the PCR jet reaction used to amplify a 297 b.p. long DNA fragment from the E.coli wt--4( ⁇ -glucuronidase) gene.
• FIG. 13 is a table summarizing high-speed PCR experiments carried out using the pressurized gas jet thermocycler.
• FIG. 14 is a list of scientific nomenclature used herein. DETAILED DESCRIPTION OF THE INVENTION
• the overall speed of DNA amplification is limited by the biochemistry rather than the heating/cooling time of the apparatus.
• the rate of Taq Polymerase elongation (-80 nucleotides/sec at 72°C) is rate-limiting. If faster (>200 nt/sec), thermostable DNA Polymerases can be found, then thermocycling times of less than 30 seconds for 30 PCR cycles are possible.
• High-speed pressurized gas PCR is generally more accurate than slower methods, probably because false reaction products have so little time to anneal and/or elongate.
• the pressurized gas jet thermocycler is compatible with on-line, fluorescent dye- based DNA detection optics.
• the pressurized gas j et PCR process and thermocycler should prove especially useful in the diagnosis of life-threatening diseases where speed is essential.
• the present invention can be (and has been) practiced to carry out rapid (about 2 to 10 minute) DNA- based tests used in biomedical research, genetics, molecular medicine, agriculture, veterinary science, forensics, and the detection of biological warfare agents.
• a prefened embodiment of the invention is a pressurized gas thermocycler which carries out high-speed PCR amplification of DNA by injecting hot or cold gas into a thennostated reaction chamber under the control of fast ( ⁇ 20 msec/cycle) electronic valves, actuators which accept digital and/or analog signals, and a microprocessor controller.
• this device would contain a minimum number of moving parts and also be compatible with optics used for detection of fluorescent dye-labeled DNA (Higuchi et al, 1992; Haugland, 1996; Spears et al, 1997).
• the temperature can be rapidly changed by injecting hot and cold pressurized gas into a reaction chamber under electronic control, using electronically programmable "hot gas” and “cold gas” valves.
• the pressure in the reaction chamber is maintained at a relatively constant value by means of a mechanical relief valve, although the temperature can be changed upon demand.
• Reaction samples (10 to 25 microliters in volume) are sealed in thin- walled glass capillary tubes as described by Wittwer and Garling (1991), or similar thin- walled plastic vessels, as described by Tret'yakov et al. (1994).
• a pressurized gas thermocycler differs from a hot-air thermocycler (U.S. Pat. No. 5,455,175 to Wittwer et al.) in several important respects, as shown in FIG. 2.
• a hot-air thermocycler U.S. Pat. No. 5,455,175 to Wittwer et al.
• heat chamber reaction chamber
• pressurized gas thermocycler FIG.
• a heating chamber up to six separate chambers are used: a heating chamber, a cold gas supply chamber, a hot gas flow conditioner, a cold gas flow conditioner, a mixing chamber, and a reaction chamber.
• preheated gas >95°C
• cold gas hi order to cool samples in the reaction chamber, cold gas ( ⁇ 20°C) is delivered under pressure to the reaction chamber via the cold gas flow conditioner and the mixing chamber by electronic valve Nc (cold gas valve 36).
• a mechanical relief valve V (40 in FIG. 3 a) allows exit of pressurized gas from the reaction chamber. Efficiency is improved by recovery of heat from exit streams and electronic valve V H when it is set to by-pass the hot gas flow conditioner.
• FIG. 3a, 3b, 3c, 3d and 3e Schematic drawings of the pressurized gas thermocycler, flow conditioners, and its reaction chamber are shown in FIG. 3a, 3b, 3c, 3d and 3e. Fabrication and Optimization of a Pressurized Gas Thermocycler.
• the thermal cycling device 10 includes a reaction chamber made of insulating (low k) material, generally designated at 38.
• reaction chamber 38 has k value of less than 0.5 Watts/centimeter-degree K.
• Reaction chamber 38 can be comprised of a number of different materials, such as stainless steel or titanium.
• Polyurethane or polyimide plastics with ceramic binders, sold under trade names such as VESPEL, TORLO ⁇ , and BUTTERBOARD (which is manufactured by Golden West of California), can also be used as a reaction chamber 38 material.
• Cold gas supply 14 is separated from reaction chamber 38 by a pressure regulator 16, a one-way check valve 18 (available from Linweld of Lincoln, ⁇ E), and a two-way electronic valve 34 (a 38 VDC valve manufactured by Peter Paul Electronics of New England, CT) which is actuated by a digital relay operated by controller 42.
• Controller 42 can be a multifunction data acquisition board, such as a KPCI-3107 manufactured by Keithley of Cleveland, OH.
• Hot gas supply 12 is connected to a preheater 20 through a pressure regulator 16 and a one-way check valve 18.
• Preheater 20 is a heat exchanger which cools the exhaust gas from reaction chamber 38 and heats the incoming gas from gas supply 12.
• Preheated gas exiting preheater 22 then enters preheater 26 and then process heater 26 (500 Watt process heater manufactured by Hotwatt of Danvers, MA).
• Process heater 26 is actuated by a heavy-duty relay operated by controller 42.
• Hot gas exiting the heater 26 flows to a three-way electronic valve 28 (operated by controller 42).
• the process heater requires a continuous flow of gas; therefore electronic valve 28 exhausts gas into preheater 22 and finally through muffler 24 (manufactured by Exair of Cincinnati, OH) whenever the reaction chamber 38 is cooling.
• the hot gas flows from electronic valve 28 into flow conditioner 30.
• Flow conditioner 30 smoothes gas flow pulses into a steadier flow of hot gas.
• the conditioned hot gas then enters mixing chamber 32.
• Cold gas from electronic valve 36 also flows through a flow conditioner 34 and into mixing chamber 32.
• the mixed gas then flows into reaction chamber 38.
• Reaction chamber 38 is kept at an elevated pressure by mechanical relief valve 40.
• Mechanical relief valve 40 has a cracking pressure of 1.3 bar (20 p.s.i.). Exhaust gas from mechanical relief valve 40 cools in preheater 20 and exits through muffler 24.
• electronic valves 28 and 36 can be opened or closed upon demand, using controller 42, based upon the temperatures in the mixing chamber 32, hot gas flow conditioner 30, and cold gas flow conditioner 34 measured by the thermocouples 44, 46, and 48 respectively.
• Thermocouple 50 is used by controller 42 to keep process heater 26 at a constant temperature.
• the reaction chamber 38 contains an elliptical cavity 52, which contains reaction samples sealed in thin-walled capillary tubes. These tubes enter through staggered, sealed holes 54.
• pressurized gas thermocycler was tested with various combinations of hot and cold supply gases. Pressurized air is available, inexpensive, and easy to control. On the other hand, pressurized helium gas is a superior heat transfer gas (FIG. 1), but is more problematic with respect to thermal control (cf. FIG. 7b).
• Operation of the pressurized gas thermocycler requires three operating modes, which are controlled by activating relays wired to the valves.
• the three operating modes are: 1) heating the reaction chamber, (2) cooling the reaction chamber, and (3) holding the temperature of the reaction chamber.
• the hot gas valve 28 is OPENED to flow conditioner 30, so that hot pressurized gas is delivered to the chamber.
• the cold gas valve 36 remains CLOSED.
• the hot gas valve 28 is CLOSED to flow conditioner 30 and the cold gas valve 36 is OPEN.
• the cold gas valve 36 and hot gas valve 28 flutter OPEN and CLOSED on demand based on the temperature measured by the thermal sensor located in the mixing chamber 32.
• This valve configuration provides superior thermal control for holding the temperature near a preset value.
• the opening and closing of hot and cold valves is controlled by electrical signals from the system controller to electrical relays which actuate the valves.
• the system control software determines the opening and closing of these valves.
• high-speed gas jet amplification of DNA can be carried out using pressurized air (or helium) as a 'hot' gas, and a pressurized 'cold' gas (CO 2 or air). Thirty cycles of PCR amplification can be achieved in 78 to 617 seconds, depending upon the heat transfer gases chosen, the size of the thermal gradient, and the length of the DNA fragments amplified.
• amplified DNA was detected using a relatively slow (-1 hour) process: gel electrophoresis followed by Ethidium bromide (EfBr) staining, a standard method of DNA detection known to those of ordinarily skill in the art.
• pressurized gas jet PCR is compatible with on-line fluorescence-based detection (Higuchi et al, 1992) using dyes such as SYBR Green, which bind selectively to double-stranded DNA (Haugland, 1996).
• dyes such as SYBR Green
• the double-stranded DNA reaction products of high-speed gas phase PCR can be rapidly detected on-line using reporter dyes like SYBR Green and inexpensive photodiode detectors.
• a pressurized gas thermocycler could be fitted with photodiode detectors and either a light-emitting diode (LED) or laser diode dye excitation source.
• LED light-emitting diode
• Such optics, or equally inexpensive fluorescence polarization detection optics (Spears et al, 1997) and fluorescein-labeled primers would allow amplification/detection of DNA on an unprecedented time scale.
• ⁇ 'D' gene amplicon (c) a 364 b.p Human Platelet Antigen HPA-4 amplicon, and (d) a human ⁇ -globin 536 b.p. amplicon were amplified using 30 cycles of [0 sec 92°C/0 sec 55°C/5 sec 72°C] with pressurized air as a hot gas and CO cooling.
• the tri-valve apparatus is a functional thermocycler. Not only is it very fast, but it exhibits good thermal control ( ⁇ 1°C). However, the performance of the device was limited by its software, which was written in BASIC code. Every time it needed to execute commands in response to thermal changes in the reaction chamber or process heater, it needed to translate from BASIC (interpreted program) to assembly code (compiled program).
• FIG. 6 shows that a short 85 b.p E.coli O157:H7 Stx amplicon could be amplified through 30 PCR cycles in high yield in ⁇ 2 minutes; and a considerable improvement in performance was achieved by writing instrument commands in assembly code.
• helium gas Ubbink, 1947; Bosworth, 1952; FIG. 1
• a small (85 b.p.) amplicon from the E.coli O157:H7 Stx gene was PCR-amplified using "hot" pressurized helium gas and CO 2 cooling.
• primer lengths were increased to 30 nt, so that higher annealing temperatures (62°C to 63°C) could be employed.
• DNA denaturation temperatures were slightly reduced (86°C to 89°C) from those used in previous experiments.
• thermocycling protocols (a) [0 sec 89°C/0 sec 62°C/0 sec 72°C]; (b) [0 sec 87°C/0 sec 62°C/0 sec 72°C]; and (c) [0 sec 86°C/0 sec 63°C/0 sec 72°C].
• PCR reactions (10 ⁇ l) were carried out in thin-walled glass capillary tubes containing 50 mM Tris (pH 8.5 at 25°C), 250 ⁇ g/ml BSA, 3 mM MgCl 2 , 0.2 mM dNTPs, 50 pmol of forward and reverse primers, 20 picograms of E.coli O157:H7 DNA, and 5 U of Taq Polymerase (Promega, Madison, WI).
• reaction products were separated on 3% agarose gels with EfBr staining. Molecular weight markers were 67-501 b.p. long (pUC19/Msp/ digest). As shown in FIG. 7a, a high yield of the expected 85 b.p. Stx amplicon was seen in all three helium/CO 2 gas jet PCR reactions. FIG. 7a and 7b demonstrate the three fastest DNA amplification reactions which have ever been carried out.
• thermocycling was considerably faster when pressurized helium gas was used rather than air.
• High-speed gas jet PCR also resulted in a very low background of non-specific "haze" or false reaction products. Presumably, false priming or elongation are rare when such fast thermocycling parameters are used; there is simply no time for spurious reaction products to accumulate.
• anthracis Sterne strain DNA (10 pg) was mixed with 25 pmol forward primer (BAN23MS1) and reverse primer (BAN23MA1) plus 200 ⁇ M dNTPs in a 24 ⁇ l reaction volume containing 1.25 Units Taq Pol in (50 mM Tris-Cl, 5.5 mM MgCl 2 , pH 8.8, 500 ⁇ g/ml BSA).
• High-speed PCR was carried out in a Model 1.62 Heliac pressurized gas thennocycler, which was programmed in 100 msec time intervals, followed by gel electrophoresis on 2% agarose gels with ethidium bromide staining. Samples were as follows: [a] 100 b.p. ladder molecular weight markers, [b] 30 PCR cycles using 2.6 bar (38 p.s.i.) hot compressed air with (3.0 bar) CO 2 cooling (0 sec 92°C, 0 sec 55°C, 4.0 sec
• FIG. 8a shows a companion temperature versus time profile.
• bacterial gene fragments 145 to 486 b.p. long and single-copy human gene fragments 147 to 191 b.p. long were amplified through 30 to 35 PCR cycles in -2.4 to 4.1 minutes (144 to 247 seconds) using 20 pg of DNA, 1 ⁇ M primers, 4.5 to 5.5 mM MgCl 2 , 100 mM dNTPs, 500 ⁇ g/ml BSA, and 1.25 U of Taq Pol.
• FIG. 9a shows a picture of DNAs amplified from the human gene responsible for Accute Intermittant Porphyria (191 b.p.), ABO blood type A-transferase gene fragment
• E.coli uidA DNA fragment were carried out using: (1) 20 cycles of [200 msec 88°C, 200 msec 56°C, 2.8 sec 72°C], followed by (2) 20 cycles of [200 msec 88°C, 200 msec 65°C, 1.8 to 2.0 sec 72°C].
• the first 20 cycles allow annealing of all 4 oligonucleotide primers at the pennissive 56°C annealing temperature using a relatively long 2.8 second Taq Pol elongation time.
• the second 20 cycles selectively amplify the inner 186 b.p. DNA fragment due to the higher 65°C annealing temperature and the shorter 1.8 to 2.0 second elongation step.
• PCR reactions (15 ⁇ l) were carried out in thin-walled glass capillary tubes containing 50 mM Tris (pH 8.5 at 25°C), 250 ⁇ g/ml BSA, 3 mM MgCl 2 , 0.2 mM dNTPs, 50 pmol of forward and reverse primers, 4.5 pg of E.coli K12 DNA, and 1.5 U ofZ-Taq Pol (Takara Shuzo Ltd).
• reaction products were separated on 2.0% MetaPhor agarose gels with EfBr staining.
• M.W. markers were pUCl9/MspI digest fragments.
• Total time for 40 high-speed PCR cycles was 4 minutes and 45 seconds (285 seconds).
• the expected 186 b.p. inner amplicon could be amplified through 40 cycles of two-step nested PCR in less than 5 minutes when the second 20 cycles were [200 msec 88°C, 200 msec 65°C, 2.0 sec 72°C].
• the amplification factor may be calculated as follows: the E.coli genome contains -4.5 x 10 6 b.p.
• FIG. 11a shows a picture of a 2206 b.p. bacteriophage ⁇ DNA fragment which was amplified using 20 pg of DNA, 1 ⁇ M primers, 4.5 mM MgCl 2 , 100 mM dNTPs, 500 ⁇ g/ml BSA, and 1.5 U o ⁇ Z-Taq Pol (Takara Shuzo Ltd) and 30 cycles of [800 msec 89° to 91°C, 800 msec 61°C, 13 sec 72°C] in a gas jet thermocycler, using 2.6 bar (42 p.s.i.) compressed air (from a pneumatic airtool compressor) and 2.8 bar (46 p.s.i.) CO 2 cooling.
• Molecular weight markers were bacteriophage ⁇ Hindrfl digest fragments.
• FIG. lib shows a picture of a 2331 b.p. bacteriophage ⁇ DNA fragment which was amplified using 20 pg of DNA, 1 ⁇ M primers, 4.5 mM MgCl 2 , 100 mM dNTPs, 500 ⁇ g/ml BSA, and 1.5 O Z-Taq Pol (Takara Shuzo Ltd) and 30 cycles of [500 msec 91°C, 500 msec 61°C, 11 to 14 sec 72°C] in a gas jet thermocycler, using 2.6 bar (42 p.s.i.) compressed air (from a pneumatic airtool compressor) and 2.8 bar (46 p.s.i.) CO 2 cooling.
• the reaction yield was clearly higher when elongation times of >12 seconds were employed, as shown in lanes (a) and (b). This result indicated that, for phage ⁇ DNA fragments 2206 to 2331 b.p. long, Z-Taq Pol had an apparent elongation rate of >194 nt/sec.
• FIG. 11a and lib demonstrate that DNA fragments 2206 to 2331 b.p. long have been successfully amplified in high-yield using the pressurized gas jet thermocycler.
• Figure 13 shows a summary of the high-speed DNA amplification processes using an embodiment of the invention which employs the tri- valve gas jet thermocycler.
• FIG. 13 shows a picture of a 297 b.p. E. coli uidA DNA fragment which was amplified using 1 ng DNA, 150 pM primers, 3 mM MgSO4, 200 ⁇ M dNTP, 400 ⁇ g/ml BSA, and 0.625 U KOD Hot Start polymerase.
• Lane 1 was 35 cycles of [30 sec 90°C (hot start), 0 sec 90°C; 0.2 sec 58°C; 2 sec 72°C]
• Lane 2 was 40 cycles of [30 sec 90°C (hot start), 0 sec 90°C; 0.2 sec 58°C; 2 sec 72°C]
• Lane 3 was 30 cycles of [30 sec 90°C (hot start), 0 sec 90°C; 0.2 sec 58°C; 2 sec 72°C] in a bi-valve gas jet thennocycler, using 2.4 bar (35 p.s.i.) compressed air.
• 25 ⁇ l of sample were placed in a Roche glass capillary tube.
• the invention decreases the time needed for amplification of DNA using the Polymerase Chain Reaction by one to two orders of magnitude over any previously described process or device (see FIG. 12).
• the gas jet process is inherently more accurate than slower procedures, since false reaction products have practically no time to anneal and/or elongate (see FIG. 5a, 6, 7a, 8a, 9a, 11a and lib).
• the pressurized gas jet thennocycler (FIG. 3a) has superior timing control as compared to heat block or hot-air devices; the gas jet device is programmable in time intervals of 100 milliseconds.
• high-speed gas jet PCR is a very reliable process.
• the high-speed gas jet PCR process is fully compatible with on-line detection optics, so that timely DNA amplification/detection can be carried out.
• the invention therefore allows fast, accurate, and reliable DNA amplification/detection to be achieved on an unprecedented time scale.
• thermocyclers Comparison to 'Ultrafast' PCR Thermocyclers. The fastest experimental thermocyclers built by Lawrence Livermore National
• Oda et al. is incompatible with fluorescent optical detection using >450 nm dyes and photodiode detectors.
• the pressurized gas device shown in FIG. 3a requires less than 2.6 seconds/cycle; and the reaction yield is over 200-fold greater (see FIG. 5a, 6, 7a, 8a, 9a, 11a, and lib).
• a single copy bacterial gene fragment 368 b.p. long was amplified 1.35 x 10 8 -fold in a total of 233 seconds for 30 PCR cycles.
• thermocycler The fastest commercially available thermocycler is manufactured by the fastest commercially available thermocycler is manufactured by the fastest commercially available thermocycler is manufactured by the fastest commercially available thermocycler is manufactured by the fastest commercially available thermocycler is manufactured by the fastest commercially available thermocycler is manufactured by the fastest commercially available thermocycler is manufactured by the fastest commercially available thermocycler is manufactured by the fastest commercially available thermocycler is manufactured by the fastest commercially available thermocycler is manufactured by the
• a pressurized gas PCR process can be used to amplify DNA to detectable quantities in as little as 78 seconds.
• the high-speed pressurized gas jet process is therefore >15 times faster than the Roche machine and is compatible with online fluorescent dye-based DNA detection optics.
• Nicoll S, Brass A, and Cubie HA (2001) "Detection of he ⁇ es viruses in clinical samples using real-time PCR.” J. Virol. Methods 96: 25-31. Northrup MA, Benett B, Hadley D, Landre P, Lehew S, Richards J, and Stratton P (1998)
• Oda RP Strausbauch MA, Huhmer AF, Jurrens SR, Craighead J, Wettstein PJ, Eckloff B,

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