KR20050071657A - Gas jet process and apparatus for high-speed amplification of dna - Google Patents

Abstract

Processes and apparatus for performing fast, precise, and reliable gas jet thermocycling on a sample using of pressurized gases delivered to a thermostated reaction chamber at high velocity using computer controlled electronic multiport valves are disclosed. Apparatus for achieving these processes are also disclosed.

Description

Gas injection method and apparatus for high speed amplification of DNA {GAS JET PROCESS AND APPARATUS FOR HIGH-SPEED AMPLIFICATION OF DNA}

Technical Field

The present invention relates to a method and apparatus for performing fast amplification of a sample. More specifically, the present invention relates to a method and apparatus for carrying out a pressurized polymerase chain reaction in which each cycle can be completed in a short time of several seconds.

Background

Polymerase chain reaction . Polymerase chain reaction (PCR) is one of the most widely used techniques in molecular biology (Mullis, US Pat. No. 4,683,202; Saiki et al., 1985; Erlich, 1989; Mullis) Et al., 1994). PCR amplified DNA can be used to diagnose mutations that lead to human genetic diseases in blood and tissue typing (Saiki et al., 1989a) (Saiki et al., 1985; Kogan et al., 1987), or important infectious diseases. It can be used to detect pathogens that cause (Persing et al., 1993; Nicoll et al., 2001).

In a typical PCR reaction, the template DNA sequence lying between the ends of two defined oligonucleotide primers can be amplified in 1 to 2 hours (FIG. 4A). Three kinds of sequential steps that are commonly used: (1) a step of a double-stranded DNA at elevated temperature (90 ℃ to 95 ℃) modified (D) into a single stranded form, (2) and the resulting single-stranded DNA strand to about 45 ℃ (A) annealing to the oligonucleotide primer at 65 ° C., (3) the thermostable DNA polymerization of the primer: template complex at about 72 ° C. such as Thermus aquaticus ( Taq ) polymerase. Kidney (E) using an enzyme (Saiki, 1989b).

One cycle of these three steps (denaturation / binding / extension) results in double fold amplification of DNA fragments whose 5 ′ and 3 ′ ends are defined by sequence specific binding of oligonucleotide primers to the DNA template. Thus, 30 fully effective PCR cycles result in 2 ³⁰ times (about 10 ⁹ times) amplification of specific DNA sequences. Thus, DNA is amplified in an amount of less than 1 picogram to micrograms, which can be detected by standard analytical methods such as gel electrophoresis, DNA hybridization or fluorescence based optical detection.

Automated PCR equipment . A variety of devices have been manufactured that automate the three-step PCR amplification method (Oste, 1989; Oste, 1994; Newton, 1995; Johnson, 1998). In general, such devices can be classified into two categories: robotic devices for moving DNA samples into columns; And a thermocycler to take the heat into the sample.

Robotic devices, such as Stratagene's ROBOCYCLER, move tubes containing PCR reaction samples into a series of heat baths that are thermostated at various temperatures or from these baths. Although these devices may be useful in certain areas of research, high speed PCR cannot be performed. These devices require more than 60 minutes for 30 amplification cycles, which is equivalent to more than 2 minutes per PCR cycle.

Since the late 1980s, thermocyclers have become a familiar device in many biochemistry laboratories. Most of the commercially available PCR devices (Perkin-Elmer, MJ Research, Ericomp, Techne, Eppendorf, BioRad, Hybaid) are thermocyclers (Johnson, 1998). In general, two types of thermocyclers are used: programmable heat blocks and forced hot thermocyclers.

Programmable block of columns . Most thermocyclers are thermal blocks with holes inside which plastic reaction tubes are heated and cooled under electronic control. Many such devices have been reviewed by Johnson (1998). The problem with programmable thermal block designs is that most of the time is spent waiting to heat or cool large pieces of metal. For example, for the MJ Research PTC-150 Thermocycler (Watertown, Mass.), 14 seconds per cycle are lost at transitions between D, A, and E temperatures (about 94 ° C, about 55 ° C, and 72 ° C). A single heating / cooling cycle from 94 ° C. to 55 ° C. wastes 28 seconds per cycle, or about 14 minutes for 30 PCR cycles.

Many commonly used PCR protocols consume 1 minute (denaturation) at 94 ° C., 1 minute (binding) at about 55 ° C., and 1 minute (height) at about 72 ° C. For example, for the first PCR method used by Cetus employees (Saiki et al., 1989b), 536 bp human β-globin DNA fragments (1 min at 94 ° C., 55 ° C.) were used. Amplification using 30 cycles of 1 minute, 1 minute at 72 ° C. The active duty time for this thermocycling protocol is only about 3.5 minutes = 210 seconds. This time is the amount required for enzymatic 30-fold replication of the 536 bp template at a Taq DNA polymerase extension rate of about 80 nt / sec (Innis et al., 1988; Gelfand and White, 1990).

Commercially available thermal block thermocyclers (Perkin-Elmer, Ericomp, MJ Research, Eppendorf, Techne, BioRad, Snark Technologies) require 20 to 25 seconds to cool from 94 ° C to 55 ° C and from 55 ° C to 94 ° C. An additional 14 to 20 seconds is required to heat the furnace (Johnson, 1998). Thus, the "dead time" for each PCR cycle is an additional 40 ± 5 seconds per cycle. As shown in FIG. 4A, a commonly used thermocycling protocol requires (220 seconds / cycle x 30 cycles) = 6600 seconds = 110 minutes (Saiki et al., 1989b). Only about 3.5 of these 110 minutes in the PCR method are focused on productivity.

Sobczak et al. (1995) found that a faster PCR protocol can be used in thermal block thermocyclers when the denaturation, binding and stretching steps are shortened to 1-2 seconds per cycle. For example, 139 base pair long human p53 gene fragments were amplified through 35 cycles (1 second at 94 ° C., 1 second at 58 ° C., 1 second at 72 ° C.). However, the heating / cooling time of the thermocycler was still longer than 25 seconds per cycle. Thus, the total thermocycling time was about 30 seconds per cycle or about 15 minutes for 30 PCR cycles.

Forced open thermocycler . To overcome the long transition dead time of the thermal block, a forced hot thermocycler has been constructed that allows 30 PCR amplification cycles to be performed in a short time of about 10 to 30 minutes. Wittwer and his colleagues have laid a significant engineering foundation for optimizing rapid DNA amplification in hot air PCR thermocyclers (Wittwer et al., 1989; Wittwer and Garling, 1991; Wittwer et al., 1994). The rate limiting step in the three step PCR reaction sequence (denaturation / binding / extension) is the rate of DNA polymerase extension. At elongation rates of 80 nucleotides per second by Taq polymerase (Innis et al., 1988; Gelfand and White, 1990), theoretically, DNA fragments shorter than 100 bp with about 20-mer primers per second per cycle were used. It is necessary to amplify. For example, only about 5 seconds per cycle was needed to replicate 536 bp β-globin amplicons through 30 PCR cycles (Idaho Technology, 1995; see FIG. 4B).

For the first commercial hot air thermocycler manufactured by Idaho Technology (Idaho Falls, Idaho, USA), the reaction time required for a single PCR cycle of denaturation / binding / extension was substantially reduced. 1) the device has a very low thermal mass, (2) gas phase heat transfer from hot air to an aqueous biochemical reaction sample is carried out in a thin-walled capillary, and (3) denaturation and binding times are minimized during the PCR cycle. It was.

For example, 536 bp of human β when using a 30 cycle PCR protocol of [0 ° C (denatured) at 94 ° C, 0s (bound) at 55 ° C, 5s (extended) at 72 ° C). Globin DNA fragments were amplified in 9.9 minutes or 19.8 seconds per cycle (Idaho Technology, 1995). Thus, this forced open thermocycling protocol uses 536 b.p. p. It was about 220 / 19.8 = 11 times faster than Saiki et al.'s original method (1989b) for PCR amplifying β-globin DNA fragments.

For routine laboratory diagnostic PCR, the hot thermocycler is not so fast as described above. For example, Nicoll et al. (2001) used Roche LightCycler ^™ [0 sec (denature) at 94 ° C., 3 sec (bound) at 58 ° C. to 67 ° C., at 72 ° C. 6 seconds (kidney)] amplified short (96-220 bp long) herpes virus DNA fragments in about 25 minutes or 30 seconds per cycle.

Hot air thermocyclers rely on suboptimal convective heat transfer . Design of a forced hot air thermocycler (US Pat. No. 5,455,175 to Wittwer; Wit et al., 1990; 1994; US Pat. No. 5,576,218 to Zurek; US Pat. 6,200,781) is an improvement over block heaters, but attention is not drawn to the important role of fluid mechanics in the heat transfer method. The inventors of the present invention describe the use of "blowers" (US Pat. No. 5,576,218 to Jurek et al.) And atmospheric air (US Pat. No. 5,455,175 to Whitworth et al .; Whitworth et al., 1990; 1994; US Pat. No. 5,576,218) Suggested as the medium. Blowers and fans move the atmosphere (compared to the source of compressed gas used in the present invention), whereby the gas velocity is much slower than in compressed gas flow. In addition, the prior inventions did not consider using refrigerated or cooled air or other refrigerated gas for the cooling portion of the PCR cycle, depending only on ambient air.

Since conventional inventions did not use compressed gas flows, the speeds used were not very high (less than 3 m / s), and as a result these inventions achieved lower convective heat transfer rates compared to those obtainable with high speed turbulent gas flows. . Turbulent flow at high speeds includes compressible nonisothermal fluid dynamics. Forced air heating and ambient air cooling at atmospheric pressure (P = 1 bar) are a unique and suboptimal solution to the fluid dynamics of biochemical reaction chambers. Whitworth et al. (US Pat. No. 5,455,175; Whitworth et al., 1990; 1994), Jurek et al. (US Pat. No. 5,576,218), or Tal et al. (US Pat. No. 6,200,781). No pressure, cooling gas cooler than 22 ° C., or time constants faster than 1 second, and they did not use flow conditioners to mix hot and cold gases and / or increase gas pressure and velocity. They did not consider the possibility that various gases could be used at various stages of the PCR method, nor did they consider the role of fluid mechanics, heat transfer physics, or flow conditions for optimal heat transfer.

For example, "Air is an ideal heat transfer medium that can change temperature rapidly due to its low density." (Wittwer et al. (1990)) is not only inaccurate but also ignores the complexity of the problem: convective heat transfer in the gas phase depends on various factors (Welty et al., 1976; Chapman, 1984). The gas heat transfer conditions required for high speed amplification of DNA using hot / cold pressurized gas jets are more accurately described as turbulent and nonisothermal compressible gas flows; Substantially none of the optimum gas parameters or flow conditions are conventionally optimized. As schematically shown in FIGS. 2A and 2B, the pressurized gas injection thermocycler is not only previously considered as shown in FIGS. 5-12, but also high speed DNA as well as gas pressure, velocity, turbulent and non-isothermal flow conditions. It operates on geometry that has not been performed in the amplification experiment.

Further referring to Whitworth et al. (1990), heat transfer across stagnant layers of air (eg, boundary layers) is very poor. This inefficient heat transfer explains why double glazing windows have good insulation properties. The only reason that a forced hot thermocycler performs better than a block heater is that the flow of air thins the insulating boundary layer surrounding the DNA sample. Thus, despite the low coefficient of thermal conductivity of air, the heat flux across the boundary layer is faster than with the block heater.

Comparison of Heat Transfer in Hot and Pressurized Gas Thermocyclers . The difference between a hot air thermocycler and a pressurized gas thermocycler is best explained by a simple analysis of heat transfer around a thin cylinder (capillary tube). Cylinders are selected because both of these thermocyclers use capillaries to support DNA samples, but the analysis will be similar for any other form of sample holder.

d _o and d _i mean the outer and inner diameters of the capillary. Natural convective heat transfer and internal heat gradients in the reacting liquid mixture are negligible due to the predominant role of cohesion and small inner diameters. Therefore, the thermal energy resin of the aqueous solution inside the capillary is represented by the following formula:

In the above formula, ρ _m and Cp _m represent the density and specific heat capacity of the mixture. This equation explains how quickly the temperature (expressed in T _s ) of the sample in the capillary changes as the gas flows across the capillary at temperature T _g . For example, assuming the sample is 94 ° C., it must be cooled to 56 ° C. by flowing a low temperature gas of 0 ° C. across the capillary. If the time t at which the cold gas starts to flow is set to 0, the time required for the aqueous sample inside the capillary to cool can be obtained by solving the above formula (1):

When solving equation (2a) for time t, we obtained the following equation:

In a similar manner, any heating or cooling of the biochemical reaction sample can be described. From equation (2b) it becomes clear that the time t is shortened when the effective heat transfer coefficient h _eff is maximized. These coefficients combine the performance of the cycler with heat transfer and fluid dynamics. The coefficient h _eff includes heat transfer through the capillary wall ( h _c ) and the boundary layer between the gas and the capillary outer surface:

The heat transfer coefficient for the capillary depends on the thermal conductivity coefficient ( k _c ) of the capillary material and its dimensions: . The heat transfer coefficient ( h _bl ) through the boundary layer is the following correlation proposed by (1) Hsu and (2) Douglas and Churchill (both cited by Welty et al. (1976)). Expressed as a relationship. These coefficients are greatly affected by fluid dynamics. However, first we define two dimensionless numbers. Reynolds number , Where ν , μ _gas , and ρ _gas represent the average gas velocity, the dynamic viscosity of the gas, and the gas density. Nusselt number Nu is Where k _gas is the gas thermal conductivity coefficient. Nussel number is defined for two flow modes: laminar and turbulent.

Laminar Flow:

For Re <500, Nu = 0.43 + 0.48 Re ^0.5 (Hsu correlation).

turbulence:

For Re> 500, (Douglas and Churchill Correlation)

If the gas velocity is slow (small Re), the Nussel number Nu will be small and h _bl will also be small. As a result, when h _bl is small compared to h _c , the effective heat transfer coefficient h _eff becomes approximately equal to h _bl . If the gas velocity increases to the point of h _bl >> h _c , the effective heat transfer coefficient approaches the limit ( h _eff is approximately equal to h _c ).

The following typical conditions exist for a forced hot thermocycler: P = 1 bar, ν = 2 m / sec and density, dynamic viscosity and thermal conductivity are respectively ρ = 0.98 kg / m ³ , μ = 2.12 x 10 ^-5 kg / msec, k _gas = 0.03 W / mK . Capillary outer diameter d _o = 0,001 m . Reynolds number to be. Since Re <500, the correlation of Hsu (1963) is used: Nu = 0.43 + 0.48 x Re ^0.5 = 5, and the rate limiting heat transfer coefficient is as follows:

The heat transfer coefficient through a glass capillary with a thermal conductivity of kc of 1.1 W / mK, an outer diameter of 1 mm, and an inner diameter of 0.8 mm is:

Thus, the effective heat transfer coefficient is as follows:

This result can be compared with a pressurized air thermocycler using a value of P = 3 bar, ν = 20 m / sec. Pressurized gas density, dynamic viscosity and thermal conductivity, respectively,

p = 2.95 kg / m ³ , μ = 2.12 x 10 ^-5 kg / msec, k _gas = 0.03 W / mK . Capillary outer diameter is equal to d _o = 0.001m. Thus, the Reynolds number is calculated as follows:

Since Re> 500, Douglas and Churchill's correlation Nu = 0.00128Re + 0.46 x Re ^0.5 = 27.92 is used, and the rate limiting heat transfer coefficient is:

The heat transfer through the capillary is the same and the effective heat transfer is as follows:

By comparing equations (4c) (hot thermocycler) and equations (5b) (pressurized air thermocycler), it can be seen that the rate limiting heat transfer coefficient through the boundary layer is at least five times greater for the pressurized air thermocycler. have. The cooling time of the comparative experiment (Equation (2)) will be 5 times shorter for the pressurized air thermocycler than for the hot thermocycler. In addition, this ratio will also be true in heating or cooling during any other step of the PCR process.

When the working gas is converted to helium in the pressurized gas thermocycler, the properties are p = 0.4 kg / m ³ , μ = 2.31 x 10 ^-5 kg / msec, k _gas = 0.17 W / mK . Now the Reynolds number is:

,

Nu = 0.00128 x 693 + 0.46 x 693 ^0.5 = 13.0.

When using pressurized helium gas, the rate limiting heat transfer coefficient is as follows:

The effective heat transfer coefficient is now as follows:

By comparing Eq. (4c) with Eqs. (5b) and (6b), there is a five-fold expected improvement in the pressurized air injection compared to the forced hot thermocycler, when pressurized helium is used as the working gas. There are over 12 times improvement. This example demonstrates that good heat transfer means faster PCR. Improved heat transfer is achieved by using pressurized gas, preferably helium, but even suboptimal gases such as air perform much better than unpressurized devices.

Advantages of Pressurized Gas Injection Thermocyclers . Based on the theoretical analysis, a rate of 3 m / sec < v <30 m / sec (Mach 0.03 < v <Mach 0.30) to rapidly change the temperature of the enzymatic reaction from about 50 ° C. to about 90 ° C. in a thin-walled vessel. It is expected that it will be possible to deliver hot / cold pressurized gas jets of to the biochemical reaction chamber. However, the required geometry, gas dynamics, flow conditions, methods for hot / cold gas mixing, and timing of gas ejection are not minor engineering issues. For those skilled in the field of fluid mechanics, the solution to this problem is solved using Navier-Stokes equations and computational fluid dynamics (CFD) modeling (Anderson, 1995).

Pressurized gas thermocycles differ from hot air thermocyclers in six aspects:

(1) A pressure higher than atmospheric pressure is used (increased gas density): P ≧ 1.3 bar.

(2) Much faster speed (reduced boundary layer thickness): ν ≧ 5 m / sec.

(3) Gas other than air may be used, for example helium may be used for rapid heating, and CO ₂ may be used for good thermal control and efficient cooling.

(4) A reaction with one or more gas flow conditioners attached is used for gas mixing.

(5) A digitally programmable solenoid valve is used to deliver hot and cold gas jets to the reaction chamber through the conditioning chamber to regulate and mix the gas flow.

(6) DNA denaturation, primers: template binding, and enzyme extension can be programmed at time intervals of 100 milliseconds or less.

It is well known that convective heating / cooling of a subject is much more efficient than thermal conduction. For example, windchill temperatures represent the additional cooling effect of wind movement, ie "convection". In this sense, efficiency refers to the time it takes for a subject to change its temperature by a certain amount. The convective cooling / heating rate is based 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 surrounded by a thin layer of gas fixed around the object called the "boundary layer"). Depends. The thickness of this layer is highly correlated with the velocity of the gas relative to the object. At higher gas velocities, the boundary layer becomes thinner and the heat transfer rate (named "heat flux") is improved. High speed can be achieved by using pressurized gas, where molecules will naturally flow from the high pressure region to the low pressure (atmospheric) region, which is controlled by opening and closing of the electronic and / or mechanical control valves. The transfer of thermal energy (heat) across the boundary layer is also greatly influenced by the type of gas used. Helium gas is particularly efficient for rapid heating of the object. Carbon dioxide (CO ₂ ) gas is cooled to −24 ° C. at constant enthalpy expansion, making it an efficient and inexpensive cooling gas. This property depends on the polytropic coefficient of the gas, which is a unique and unique thermodynamic property for each gas or gas mixture.

If the gas velocity approaches Mach 0.3 (0.3 times the speed of sound of the gas), the gas becomes compressible. This compressibility implies a stronger coupling between gas temperature (thermal internal energy) and kinetic energy (gas velocity), which in turn implies turbulence. The rapid heat transfer required for high speed amplification of DNA is more accurately described as a turbulent and nonisothermal compressible gas stream. In general, these reaction conditions are rarely used in biochemistry. Thus, it is not clear to those skilled in the biochemistry how to use these complex gas flow conditions to conduct temperature sensitive biochemical reactions.

Experts in heat transfer always contrast the macroscopic nature of convective heat transfer with the microscopic nature of heat conduction. Since the heat flux in the block heater has a thermal conductivity type (micro), the speed is determined by Fourier's law Where q _cond is the heat flux due to heat conduction in the x-direction, k is the thermal conductivity (the property of the material from which the block heater is made), Is the temperature gradient in the x-direction. Velocity is affected by (1) the distance (in centimeters) at which heat is conducted, (2) the temperature difference limited by the material properties, and (3) the thermal conductivity coefficient.

The basic laws governing heat transfer in gas phase physical principles have been known since Fourier (1822) and Navier (1856) and are taught in standard mechanical engineering textbooks (Bosworth, 1952; Azbel, 1984; Chapman, 1984). However, the fluid dynamics of the turbulent and non-isothermal compressible gas flows used in the novel methods and apparatus described herein are technical topics that are not generally known to those in the biochemical arts.

The thermal conductivity coefficient of helium was measured by Ubbink (1947), Johnston and Grilly (1946), Bosworth (1952), Azbel (1984). And in a table published by Chapman (1984). As shown in Figure 1, helium has a thermal conductivity coefficient that corresponds to seven times that of air. Neon transfers heat about twice as fast as air. Thus, hot thermocyclers have used inadequate gas for rapid heat transfer. Among non-combustible gases, air is available; Helium, especially ³ He, an isotope of helium, is optimal. U.S. Patent No. 5,455,175 to Whitworth et al. Assumes that the same gas (air) must be used to heat the reaction chamber, cool the chamber, or maintain its temperature at a fixed value. Indeed, as shown in the temperature vs. time profile of FIGS. 8B and 9B, gases with high thermal conductivity such as helium (large k gases) are superior in heating / cooling the reaction chamber, while air or CO ₂ A low k gas, such as, provides better thermal control when maintaining the temperature for about a few seconds during enzymatic stretching at about 72 ° C. (see FIGS. 5B, 9B, 10B). In other words, various gases are optimal for various stages of the PCR method.

Second, US Pat. No. 5,455,175 to Whitworth et al., US Pat. No. 5,576,218 to Jurek et al., And US Pat. No. 6,200,781 to Tahl et al. Provide air (other gases) at atmospheric pressure (other pressures not listed) for thermocycling. Is not described). Air is not only a relatively poor gas heat transfer medium but also need not be used at atmospheric pressure. PCR amplification of DNA using air at atmospheric pressure (P = 1 bar) is convenient. However, this method is arguably faster at elevated pressure (P ≧ 1.3 bar) and speed ( ν ≧ 5 m / s) (see FIGS. 4, 5b, 7b, 8b, 9b and 12b).

In the forced hot thermocycling method of Jurek et al., Described in US Pat. No. 5,576,218, “The sample could be heated from 50 ° C. to 85 ° C. within 12 to 15 seconds and at a temperature substantially lower than the target temperature, for example The sample was cooled from 85 ° C. to 50 ° C. in about 60 to 75 seconds by injecting 22 ° of cooling air. ” Thus, Jurek et al., Described in US Pat. No. 5,576,218, require at least (12 + 60) = 72 seconds per cycle to heat and cool the gas (air) only, regardless of whether any useful biochemistry occurs. do. Jurek et al. Provide no evidence that their methods or devices can actually be used to amplify DNA. In addition, Jurek et al. Do not consider the possibility of using a gas pressure higher than atmospheric pressure (P> 1 bar), since no gas pressure in their apparatus has been measured. For the present invention using a gas pressurized at about 2.5 bar, 30 cycles of PCR amplification are achieved in a short time of 78 seconds, which is achieved in one cycle using the Jurek et al. Method (US Pat. No. 5,576,218). Almost the same time as needed.

Third, it was assumed by Witwer et al. (US Pat. No. 5,455,175; Whitworth et al. 1990; 1994) that the heating chamber and the reaction chamber were identical (see FIG. 2). In fact, much faster convective heat transfer is possible when the reaction chamber is separated from the hot gas source by one or more solenoid valves and flow conditioners and / or gas accumulation chambers.

Fourth, Whitworth et al. (US Pat. No. 5,455,175), Jurek et al. (US Pat. No. 5,576,218) and Tal et al. (US Pat. No. 6,200,781) assumed that air at ambient temperature was the optimal cooling medium. Indeed, biochemical reaction chambers can be cooled much faster using gases other than air at temperatures below 25 ° C. The temperature vs. time profiles of FIGS. 5B, 7B, 8B and 9B show that the pressurized CO ₂ gas (P ≧ 2.3 bar, T = 5 ° C.) is a gas cooling medium compared to the ambient air (P = 1 bar, T = 23 ° C.). It clearly demonstrates that it is much better.

In theory, using a pressurized gas containing air (P> 5 bar) at a relatively high pressure supplied by a vortex tube such as those described by Ranque (1934) and Hilsch (1947). Thereby rapidly cooling the biochemical sample. However, Whitworth (US Pat. No. 5,455,175) or Jurek (US Pat. No. 5,576,218) did not consider using pressurized gas or Lanck-Hilsch's vortex tubes for cooling.

Fifth, Wittwer et al . (US Pat. No. 5,455,175; Wittwer et al . 1990; 1994), Jurek et al . (US Pat. No. 5,576,218), and Tal et al . (US Pat. No. 6,200,781) were applied to heat / cool DNA reaction samples. It is assumed that the appropriate time constant programmed for is an integer of 0, 1, 2, 3 ... seconds. They do not consider that faster PCR is possible when the device is programmed at a time interval of 1 second of minutes. In the present invention, the time parameter can be programmed to a tenth of a second, thus allowing much faster denaturation, coupling, and stretching (FIG. 13).

In short, for effective gas heat transfer, the forced hot thermocycler uses the wrong gas, wrong gas pressure, and misplacement of the heat chamber into the reaction chamber for heating and cooling. The time constant used (≧ 1 second) is excessively slow for high speed thermocycling (see FIG. 2), and the gas of the forced hot thermocycler lacks the gas power of the optimal CFD-model and does not use flow conditions. A much faster thermocycler can be devised using pressurized gas, in particular a helium / air / CO ₂ combination, which is delivered to a thermostatic reaction chamber using a flow conditioner (FIG. 3e) to actuate the valve electronically. Let's do it.

To explain the speed and performance of the present invention in a correct view, Chiou et al . (2001) of the Massachusetts Institute of Technology recently described the "status" of the thermocycler. They point out that "the purpose of this investigation is to produce a PCR machine that amplifies samples in the shortest possible time ..." (p. 2018). The device described by Chiwoo et al. (2001) allowed a 500 base pair long bacteriophage λ DNA fragment to start with ^˜1 nanogram (10 ⁻⁹ g) of DNA and amplified through 30 PCR cycles after 23 minutes; The efficiency was 78%. Thus, for 30 PCR cycles, the amplification factor is 1.58 ³⁰ = 6.0 x 10 ⁵ or 600,000 times.

The present invention allows for the rapid amplification of viruses, bacteria and single-copy human gene fragments on a time scale at least ten times faster than the method described by Chiwoo et al. (2001).

Moreover, as shown in FIG. 8A, the efficiency of gas injection PCR is much higher at ˜93%. The present invention provides DNA amplification factors of 1.86 ³⁰ = 1.35 × 10 ⁸ times or 135 million times. Thus, gas injection PCR is [225 times more sensitive than [1.35 × 10 ⁸ /6.0×10 ⁵ ] = 225 times the method of Qiu et al. (2001). Pressurized gas thermocyclers routinely amplify the virus and bacterial DNA fragments 85 to 2331 base pairs in length after 1.3 to 10.3 minutes (78 to 617 seconds) through 30 PCR cycles. Single copy human gene fragments in this size range are amplified in an amount detectable after 3.5 to 6.5 minutes via 35 PCR cycles (FIGS. 5A, 9A and 13).

 Accordingly, in view of the limitations of the prior art described above, a novel gas injection amplification method and its automated apparatus using pressurized gas and solenoid valves are described herein.

Summary of the Invention

The present invention includes a high speed method of amplifying DNA. A reaction chamber containing a biological sample, a DNA polymerase, an oligonucleotide primer, and a deoxynucleotide precursor is provided. The reaction chamber receives a flow of one or more heat transfer gases. The first heat transfer gas is heated in a heating chamber that is physically separated from the reaction chamber. The heated gas is delivered to the reaction chamber at a pressure of at least 1.3 bar (P ≧ 20 p.s.i.).

Heat from the heated gas (≧ 95 ° C.) is used to denature the DNA. A second heat transfer gas, which may be of the same kind or a different kind of gas as the first heat transfer gas, is carried to the reaction chamber. The second heat transfer gas (≦ 20 ° C.) cools the reaction chamber to a temperature low enough for the denatured DNA to bind to the oligonucleotide primer. Finally, using a first high temperature (≧ 95 ° C.) heat transfer gas, the temperature of the reaction chamber is increased to a sufficient temperature (typically 68 to 75 ° C.) to enable enzymatic extension of the primer: template complex.

Extremely fast thermocycling is possible when high temperature (≧ 95 ° C.) and cooling (≦ 20 ° C.) gases are carried in a biochemical reaction chamber under the control of a binary, three-way solenoid valve, or multiple solenoid valve. Optionally, the temperature of the enzyme-catalyzed reaction can be controlled on a time scale of 10 milliseconds by delivering a jet of hot and cooling gas to the thermostatic reaction chamber via a flow conditioner (or series of flow conditioners). Ideally, the gas of choice is helium, but for convenience other gases may also be used.

Embodiments of the invention also include a high speed amplification device of DNA. The apparatus includes a reaction chamber in which compressed high temperature (≧ 95 ° C.) and low temperature (≦ 20 ° C.) gas jets are carried on a time scale of one second of moisture. The overall form of the device consists of six parts: (1) a heater that increases the temperature of the heating gas to a predetermined value (≥95 ° C), (2) mixes low and hot gases, regulates the flow and pressure pulses. One or more flow control chambers to attenuate the temperature, (3) inlet manifolds with mixing chambers to mix hot and cold gases, (4) gas reaction chambers, (5) exhaust nozzles, and (6) reactor outlets and hot gas side tubes. Heat exchanger to recover heat from the. Thus, in certain cases, pressurized gas thermocyclers are similar to jet and / or rocket engines. However, instead of mixing the fuel and the oxidant (as in an injection or rocket), hot and cold noncombustible gases are mixed under the control of the solenoid valve; Ignition does not occur. The essential method described in the present invention is thus suitably referred to as "gas injection PCR" and the device is suitably named "PCR injection". Rapid, precise and reliable thermal control of enzyme-catalyzed reactions is the goal of PCR injection.

The heating chamber physically separated from the reaction chamber is connected to the reaction chamber by a fluid. The first vessel with heating gas at a pressure higher than the pressure of the reaction chamber is connected by fluid to the heating chamber. This connection may include a flow control chamber for regulating flow and suppressing pressure pulses. The second vessel with the cooling gas (which may be the same kind of gas or a different kind of gas) is connected to the reaction chamber by a fluid. This connection may include a flow control chamber for regulating flow and suppressing pressure pulses. At least one cooling gas inlet valve is positioned between the second vessel and the reaction chamber and at least one heating gas inlet valve is positioned between the first vessel and the reaction chamber. A programmable controller with electrical inputs and outputs is used to control the opening of the inlet valve. A temperature sensor located on the reaction chamber provides the output to the controller. The controller opens or closes the inlet valve to reach or maintain the desired temperature in the reaction chamber.

If the temperature of the optimum heat transfer gas, such as helium, is increased to ≧ 95 ° C. in a chamber that is physically separated from the reaction chamber and is transported to the reaction chamber by an electronically actuated valve, extremely fast thermocycling is possible. Even cold (≤20 ° C) gas is transported to the chamber for faster heat transfer. For automated heating and cooling of the reaction chamber, preferably two or more solenoid valves (hot gas valves and cooling gas valves) and one or more mechanical relaxation valves are used.

In fact, using pressurized helium gas to heat and cool the reaction chamber is not required and is not the best method. Bottled pressurized carbon dioxide (CO ₂ ) gas expands and cools when it leaves its storage vessel (isoenthalpy 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.

For example, pressurized CO ₂ at low temperature (˜5 ° C.) and pressurized helium gas at high temperature (˜170 ° C.) delivered to a thermostatic reaction chamber using 6 V or 24 V solenoid valves and digital and / or analog signals. The gas can be used to periodically change the temperature from 92 ° C. (DNA denaturation temperature) to 55 ° C. (primer binding temperature) to 72 ° C. (DNA polymerase extension temperature) in less than 4 seconds. When using minimum (0-2 seconds) denaturation, binding and extension time, depending on the length of the DNA fragment to be amplified, 30 cycles of 92 ° C./55° C./72° C. can be performed with ˜2.0 to 4.0 minutes ( 12).

Accordingly, it is an object of the present invention to amplify DNA that can be rapidly carried out using a pre-heated and / or pre-cooled gas in which a polymerase chain reaction (PCR) is carried to a thermostatic reaction chamber using an electromagnetic valve. To provide a way. In this process, hot (≧ 95 ° C.) or cold (≦ 20 ° C.) pressurized gas is carried to a physically separated reaction chamber containing a biological sample to allow DNA denaturation, primer: template binding, and polymerase-catalyzed Control the temperature used for stretching.

It is a further object of the present invention to provide a method of rapidly thermocycling these samples by controlling the flow of hot or cold pressurized gas (P ≧ 1.3 bar) to biological samples.

It is yet another object of the present invention to provide an apparatus for performing DNA amplification using an electromagnetic valve that controls the flow of hot and cold pressurized gas into a thermostatic biochemical reaction chamber.

It is another object of the present invention to use one or more chambers that regulate the hot and cold gas flows prior to the reaction chamber to relieve pressure pulsations.

It is also an object of the present invention to use a mixing chamber before the reaction chamber to achieve a homogeneous temperature in the flow received into the reaction chamber.

It is also an object of the present invention to provide a device capable of rapidly thermally cycling a biological sample or samples using one or more gases at a pressure of at least 1.3 bar.

It is further an object of the present invention to provide a device capable of rapidly thermally cycling a biological sample or samples, wherein the device uses pressurized air, helium, carbon dioxide, nitrogen, argon or a mixture thereof as a gas heat transfer medium. The same gas or gas mixture need not be used for heating and cooling.

It is also an object of the present invention to provide a device capable of rapidly thermally cycling a biological sample or samples, wherein the time parameter for DNA denaturation, primer binding and extension can be programmed to one second of minutes.

Finally, it is an object of the present invention to provide a device in which a physically separated gas heat chamber, a cooling chamber, and a reaction chamber are used.

The above and other features, aspects, and advantages of the present invention will be better understood with reference to the following drawings, description and appended claims.

Brief description of the drawings

To better understand how the above-mentioned and other advantages and objects of the present invention are obtained, a more detailed description of the invention briefly described above will be described with reference to specific examples thereof illustrated in the accompanying drawings. will be. It is to be understood that these drawings only depict typical embodiments of the invention and are not to be considered limiting of its scope. The invention will be further described and described in further detail using the accompanying drawings:

1 is a table showing the thermal conductivity of a gas as a thermal conductivity unit (k-value) expressed in watts / meter- ° C.

2A is a schematic diagram comparing a hot air thermocycler with a pressurized gas thermocycler.

2B is a table comparing the characteristics of a hot air thermocycler with a pressurized gas injection thermocycler.

3A is a perspective view of an embodiment of an apparatus for amplifying DNA in accordance with the present invention.

3B is a perspective view of reaction chamber 38, flow conditioners 30 and 34, and thermocouples 44, 46, 48, and 50.

FIG. 3C is a diagram of the flow conditioner used in the reaction chamber 38 of FIG. 3A.

3D shows the computational fluid power mesh of the flow conditioner shown in FIG. 3C.

FIG. 3E shows a perspective view of the reaction chamber 38 shown in FIG. 3A.

4 shows temperature versus time profiles of different types of thermocyclers. Pressurized gas thermocycler is ˜10 times faster than hot thermocycler; Note that it is ˜100 times faster than conventional thermal block thermocyclers.

Figure 5a shows (a) E. coli O157: H7 Stx2 amplicon of 91 base pairs, (b) Bacteriophage λ 'D' gene fragment of 333 base pairs, (c) 364 base pairs of human platelet antigen HPA- Gel electrophoresis photographs showing high-speed gas injection PCR amplification of four allele fragments, and (d) four different DNA templates comprising gene fragments of human β-globin 536 base pairs.

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 gel electrophoresis diagram showing the reaction product of a fast gas jet PCR amplification of 85 base pairs of E. coli O157: H7 Stx DNA fragments using 2.8 bar pressurized air by cooling 2.9 bar CO ₂ . It is a photograph.

FIG. 7A shows E. coli O157: H7 Stx DNA amplicons of 85 base pairs after 85 seconds (lane a), 81 seconds (lane b), or 78 seconds (lane c) through 30 fast PCR cycles. Gel electrophoresis photographs showing He / CO ₂ gas jet PCR amplification.

FIG. 7B is the temperature versus time profile for the experiment shown in FIG. 7A (lane c). This experiment represents the fastest DNA amplification experiment performed.

FIG. 8A is a gel electrophoresis photograph showing the reaction product of fast gas jet PCT amplification of a 368 base pair DNA fragment from Bacillus anthracis .

FIG. 8B is a temperature versus time profile for PCR jet amplification of the Bacillus anthracis DNA fragment of the 368 base pairs shown in FIG. 8A (lane e). The total time for 30 high speed PCR cycles was 233 seconds.

Figure 9a shows (a) 191 base pairs of human acute intermittent porphyrinosis (AIP) amplicons, (b) 147 base pairs of human ABO blood type 'A-transferase' gene fragment, (c) 486 base pairs of E. coli ( E coli ) uidA gene fragment, (d) 297 base pair E. coli uidA gene fragment, (e) 145 base pair E. coli uidA gene fragment gel electrolysis showing fast gas injection PCR amplification Yeongdong is a picture.

FIG. 9B is a temperature versus time profile of the PCR injection reaction used to amplify the 147 base pair human ABO blood type A-transferase gene fragment shown in FIG. 9A. 35 PCR cycles were performed at 228 seconds.

FIG. 10A is a gel electrophoresis photograph showing nested PCR amplification of an E. coli uidA DNA fragment of an outer 486 base pair and a nested inner 186 base pair.

FIG. 10B is a diagram showing the DNA sequence and position of the primer used in the reduced PCR experiment shown in FIG. 10A.

FIG. 11A is a gel electrophoresis photograph showing gas jet PCR amplification of a 2206 base pair length DNA fragment from bacteriophage λ.

FIG. 11B is a gel electrophoresis photograph showing gas jet PCR amplification of a 2331 base pair length DNA fragment from bacteriophage λ.

FIG. 12A is a gel electrophoresis photograph showing gas jet PCR amplification of a 297 base pair long DNA fragment from the E. coli uidA (β-glucuronidase) gene.

FIG. 12B is a temperature versus time profile of a PCR spray reaction used to amplify a 297 base pair long DNA fragment from the E. coli uidA (β-glucuronidase) gene.

FIG. 13 is a table summarizing high speed PCR experiments performed using a pressurized gas injection thermocycler.

14 is a list of scientific nomenclature used herein.

Detailed description of the invention

The present invention will be described by applying the preferred embodiments. It is not intended to be exhaustive or to limit the invention to the described embodiments. It is intended that the present invention include all modifications and alternatives, such as using a two-way or three-way valve, which may be included within the spirit and scope of the invention.

Five characteristics of pressurized gas thermocyclers are important:

(1) This is done by fast amplification of DNA with 30 cycles of PCR amplification of 85 base pair (bp) DNA fragments performed in 78 seconds. Longer (145-2331 bp) viral and bacterial DNA fragments amplified ˜10 ⁸ fold after approximately 2-10 minutes (FIGS. 5-12).

(2) During the amplification of DNA in a pressurized gas sprayed thermocycler, the time intervals used for DNA denaturation, primer: template binding, and enzymatic elongation can be programmed to one second of minutes.

(3) The overall rate of DNA amplification is limited by biochemistry, not the heating / cooling time of the device. In particular, Taq polymerase extension rate (˜80 nucleotides / sec at 72 ° C.) is rate-limiting. If faster (> 200nt / sec) thermostable DNA polymerase can be found, it is possible for the thermocycling time to be less than 30 seconds for 30 PCR cycles.

(4) Fast pressurized gas PCR is generally more accurate than the slower method, probably because it has little time for the wrong reaction product to bind and / or stretch.

(5) The pressurized gas sprayed thermocycler is compatible with on-line fluorescent die-based DNA detection optics.

Pressurized gas injection PCR processes and thermocyclers should demonstrate their usefulness, particularly in the diagnosis of life-threatening diseases where speed is of paramount importance. The present invention may be practiced to rapidly perform (about 2 to 10 minutes) DNA-based tests used in the detection of biomedical investigations, genetics, molecular medicine, agriculture, veterinary science, forensics, and biological combat reagents. (Has been practiced).

Preferred embodiments of the invention are provided by injecting a hot or cold gas into a thermostatic reaction chamber under the control of a fast (<20 msec / cycle) solenoid valve, an actuator accommodating digital and / or analog signals, and a microprocessor controller. Pressurized gas thermocycler to perform high speed PCR amplification of DNA. Ideally, this device would be compatible with the optical device containing the fewest number of moving moieties and also used for the detection of fluorescent die-labeled DNA [Ref. Higuchi et al., 1992; Haugland, 1996; Spears et al., 1997].

In this embodiment, it is understood that the temperature can be changed rapidly by injecting hot and cold pressurized gas into the reaction chamber under electronic control using electronically programmable "hot gas" and "cold gas" valves. shall. The pressure in the reaction chamber is maintained at a relatively constant value by the mechanical relaxation valve, although the temperature can be changed as desired. Reaction samples (10-25 μl of volume) may be sealed in thin walled glass capillary tubes as described by Wittwer and Garling (1991), or by Tretakov et al . (1994). One was sealed in a similar thin walled plastic container. Thus, the pressure inside the reaction chamber varies over a range of 1.3 to 6.0 bar (20 to 90 psi) as the temperature of the gas changes, while the pressure in the capillary tube remains unchanged close to 1.0 bar (14.7 psi). .

Pressurized gas thermocyclers differ from hot air thermocyclers (US Pat. No. 5,455,175 to Whitworth et al.) In several important respects, as shown in FIG. In a hot air thermocycler (FIG. 2A), a single reaction chamber with a heating lamp is used to heat the air and perform the PCR reaction (heating chamber = reaction chamber). In a pressurized gas thermocycler (FIG. 2B), up to six separate chambers are used: heating chamber, cold gas feed chamber, hot gas flow conditioner, cold gas flow conditioner, mixing chamber and reaction chamber. To heat the sample in the reaction chamber, the preheated gas (≥95 ° C.) from the heating chamber is conveyed to the reaction chamber through a hot gas flow conditioner and mixing chamber under pressure by an electromagnetic valve V _H (hot gas valve 28). do. To cool the sample in the reaction chamber, cold gas (≦ 20 ° C.) is conveyed to the reaction chamber through a cooling gas flow conditioner and mixing chamber under pressure by an electromagnetic valve V _C (cooling gas valve 36). The mechanical relaxation valve of the valve V _R (40 in FIG. 3A) discharges pressurized gas from the reaction chamber. The efficiency is improved by recovering heat from the solenoid valve V _H installed to bypass the exhaust stream and the hot gas flow conditioner.

Reference will now be made to the drawings wherein like structures will be provided with like reference numerals. Schematics of pressurized gas thermocyclers, flow conditioners, and reaction chambers thereof are shown in FIGS. 3A, 3B, 3C, 3D, and 3E.

Fabrication and Optimization of Pressurized Gas Thermocycler

As shown in FIG. 3A, the thermal cycling apparatus 10 includes a reaction chamber made of an insulating (low k) material, generally designated 38. Preferably the reaction chamber 38 has a k value of less than 0.5 Watts / cm- ° K. Reaction chamber 38 may be composed of a number of different materials, such as stainless steel or titanium. In addition, polyurethane or polyimide plastics with ceramic binders sold under the trade names VESPEL, TORLON and BUTTERBOARD (manufactured by Golden West of California) may be used as the material of the reaction chamber 38. The cold gas feeder 14 is a dual solenoid valve 34 actuated by a pressure regulator 16, a member check valve 18 (available from Lindweld of Lincoln, NE), and a digital relay operated by a controller 42. (38 VDC valve manufactured by Peter Paul Electronics of New Britain, CT) is separated from the reaction chamber 38. Controller 42 may be a multifunction data acquisition board such as KPCI-3107 manufactured by Keithley of Cleveland, OH. The hot gas supplier 12 is connected to the preheater 20 via a pressure regulator 16 and a one-way check valve 18. The preheater 20 is a heat exchanger that cools the spent gas from the reaction chamber 38 and heats the gas coming from the gas supply 12. The preheated gas leaving preheater 22 then enters preheater 26 followed by process heater 26 (500 Watt process heater manufactured by Hotwatt of Danvers, MA). The process heater 26 is operated by a very robust relay operated by the controller 42. The hot gas leaving heater 26 flows to a three-way solenoid valve 28 (operated by controller 42). Process heaters require a continuous flow of gas; The solenoid valve 28 consumes gas through the preheater 22 and finally the muffler 24 (manufactured by the Air of Cincinnati, OH) whenever the reaction chamber 38 is cooled. When reaction chamber 38 is heated, hot gas flows from solenoid valve 28 to flow conditioner 30. Flow conditioner 30 sinks the gas flow pulses into a tighter flow of hot gas. The regulated hot gas is then introduced into the mixing chamber 32. Cooling gas from the solenoid valve 36 also flows through the flow conditioner 34 to the mixing chamber 32. The mixed gas then flows to reaction chamber 38. The reaction chamber 38 is maintained at an elevated pressure by the mechanical relaxation valve 40. The mechanical relaxation valve 40 has a decomposition pressure of 1.3 bar (20 p.s.i.). The spent gas from the mechanical relaxation valve 40 is cooled in the preheater 20 and discharged through the muffler 24.

As shown in FIG. 3B, solenoid valves 28 and 36 use conditioners 42, mixing chambers 32, hot gas flow conditioners 30, measured by thermocouples 44, 46, and 48, respectively, And on the basis of the temperature of the cooling gas flow conditioner 34. Thermocouple 50 is used by conditioner 42 to maintain process heater 26 at a constant temperature.

As shown in FIG. 3E, the reaction chamber 38 contains an elliptical cavity 52, which contains a reaction sample sealed in a thin wall capillary tube. The tube enters through staggered holes 54.

Pressurized gas thermocyclers were tested with various combinations of hot and cold feed gases. Pressurized air is available, inexpensive and easy to control. On the other hand, pressurized helium gas is an excellent heat transfer gas (FIG. 1), but has many problems with thermal control (see FIG. 7B).

Operation of the pressurized gas thermocycler requires three modes of operation, which are controlled by activating a relay wired to the valve. The three modes of operation are (1) heating of the reaction chamber, (2) cooling of the reaction chamber, and (3) maintaining the temperature of the reaction chamber.

In order to increase the temperature of the reaction chamber 38, the hot gas valve 28 is opened to the flow conditioner 30 so that hot pressurized gas is conveyed to the chamber. The low temperature gas valve 36 remains blocked .

In order to lower the temperature of the chamber, the hot gas valve 28 to the flow conditioner 30 is shut off and the cooling gas valve 36 is opened.

During the elongation phase of the PCR process, when the temperature is kept close to 72 ° C., the cooling gas valve 36 and the hot gas valve 28 meet the demand based on the temperature measured by the thermal sensor located in the mixing chamber 32. Open and shut off accordingly. This valve arrangement provides excellent thermal control to keep the temperature close to a preset value.

The opening and closing of the hot and cold valves is controlled by an electronic signal from the system controller to the electronic relays that operate the valve. System control software determines the opening and closing of the valve.

When different "hot" or "cold" feed gases are applied, the modified software is used since the gas flow and mixing in the chamber will vary depending on the gas mixture selected. Using pressurized air or helium as the "hot"gas; It is convenient to use air or C0 ₂ as the "low temperature" gas, but various different gases can be used. Using the same gas to heat, cool, or maintain the temperature of the chamber is not required and is not the best method.

As shown below (FIGS. 5A, 6, 7A, 8A, 9A, 11A, 11B and 12A), fast gas jet amplification of DNA is pressurized air (or helium) as a "hot" gas, and pressurized "low temperature" It can be carried out using gas (CO ₂ or air). Depending on the heat transfer gas selected, the size of the heat gradient, and the length of the DNA fragments to be amplified, 30 cycles of PCR amplification can be achieved after 78-617 seconds. In the examples described below, amplified DNA was detected using a relatively slow (˜1 hour) process: standard electrophoresis of DNA detection known to those skilled in the art, followed by gel electrophoresis followed by ethidium bromide (EtBr) staining.

However, pressurized gas jet PCR is compatible with on-line fluorescence-based detection (Higuchi et al., 1992) using a die such as SYBR Green, which selectively binds to double stranded DNA (Haugland, 1996). In principle, double stranded DNA reaction products of high speed gas phase PCR can be quickly detected online using a reporter die such as SYBR Green and a low cost photodiode detector. For example, the pressurized gas thermocycler may be equipped with a photodiode detector and a photo-emitting diode (LED) or laser diode die excitation source. Such optics or equivalently inexpensive fluorescence polarization detection optics (Spears et al., 1997) and fluorophore-labeled primers will allow for heavy / detection of DNA on an unprecedented time scale.

Example

The following examples illustrate the invention described herein better and are not intended to limit the invention in any way. Those skilled in the art will recognize that there are several different parameters that can be modified using conventional experimental methods and are intended to be within the scope of the present invention.

Fast Amplification of DNA Using Three-Valve Pressurized Gas Thermocycler

Initial high speed PCR experiments in a three-valve gas jet thermocycler were performed using compressed air as the "hot" gas (-170 ° C) and CO ₂ as the "low temperature" gas (-5 ° C). This configuration is not the fastest possible but is convenient and does not require active cooling of "cold" gases.

Example 1

30 PCR cycles at 5:35 (335 seconds)

Gas jet DNA amplification experiments were carried out using pressurized air (2.4 bar = 36 psi) as the “hot” gas and pressurized CO ₂ (2.6 bar = 40 psi) as the “low temperature” gas in a three-valve instrument (FIG. 3A). Was performed. Four model DNA templates of different lengths were PCR-amplified in separate 10 μl reactions, which were performed in thin walled glass capillary tubes: (a) 91 bp E. coli 0157: H7 Stx amplicon, (b) 333 bp λ [0 second 92 ° C. using 'D' gene amplicon, (c) 364 bp human platelet antigen HPA-4 amplicon, and (d) human β-globin 536 bp amplicon as pressurized air and CO ₂ cooling / 0 second 55 ° C./5 seconds 72 ° C.] at 30 cycles. Gas jet PCR reactions (10 μl) were subjected to 50 mM Tris (pH 8.5, 25 ° C.), 250 μg / ml BSA, 3 mM MgCl ₂ , 0.2 mM dNTP, 50 pmol of forward and reverse primer, 20 picograms of template DNA, and 5 U The thin walled glass capillary tube containing Taq polymerase (Promega, Madison, WI) was carried out. After amplification, the reaction product was separated on 3% MetaPhor agarose gel by EtBr staining. Molecular weight markers were 67-501 bp in length (pUC19 / MspI DNA fragment).

As shown in FIG. 5A, all four amplicons were amplified in high yield using pressurized gas jet PCR. Starting with 100 pg of DNA, 30 PCR cycles were performed for 335 seconds using 2.5 U Taq polymerase with 15 μl reaction volume. Objective 536 bp β-globin gene, 364 bp HPA-4 allele, 333 bp bacteriophage λ 'D' gene and 91 bp E. coli O157: H7 Stx2 amplicon [ 0sec 92 ° C (denatured) / 0sec 55 ° C (binding) / 5sec 72 [deg.] C. (extension)]. The background of the nonspecific amplification product was observed low in all four reactants. A particularly high yield was also observed for the λ 'D' gene amplicons (lane b). The temperature versus time profile of these 335 second gas phase PCR experiments is a significant improvement in performance over the previously reported thermocycler or thermocycling methods (FIGS. 4 and 5b).

Example 2

Software improvement, 30 PCR cycles for 2:48 (168 seconds)

The three-valve device is a functional thermocycler. It is not only very fast but also shows good thermal control (± 1 ° C). However, the performance of the device is limited by its software written in BASIC code. Whenever it is necessary to perform an instruction in response to a heat change in a reaction chamber or process heater, it needs to be translated from BASIC (interpreter program) to assembly code (compile program).

Significant time (~ 1 sec / cycle) is lost due to this software limitation. In particular, more than 30 seconds of time (> 1 second / cycle) were unproductively lost during 30 cycles of vapor phase PCR amplification. As a result, system control software was rewritten in assembly code for faster operation. This modification enabled much faster gas jet PCR.

At Taq polymerase elongation rate of ≧ 80 nt / sec, ˜1 sec / cycle taken during the transition phase, A-E + ED (FIG. 4A), yielded a 85 bp DNA short template using a 30-mer PCR primer. Expected to be more than enough to replicate (T _m- 65 ° C.). Accordingly, experiments were performed with pressurized air heating / CO ₂ cooling using relatively short amplicons (85 bp) from the E. coli 0157: H7 cigartoxin gene ( Stx ).

Gas jet PCR reaction (10 μl) was performed with 50 mM Tris (pH8.5, 25 ° C.), 250 μg / ml BSA, 3 mM MgCl ₂ , 0.2 mM dNTP, 50 pmol of forward and reverse primer, 20 picograms of E. coli It was performed with 2.4 bar (36 psi) of pressurized air and 2.6 bar (-40 psi) of CO ₂ cooling in a thin walled glass capillary tube containing 0157: H7 DNA, and 5 U of Taq polymerase (Promega). After 30 cycles of amplification, the DNA fragments were separated on 3% MetaPhor ^™ agarose and stained with EtBr. Molecular weight markers were pUC19 / MspI digests 67-501 bp in length.

6 can amplify a short 85 bp E. coli 0157: H7 Stx amplicon through 30 PCR cycles in high yield for <2 minutes; It is shown that significant performance improvements have been achieved by writing device instructions in assembly code. Based on the high thermal conductivity of helium gas (Ubbink, 1947; Bosworth, 1952; FIG. 1), it was assumed that much faster thermocycling is possible when helium rather than air is used as the “hot” gas.

Example 3

30 PCR cycles for 1:12 (78 seconds)

Small amplicons (85 bp) from the E. coli 0157: H7 Stx gene were PCR amplified using “hot” pressurized helium gas and CO ₂ cooling. In order to further reduce the thermocycling time, higher binding temperatures (62 ° C. to 63 ° C.) were applied by increasing the primer length to 30 nt. In addition, the DNA denaturation temperature was slightly reduced (86 ° C. to 89 ° C.) than that used in previous experiments.

Three different high speed vapor phase thermocycling protocols were applied: (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 μl) were subjected to 50 mM Tris (pH 8.5, 25 ° C.), 250 μg / ml BSA, 3 mM MgCl ₂ , 0.2 mM dNTP, 50 pmol forward and reverse primers, 20 picograms of E.coli O157: H7 DNA and Performed in thin walled glass capillary tubes containing 5 U Taq polymerase (Promega, Madison, WI). 30 cycles of [0sec 89 ° C./0sec 62 ° C./0sec 72 ° C.]; (b) [0 sec 87 ° C./0sec 62 ° C./0sec 72 ° C.]; And [0sec 86 ° C./0sec 63 ° C./0sec 72 ° C.], the reaction product was separated on 3% agarose gel by EtBr staining. Molecular weight markers were 67-501 bp (pUC19 / MspI digest).

As shown in FIG. 7A, a high yield of target 85 bp Stx amplicon was observed in all three helium / CO ₂ gas jet PCR reactions. 7A and 7B demonstrate the three fastest reactions of DNA amplification reactions previously performed. Objective 85 bp E. Coli O157: H7 Stx amplicons were amplified in all three reactions: (a) 1: 25 = 85 seconds, (b) 1: 21 = 81 seconds, (c) 1: 18 = 78 seconds. The experiment also demonstrates that thermocycling is considerably faster when pressurized helium gas is used rather than air. High speed gas jet PCR also made the background of nonspecific "fumes" or erroneous reaction products very low. Assuming incorrect priming or stretching is rare if such rapid thermocycling parameters were used; There is no time for false reaction products to accumulate.

Further improvements of the high speed PCR jet thermocycler are substantial improvements in software, thermal control, optimization of gas operating pressure, reduction of noise, and the ability to amplify DNA fragments of different lengths from various sources. Examples of high speed PCR experiments in which these improvements are performed are illustrated in Examples 4-7 and described below.

Example 4

Bacillus anthracis at 368 bp B. anthracis)  PCR amplification of DNA fragments

30 PCR cycles for <4 minutes (233 seconds)

The ability to rapidly amplify DNA from deadly bacterial pathogens, such as the biological warfare bacillus anthracis, is particularly important (Franz et al., 1997). As shown in FIGS. 8A and 8B, high-speed jet PCR amplification of 368 bp DNA fragments from the biological warfare bacillus anthracis was performed at 167 ° C., 2.9 bar (44 psi) and CO ₂ at 3.0 bar (45 psi). It was used with gas cooling. Bacillus anthracis Sterne strain DNA supplied from the US Institute of Infectious Diseases (USAMRIID) was used as a template for PCR jet amplification using primers BAN23MS1 and BAN23MA1 (21mers). [0 msec 92 ° C., 0 msec 55 ° C., 3.2 sec 72 ° C. using 24 μl reaction containing 10 pg of DNA, 1 μM primer, 5.5 mM MgCl ₂ , 100 mM dNTP, 500 μg / ml BSA and 1.25 U of Taq polymerase ] PCR cycles were performed 30 times. 25 pmol of Bacillus anthracis Sterne strain DNA (10 pg) in a reaction volume of 24 μl containing 1.25 units Taq Pol in (50 mM Tris-Cl, 5.5 mM MgCl ₂ , pH 8.8, 500 μg / ml BSA) Mixed with forward primer (BAN23MS1) and reverse primer (BAN23MA1) and 200 μM dNTP. Fast PCR was performed in a model 1.62 Heliac pressurized gas thermocycler programmed at 10 msec time intervals and gel electrophoresed on a 2% agarose gel with ethidium bromide staining. Samples are as follows: [a] 100 bp ladder molecular weight marker, [b] (3.0 bar) CO ₂ cooling (0 sec 92 ° C., 0 sec 55 ° C., 4.0 sec 72 ° C.) and 2.6 bar (38 psi) hot compressed air 30 PCR cycles using [c] 30 bar PCR cycles using 3.0 bar CO ₂ cooling (0 sec 92 ° C., 0 sec 55 ° C., 4.0 sec 72 ° C.) and hot He gas (2.9 bar), [d] 2.9 Bar high temperature He, 30 PCR cycles using 3.0 bar CO ₂ cooling (0sec 92 ° C, 0sec 55 ° C, 3.6sec 72 ° C), [e] 2.9 bar high temperature He, 3.0 bar CO ₂ cooling [0sec 92 ° C, 0sec 30 PCR cycles using 55 ° C., 3.2 sec 72 ° C.). PCR-amplified DAN was loaded on a 20% agarose gel and electrophoresed at 80V for 45 minutes. The total elapsed time of 30 high-speed PCR cycles was [b] 4 minutes, 28 seconds, [c] 4 minutes, 20 seconds, [d] 4 minutes, 10 seconds, [e] 3 minutes, 53 seconds.

As shown in FIG. 8A (lane e), the target 368 bp fragment was amplified ˜10 ⁸ fold for a time period as short as 3 minutes 53 seconds (233 seconds) with 30 cycles of high speed gas jet thermocycling. 8B shows a pair of temperature versus time profiles.

The reaction yield can be calculated as follows. Starting with 10 pg of Bacillus anthracis DNA, about 100 ng of the 368 bp reaction product was formed (FIG. 8A). Since the genome of Bacillus anthracis contains ˜5.0 × 10 ⁶ base pairs and the 368 bp DNA fragment represents only a portion of the bacterial chromosome, the initial 10 pg of bacterial DNA is ˜ [368 / (5.0 × 10 ⁶ ) × 10 ⁻¹¹ g] = 0.74 × ^10-15 g of 368 bp amplicons can be calculated. This 0.74 × 10 ⁻¹⁵ g (= 0.74 femtogram) template DNA was amplified with ˜100 ng of 368 bp reaction product. Thus, amplification factor of (1.00 × 10 ⁻⁷ g) / (0.74 × 10 ⁻¹⁵ g) = 1.35 × 10 ⁸ fold was achieved using 30 PCR cycles.

If the polymerase chain reaction is 100% efficient, one would expect that the amount of DNA would be double after every PCR cycle. In theory, after 30 cycles of DNA doubling, 2 ³⁰ = 1.07 × 10 ⁹ fold amplification will be achieved. In this experiment, 1.35 × 10 ⁸ fold amplification was observed after 30 fast PCR cycles. It can be calculated as 1.866 ³⁰ = 1.34 × 10 ⁸ . Thus, each PCR cycle amplifies the bacterial DNA 1.866 fold, corresponding to an efficiency of 1.866-1 = 0.866. In other words, each high speed PCR cycle was -86.6% efficient.

The experiment demonstrated that starting from about 10 picograms of DNA, it was possible to amplify DNA fragments from a particular pathogen such as Bacillus anthracis after about <4 minutes.

Example 5

PCR amplification of E. coli and human DNA from 145 to 486 bp

30 to 35 PCR cycles after 2.4 to 4.1 minutes (144 to 247 seconds)

Further experiments were conducted to demonstrate the speed and versatility of the high speed gas jet thermocycler. For example, bacterial gene fragments from 145 to 486 bp and single-copy human gene fragments from 147 to 191 bp were prepared using 20 pg of DNA, 1 μM primer, 4.5 to 55 mM MgCl ₂ , 100 mM dNTP, 500 μg / ml BSA, and Amplification through 30-35 cycles for ˜2.4-4.1 minutes (144-247 seconds) using 1.25 U of Taq polymerase.

9A shows DNA (191bp), ABO blood type a-transferase gene fragment (147bp), E. coli uidA gene fragment (486bp), E. coli amplified from human genes resulting in Acute Intermittant Porphyria. uidA gene fragment (297bp) and E. coli uidA gene fragment (145bp). As shown in the temperature vs. time profile for FIG. 9A (lane a) (FIG. 9B), good thermal control ensures that pressurized air is hot gas at 2.6 bar (42 psi) with CO ₂ gas cooling at 2.8 bar (46 psi). It was possible when used.

Example 6

486 / 186bp E.coli uidA  Nested PCR Fragments of DNA Fragments

40 PCR cycles for 4.8 minutes (285 seconds)

More sensitive DNA amplification was possible when the "Nested PCR" protocol was applied. In particular, Ruzicka et al. (1992) describe a nested PCR method, in which a first pair of external primers was used for PCR in ˜20 cycles with a binding temperature of 45 ° C. to 55 ° C .; Amplification from an internal (nested) primer set with binding temperature> 60 ° C. (FIG. 10B). In this way, two steps of external / internal PCR can be performed in one closed reaction vessel.

Using primers from Juck et al, 1996, nested PCR amplification of the outer 486 bp and inner 186 bp E. coli uidA DNA fragments was performed using: (1) 20 cycles of [200 msec 88 ° C. , 200 msec 56 ° C., 2.8 sec 72 ° C.], and then (2) 20 cycles of [200 msec 88 ° C., 200 msec 65 ° C., 1.8 to 2.0 sec 72 ° C.]. The first 20 cycles allowed the binding of all four oligonucleotide primers at an acceptable 56 ° C. binding temperature using a relatively long 2.8 second Taq polymerase extension time. The second 20 cycles selectively amplified the internal 186 bp DNA fragment due to the higher 65 ° C. binding temperature and the shorter 1.8 to 2.0 second elongation step.

As shown in FIG. 10A, PCR reactions (15 μl) were subjected to 50 mM Tris (pH 8.5, 25 ° C.), 250 μg / ml BSA, 3 mM MgCl ₂ , 0.2 mM dNTP, 50 pmol of forward and reverse primers, 4.5 pg of E Performed in thin walled glass capillary tubes containing .coli K12 DNA, and 1.5 U of Z-Taq polymerase (Takara Shuzo Ltd). (a) After 20 cycles of [100 msec 89 ° C./100 msec 65 ° C./2.5 sec 72 ° C.] followed by 20 cycles of [100 msec 90 ° C./100 msec 61 ° C./1.8 sec 72 ° C.], the reaction product was subjected to EtBr staining. Isolated on 2.0% MetaPhor agarose gel. The molecular weight marker was pUC19 / MspI digest fragment. Purpose 186 bp uidA amplicons in lanes (a) and (b) run below the 190 bp molecular weight marker. The time required for 40 fast PCR cycles was 4 minutes and 45 seconds (285 seconds).

As shown in FIG. 10A (lane a), starting with 1 picogram of E. coli 0157: H7 DNA, the target 186 bp internal amplicon had a second 20 cycles [200 msec 88 ° C., 200 msec 65 ° C., 2.0 sec. 72 ° C.] could be amplified through 40 cycles of two-step nested PCR for less than 5 minutes. As shown in FIG. 10A (lane b), when shorter 1.8 sec / cycle Taq polymerase elongation was applied, 40 cycles of nested PCR could be achieved for 4 minutes 45 seconds (40 cycles). Total PCR time = 285 seconds).

Amplification factors can be calculated as follows: The E. coli genome contains ˜4.5 × 10 ⁶ bp. 186 bp E. coli DNA fragment (1.86 × 10 ² /4.5×10 ⁶ ) = 4.1 × 10 ⁻⁵ Represents the bacterial genome. Bacterial DNA of the initial 1 picogram (10 ^-12 g) were amplified in an amount of ^{~50ng (5 × 10 -8 g)} ( Fig. 10a). Therefore, 186 bp E. coli uidA DNA fragment was amplified at (1 / 4.1 × 10 ⁻⁵ ) × (5 × 10 ⁻⁸ / 10 ⁻¹² ) = 1.2 × 10 ¹¹ times in a total of 285 seconds of nested PCR time.

Example 7

PCR amplification of bacteriophage λ DNA 2206-2331bp

30 PCR cycles for 10.3 minutes (617 seconds)

FIG. 11A shows 20 pg DNA, 1 μM primer, 4.5 mM in a gas jet thermocycler using 2.6 bar (42 psi) compressed air (compressed air from an aired air tool compressor) and 2.8 bar (46 psi) CO ₂ cooling. MgCl ₂ , 100 mM dNTP, 500 μg / ml BSA, and 1.5 U of Z-Taq polymerase (Takara Shuzo Eltidi) and 30 cycles [800 msec 89 ° C. to 91 ° C., 800 msec 61 ° C., 13 sec 72 ° C.] A photograph of a 2206 bp bacteriophage λ DNA fragment amplified using is shown. Lane (a) shows the target reaction product of 2206 bp using a denaturation temperature of 91 ° C. (total PCR time = 10 minutes, 49 seconds). Lane (b) shows the reaction product at 2206 bp using a denaturation temperature of 90 ° C. (total PCR time = 10 min 28 sec). Lane (c) shows the reaction product at 2206 bp using a denaturation temperature of 89 ° C. (time required between 30 PCRs in total: 10 minutes, 3 seconds = 603 seconds). The molecular weight marker was a bacteriophage λ HindIII digested fragment.

FIG. 11B shows 20 pg DNA, 1 μM primer, 4.5 mM in a gas jet thermocycler using 2.6 bar (42 psi) compressed air (compressed air from an aired air tool compressor) and 2.8 bar (46 psi) CO ₂ cooling. MgCl ₂ , 100 mM dNTP, 500 μl / ml BSA, and 1.5 U Z-Taq polymerase (Takara Shuzo Eltidi) and 30 cycles of [500 msec 91 ° C., 500 msec 61 ° C., 11-14 sec 72 ° C.] The photograph of the amplified 2331bp bacteriophage λ DNA fragment is shown. Lanes (a) to (d) are (a) 14 seconds at 72 ° C (total PCR time = 10 minutes 50 seconds), (b) 13 seconds at 72 ° C (total PCR time = 10 minutes 17 seconds), (c A) the desired 2331 bp reaction product obtained using an elongation time of 12 seconds at 72 ° C. (total PCR time = 9 minutes 42 seconds), and (d) 11 seconds at 72 ° C. (total PCR time = 9 minutes 8 seconds). As shown in lanes (a) and (b), the reaction yield was certainly higher when an elongation time of> 12 seconds was applied. This result suggests that for phage λ DNA fragments of 2206 to 2331 bp, Z-Taq polymerase has a clear elongation rate of ≧ 194 nt / sec.

11A and 11B demonstrate that DNA fragments of 2206-2331bp were successfully amplified in high yield using a pressurized gas jet thermocycler. FIG. 13 shows a summary of a high speed DNA amplification method using an embodiment of the invention using a three-valve gas jet thermocycler.

Example 8

In a two-valve jet thermocycler E.coli uidA  PCR amplification of DNA fragments

FIG. 13 is an illustration of 297 bp E. coli uidA DNA fragments amplified using 1 ng DNA, 150 pM primer, 3 mM MgSO ₄ , 200 μM dNTP, 400 μg / ml BSA, and 0.625 U KOD Hot Start polymerase To show. Lane 1 is [30sec 90 ° C. (hot start), 0sec 90 ° C. in a two-valve gas jet thermocycler using 2.4 bar (35 psi) compressed air; 0.2 sec 58 ° C .; 2 sec 72 [deg.] C.], 35 cycles, lane 2 is [30sec 90 [deg.] C. (hot start), 0sec 90 [deg.] C .; 0.2 sec 58 ° C .; 2 sec 72 ° C.], 40 cycles, and lane 3 is [30sec 90 ° C. (hot start); 0 sec 90 ° C .; 0.2 sec 58 ° C .; 2 sec 72 ° C.]. 25 μl of sample was placed in a Roche glass capillary tube. This result means that high speed jet PCR can be performed in a reaction vessel suitable for on-line fluorescence based DNA detection. In addition, good thermal control (plus or minus 0.7 ° C.) was possible using a two stage flow conditioner and CFD-optimized chamber structure.

Some of the major advantages of the present invention are as follows: First, the present invention reduces the time required for amplification of DNA using polymerase chain reaction by one to two orders of magnitude as compared to the previously described method or apparatus (FIG. 12). Second, the gas jet method was inherently more accurate than the later method because the wrong reaction product did not have time to substantially bind and / or elongate (see FIGS. 5A, 6, 7A, 8A, 9A, 11A, and 11B). ). Third, the pressurized gas jet thermocycler (FIG. 3A) shows better timing control compared to thermal blocks or hot-air devices, and the gas jet devices are programmed at time intervals of 100 ms. Fourth, high speed gas jet PCR is a very reformative method. Except for electromechanical relays and valves, moving parts are not used. Fifth, the high speed gas jet PCR method is fully compatible with the on-line detection optics so that timely DNA amplification / detection can be performed. Thus, the present invention allows rapid, accurate and reproducible DNA amplification / detection to be achieved on an unprecedented time scale.

Comparison with "Ultrafast" PCR Thermocyclers

Lawrence Livermore National Laboratory, Northrup et al., 1998, the University of Pittsburgh, Oda et al., 1998, the University of Washington, Freidman and Meldrum, 1998), and the fastest experimental thermocycler made by the Massachusetts Institute of Technology, Chiou et al., 2001, required 8.5 to 23 minutes for 30 PCR cycles.

For example, Oda et al, 1998 describe "ultrafast PCR" that "a cycle time as fast as 17 seconds can be achieved." Unfortunately, the infrared heating method used by Oda et al. Is incompatible with fluorescent optical detection using> 450 nm die and photodiode detectors.

Chiou et al., 2001, described that "30 cycle PCR operations were performed, allowing up to 78% efficiency for 30 cycle PCR of 500 bp target from genomic λ DNA for 23 minutes." . At this efficiency, 30 PCR cycles were amplified by 1.56 ³⁰ = 6.2 × 10 ⁵ times at a rate of 23 minutes / 30 cycles = 46 seconds / cycle.

The pressurized gas unit shown in FIG. 3A required less than 2.6 seconds / cycle and the reaction yield was at least 200 times greater (FIGS. 5A, 6, 7A, 8A, 9A, 11A and 11B). For example, as shown in FIG. 8A, one copy bacterial gene fragment 368bp was amplified 1.35 × 10 ⁸ fold in 233 seconds for 30 PCR cycles.

Kopp et al. (1998) described that small scale continuous-flow PCR devices can amplify 176 bp DNA fragments over 20 cycles for 3-4 minutes. However, ˜10 ⁸ copies of template DNA are required as starting material (Zorbas, 1999). Presumably from the data of Kopp et al., 1998, 30 cycles of PCR amplification took 4.5-6 minutes, and the yield of amplified DNA was obtained using the pressurized gas apparatus shown in FIG. 3A. 10 ³ to 10 ⁴ times lower.

Comparison with commercial devices of current technology level

The fastest commercially available thermocycler is licensed from Idaho Technologies (US Pat. No. 5,455,175, Wittwer et al.), Boehringer-Mannheim Division of Roche's Modular System. Is prepared by. This hot-air thermocycler requires about 9.5 minutes for 30 cycle amplifications of 536 base pairs of β-globin DNA fragments. However, their on-line to the sensor device, between the light cluster ^TM (Lightcycler ^TM) will require 30 to 30 minutes in the PCR cycle.

As shown in FIGS. 7A and 7B, a pressurized gas PCR process can be used to amplify DNA in detectable amounts as fast as 78 seconds. The high speed pressurized gas jet process is therefore> 15 minutes faster than on-roche machines and compatible with on-line fluorescent die based DNA detection optics.

Higher yields of amplification (> 10 ⁸ fold) were achieved with longer elongation times (3.2 sec / cycle at 72 ° C.). For example, 30 PCR cycles in less than 4 minutes were achieved using a 368 bp Bacillus anthracis amplicon (FIGS. 8A and 8B). Shorter (145-486 bp) bacterial DNA fragments were amplified ˜10 ⁸ fold for 144-280 seconds. The overall description of the invention as well as the preferred embodiments are as mentioned above. Those skilled in the art will recognize and practice further variations of the methods and apparatus within the teachings of the present invention. Accordingly, all such modifications and additions are within the scope of the present invention, which is limited only by the following claims.

references

US patent literature

Other literature

SEQUENCE LISTING <110> Megabase Research Products <120> A Gas Jet Process and Apparatus for High-Speed Amplification of DNA <130> P05668WO0 <140> PCT <141> 2002-11-01 <160> 5 <170> PatentIn version 3.1 <210> 1 <211> 24 <212> DNA <213> Escherichia coli <400> 1 atcaccgtgg tgacgcatgt cgcg 24 <210> 2 <211> 24 <212> DNA <213> Escherichia coli <400> 2 caccacgatg ccatgttcat cgcc 24 <210> 3 <211> 21 <212> DNA <213> Escherichia coli <400> 3 tatgaactgt gcgtcacagc c 21 <210> 4 <211> 21 <212> DNA <213> Escherichia coli <400> 4 catcagcacg ttatcgaatc c 21 <210> 5 <211> 486 <212> DNA <213> Escherichia coli <400> 5 atcaccgtgg tgacgcatgt cgcgcaagac tgtaaccacg cgtctgttga ctggcaggtg 60 gtggccaatg gtgatgtcag cgttgaactg cgtgatgcgg atcaacaggt ggttgcaact 120 ggacaaggca ctagcgggac tttgcaagtg gtgaatccgc acctctggca accgggtgaa 180 ggttatctct atgaactgtg cgtcacagcc aaaagccaga cagagtgtga tatctacccg 240 cttcgcgtcg gcatccggtc agtggcagtg aagggcgaac agttcctgat taaccacaaa 300 ccgttctact ttactggctt tggtcgtcat gaagatgcgg acttgcgtgg caaaggattc 360 gataacgtgc tgatggtgca cgaccacgca ttaatggact ggattggggc caactcctac 420 cgtacctcgc attaccctta cgctgaagag atgctcgact gggcagatga acatggcatc 480 gtggtg 486

Claims (40)

As a fast amplification method of DNA, (a) providing a reaction chamber containing a biological sample having DNA, a DNA polymerase, an oligonucleotide primer, and a deoxynucleotide precursor and containing one or more heat transport gas streams; (b) heating the first heat transport gas in a heat chamber that is physically separate from the reaction chamber; (c) deliver a first heat transport gas to the reaction chamber at a pressure greater than the pressure of the reaction chamber, wherein heat is transported from the first heat transport gas to the DNA to denature the DNA; (d) delivering a second heat transport gas to the reaction chamber to cool the reaction chamber to a temperature at which denatured DNA is annealed to the oligonucleotide primers; (e) increasing the temperature of the reaction chamber to a temperature sufficient to permit enzyme-catalyzed elongation of the primer: template complex. The method of claim 1, further comprising repeating steps (c) to (e) until a desired amount of amplified DNA is produced. The method of claim 1, further comprising regulating the flow of the first heat transfer gas from the heat chamber to the reaction chamber using one or more multiport valves. The method of claim 1 wherein the first heat transport gas and the second heat transport gas are gases of the same kind. The method of claim 1 wherein the first heat transport gas and the second heat transport gas are different gases. The method of claim 1 wherein the pressure of the first heat transport gas is at least 1.3 bar (≧ 20 pounds per square inch). The method of claim 1 wherein the pressure of the second heat transport gas is at least 1.3 bar (≧ 20 pounds per square inch). The method of claim 1 wherein the first heat transport gas is air. The method of claim 1 wherein the first heat transport gas is helium or an isotope mixture thereof comprising helium-3. The method of claim 1 wherein the first heat transport gas is selected from the group consisting of hydrogen, neon, methane, nitrogen, argon, krypton, carbon dioxide and mixtures thereof. The method of claim 1 wherein the second heat transport gas is carbon dioxide. The method of claim 1 wherein the second heat transport gas is air cooled to a temperature below 20 ° C. by a Ranque-Hilsch Vortex tube before being delivered to the reaction chamber. The method of claim 1 wherein the second heat transport gas is selected from the group consisting of air, hydrogen, neon, methane, argon, krypton, carbon dioxide and mixtures thereof. The method of claim 1, wherein the second heat transport gas is cooled to a temperature below 20 ° C. before entering the reaction chamber. The method of claim 1, further comprising adjusting the flow of the first and second heat transport gases to the reaction chamber using one or more electronic multiport valves. 2. The method of claim 1, further comprising suppressing pressure pulsations and temperature fluctuations using one or more flow conditioners. The method of claim 1, wherein the first and second heat transport gases flow from their respective flow conditioners into the mixing chamber to reach a constant temperature. 17. The method of claim 16, further comprising operating the electronic multiport valve via an electronic signal. The method of claim 1, further comprising mixing the first and second heat transfer gases into the reaction chamber using one or more gas flow conditioners. The method of claim 1, wherein the time required for DNA denaturation, the time required for primer binding, and the time required for extension by the enzyme are programmed at time intervals of one second of minutes. As a device for high speed amplification of DNA, (a) a reaction chamber having an operating pressure of at least 1.3 bar; (b) a heating chamber physically separated from the reaction chamber and connected by a fluid, the heating chamber comprising a first vessel connected by the fluid and having a heating gas at a pressure higher than the reaction chamber pressure; (c) a flow conditioner connected to the heating chamber and the mixing chamber by fluid to suppress pressure pulsations; (d) a second vessel fluidly connected to the reaction chamber and having a cooling gas; (e) a flow conditioner connected by fluid to the cold gas supply chamber and the mixing chamber to suppress pressure pulsations in the cold gas flow; (f) a heated gas inlet valve positioned between the heating chamber and the hot gas flow conditioner; (g) a cooling gas inlet valve positioned between the second vessel and the cold gas flow conditioner; (h) having an input and an output, the output device being connected to a heating gas inlet valve and a cooling gas inlet valve to control the opening and closing of the valve; And a first temperature sensor coupled to the reaction chamber and connected to the input of the programmable controller, A device in which the controller responds to a temperature sensor to open and close the heating gas inlet valve and the cooling gas inlet valve to maintain the temperature of the reaction chamber at the desired setting. The apparatus of claim 21 wherein the first heat transport gas pressure is at least 1.3 bar. The apparatus of claim 21 wherein the first heat transport gas is air. 22. The apparatus of claim 21 wherein the first heat transport gas and the second heat transport gas are gases of the same kind. 22. The apparatus of claim 21 wherein the first heat transport gas and the second heat transport gas are different gases. 22. The apparatus of claim 21, wherein the first heat transport gas is helium and the second heat transport gas is carbon dioxide. The apparatus of claim 21 wherein the first heat transport gas is air and the second heat transport gas is carbon dioxide. 22. The apparatus of claim 21, wherein the first heat transport gas is air and the second heat transport gas is air cooled using a Rank-Hilsch Vortex tube. 22. The apparatus of claim 21, further comprising a flow conditioner connected to the mixing chamber for mixing hot and cold gases. 22. The apparatus of claim 21, further comprising one or more solenoid valves for controlling the gas flow and pressure to the reaction chamber. 22. The apparatus of claim 21, further comprising a noise-reducing muffler assembly operatively connected to the exhaust gas outlet. 22. The apparatus of claim 21, wherein at least one output of the programmable controller is connected to a heating element of the heating chamber to turn the heating element on and off. 22. The apparatus of claim 21, wherein the reaction chamber has a heat transfer coefficient of less than 0.5 Watts / centimeter-Kelvin. The apparatus of claim 21 wherein the reaction chamber comprises polyurethane plastic. 22. The device of claim 21, further comprising an optical detection device for facilitating DNA detection by fluorescence. 36. The apparatus of claim 35, wherein the optical detection device comprises a light emitting diode, a parallelizing lens, a filter, a photodiode. 36. The apparatus of claim 35, wherein the optical detection device comprises a laser diode, a parallelizing lens, a filter and a photodiode. As a fast amplification method of DNA, (a) providing a reaction chamber containing a biological sample with DNA, a DNA polymerase, an oligonucleotide primer, and a deoxynucleotide precursor; (b) heat the helium gas in a heating chamber that is physically separate from the reaction chamber; (c) transfer the heated helium gas to the reaction chamber, where heat is transported from the helium gas to the DNA to denature the DNA; (d) delivering a cooling gas to the reaction chamber to cool the reaction chamber to a temperature at which the modified DNA binds to the oligonucleotide primer; (e) increasing the temperature of the reaction chamber to a temperature sufficient to permit extension of the primer: template complex. 39. The method of claim 38, wherein the cooling temperature is carbon dioxide. The method of claim 38, wherein the second heat transport gas is air cooled to a temperature below 20 ° C. by a Rank-Hilsch Vortex tube before being delivered to the reaction chamber.