Patent Application
20080057544
The invention will be better understood by reference to the appended figures of which:
In describing and claiming the present invention, the following definitions apply:
“Nucleic acid,” “DNA,” and similar terms also include nucleic acid analogs, i.e. analogs having other than a phosphodiester backbone. For example, the so-called “peptide nucleic acids,” which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention.
“Amplification” of DNA denotes the use of polymerase chain reaction (PCR) to increase the concentration of a particular DNA sequence within a mixture of DNA sequences.
An “amplicon” is a product of the amplification of a target genetic sequence.
A “PCR reaction mixture” denotes a mixture adaptable for simultaneously amplifying multiple genetic targets under suitable conditions for PCR.
A “genetic target” denotes a genetic sequence capable of amplification by polymerase chain reaction (PCR). A genetic target in accordance with the present invention includes any DNA sequence, including bacterial, viral, fungal, human, plant, and animal DNA, for example.
“Continuous monitoring” and similar terms refer to monitoring multiple times during a cycle of PCR, preferably during temperature transitions, and more preferably obtaining at least one data point in each temperature transition.
“Fluorescence detection” and similar terms refer to labeling nucleic acids with a fluorescence indicator. The fluorescence indicator can be a nucleic acid intercalating dye such as Ethidium Bromide, Thiazole orange, Pico™ Green or SyBr™ Green. As well, labeled hybridization probes using FRET, Taq-Man™ or other chemistries such as molecular beacons can also be used as fluorescence detection tools.
“Effective amount” means an amount sufficient to produce a selected effect. For example, an effective amount of PCR primers is an amount sufficient to amplify a segment of nucleic acid by PCR provided that a DNA polymerase, buffer, template, and other conditions, including temperature conditions, known in the art to be necessary for practicing PCR are also provided.
“Probe”, refers to a nucleic acid oligomer that hybridizes specifically to a target sequence in a nucleic acid, which, in the context of the present invention, is an amplicon, under standard conditions that promote hybridization. This allows detection of the amplicon. Detection may either be direct (i.e., resulting from a probe hybridizing directly to the amplicon sequence) or indirect (i.e., resulting from a probe hybridizing to an intermediate molecular structure that links the probe to the target amplicon). A probe's “target” generally refers to a sequence within (i.e., a subset of) an amplified nucleic acid sequence which hybridizes specifically to at least a portion of a probe oligomer using standard hydrogen bonding (i.e., base pairing). A probe may comprise target-specific sequences and other sequences that contribute to three-dimensional conformation of the probe (e.g., as described in Lizardi et al., U.S. Pat. Nos. 5,118,801 and 5,312,728).
By “sufficiently complementary” is meant a contiguous nucleic acid base sequence that is capable of hybridizing to another base sequence by hydrogen bonding between a series of complementary bases. By definition, this allows stable hybridization of a probe oligomer to a target sequence in the amplicon even though it is not completely complementary to the probe's target-specific sequence. Complementary base sequences may be complementary at each position in the base sequence of an oligomer using standard base pairing or may contain one or more residues that are not complementary using standard hydrogen bonding (including a basic “nucleotides”), but in which the entire complementary base sequence is capable of specifically hybridizing with another base sequence in appropriate hybridization conditions. Contiguous bases are preferably at least about 80%, more preferably at least about 90%, and most preferably greater than 95% complementary to a sequence to which an oligomer is intended to specifically hybridize. To those skilled in the art, appropriate hybridization conditions are well known, can be predicted based on base composition, or can be determined empirically by using routine testing (e.g., see Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) at § 1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57 particularly at §9.50-9.51, 11.12-11.13, 11.45-11.47 and 11.55-11.57).
The terms “label” and “detectable label” refer to a molecular moiety or compound that can be detected or can lead to a detectable response. A label is joined, directly or indirectly, to a nucleic acid probe. Direct labeling can occur through bonds or interactions that link the label to the probe, including covalent bonds or non-covalent interactions (e.g., hydrogen bonding, hydrophobic and ionic interactions) or through formation of chelates or co-ordination complexes. Indirect labeling can occur through use of a bridging moiety or “linker,” such as an antibody or additional oligonucleotide(s), which is either directly or indirectly labeled, and which can amplify a detectable signal. A label can be any known detectable moiety, such as, for example, a radionuclide, ligand (e.g., biotin, avidin), enzyme or enzyme substrate, reactive group, chromophore, such as a dye or particle that imparts a detectable color (e.g., latex or metal particles), luminescent compound (e.g., bioluminescent, phosphorescent or chemiluminescent labels) and fluorescent compound.
PCR techniques applicable to the present invention include inter alia those described in “PCR Primer. A Laboratory Manual”, Dieffenback, C. W. and Dveksler, G. S., eds., Cold Spring Harbor Laboratory Press (1995); “Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia”, Saiki R K, Scharf S, Faloona F, Mullis K B, Horn G T, Erlich H A, Amheim N, Science (1985) December 20; 230(4732): 1350-4.
The PCR of the present invention is performed using a modified 2-step cycling profile as compared to standard PCR, namely successive cycles of denaturation of double stranded target nucleic acid and annealing/extension of the primers to produce a large number of copies of segments of the target DNA. Each cycle is a thermal cycle in which the reaction temperature is raised to denature the double stranded DNA and lowered to allow annealing and extension.
In one embodiment of the present invention, the PCR makes use of successive two-step cycles in which the temperature is raised to a first temperature for denaturation of the double stranded DNA and lowered to a second temperature to allow annealing and extension of the primers.
Following amplification of a nucleic acid using the system described herein, the amplicons may be detected using any method known in the art.
Preferably, the label on a labeled probe is detectable in a homogeneous assay system, i.e., where, in a mixture, bound labeled probe exhibits a detectable change, such as stability or differential degradation, compared to unbound labeled probe, without physically removing hybridized from non-hybridized forms of the label or labeled probe. A “homogeneous detectable label” refers to a label whose presence can be detected in a homogeneous fashion, for example, as previously described in detail in Arnold et al., U.S. Pat. No. 5,283,174; Woodhead et al., U.S. Pat. No. 5,656,207; and Nelson et al., U.S. Pat. No. 5,658,737. Examples of labels that can be used in a homogenous hybridization assay include, but are not limited to, chemiluminescent compounds (e.g., see U.S. Pat. Nos. 5,656,207, 5,658,737 and 5,639,604), such as acridinium ester (“AE”) compounds, including standard AE or derivatives thereof. Synthesis and methods of attaching labels to nucleic acids and detecting labels are well known in the art (e.g., see Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), Chapter 10; U.S. Pat. Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, 4,581,333 and European Patent Application No. 0 747 706).
In accordance with another embodiment of the present invention, where the amplicons are detected using an assay without prior separation of the amplicons, they are detected using different detectable molecules to allow the amplicons from the different primer pairs to be distinguishable. For example, probes used to hybridize to the various amplicons can be labeled with labels that are detectable at different wavelengths.
In accordance with another embodiment of the present invention, the amplicon production is monitored in real-time using procedures known in the art (e.g. see U.S. Pat. No. 6,569,627) and using the detection apparatus of the present invention.
It has now been found that rapid thermal cycling within a temperature spectrum can successfully achieve a PCR, and cycling between exact temperatures is not required, In the past PCR has been understood to be a combination of three sequential reactions (i.e. denaturation, annealing, extension) occurring at three different temperatures for three time periods. The present invention makes use of the fact that each of the reactions within PCR can occur over a range of temperatures and these temperatures overlap. Denaturation and annealing each occur so rapidly that no holding time at a particular temperature is necessary for these reactions to occur. Extension occurs over a range of temperatures at varying rates and can occur between the annealing temperature and the denaturation temperature. As a result, the method of the present invention makes use of a single temperature for both the annealing and extension portions of PCR.
Some advantages of the techniques contained herein are based on rapid cycling, with its advantages in speed and specificity.
The specification describes the apparatus positioned in a convenient horizontal orientation as illustrated herein. Accordingly, directional references such as “horizontal” refer to the apparatus when in this orientation. The device may be operated in other orientations, with suitable modifications. For convenience of description, terms such as “horizontal” are used but understood as being capable of suitable modification.
The present invention provides a thermal cycler device for performing reactions, such as PCR, using at least two spaced apart temperature blocks, each maintained at a different temperature. Each temperature block is configured to receive one or more reaction vessels such that only a portion of the outer surface of the one or more reaction vessels is in direct contact with the temperature block. As a result of this configuration, the reaction vessel or vessels can be moved from one temperature block to the other using a one-dimensional (e.g. lateral) movement of either the reaction vessel(s) or the temperature blocks, or a combination thereof.
In the past, thermal cyclers having more than one temperature block have required that the reaction vessels be lifted from a receptacle in one temperature block, moved over and lowered into a receptacle in the second temperature block. In the past this design has been used for performing PCR because it was previously believed that the most effective way to achieve precise temperature control of the reaction mixture was to maximize the surface area of the reaction vessel in direct contact with the temperature source. It has now been found that this is not required for efficient PCR. In fact, only a portion of the outer surface of the reaction vessel surface needs to be in direct contact with the temperature block. It is only necessary that a limited amount of outer surface be in contact with the temperature block.
The amount of surface area contact between the heating block and the reaction vessel will influence the rate at which a sufficient portion of the reaction mixture is heated or cooled sufficiently to drive the reaction. The higher the amount of surface area contact, the faster the speed at which the reaction is driven, and vice versa. Faster thermal transfer can also be influenced by other factors, including, but not limited to, the thickness of the reaction vessel wall, the thermal conductivity of the material of the reaction vessel, the thermal conductivity of the heating block, the shape and size of the reaction vessel, the shape of the reaction vessel receiving groove within each temperature block and the volume of the reaction mixture.
The above factors can be altered depending on the intended application of the device of the present invention. Similarly, the amount of direct surface area contact between the outer surface of the reaction vessel and the temperature contact can be varied depending on the intended application. For example, if faster thermal transfer is required then a larger direct surface area contact is required than if a slow thermal transfer is satisfactory.
A standard PCR tube may be used as the reaction vessel and in one version at least about 40% of the outer surface of the reaction vessel (wherein the outer surface does not include the surface of any lid present on the reaction vessel) must be in direct contact with the temperature block. As seen in the figures, an effective region of contact is about 50% of the vessel wall.
In one embodiment, the system contains two temperature blocks mounted in side-by-side spaced apart relation. Transfer of the reaction vessel or vessels between the two reaction blocks is achieved either by one-dimensional (lateral) movement of the vessel or vessels or by one-dimensional movement of the temperature blocks. The reaction vessel or vessels may be held by holding means attached to a horizontal transfer means, such as a robotic arm, that transfers the vessels or vessels via a substantially horizontal movement between the two temperature blocks.
An alternative embodiment composes three or more temperature blocks. As the two-block system, the reaction vessel(s) is moved, or the blocks are moved, such that the reaction vessel is in direct contact with only one temperature block at a time.
As shown in
The reaction vessel can be a standard conical PCR reaction tube 23 (e.g. 200 μl volume) or alternatively can be a rectangular cuvette. The reaction vessel is preferably designed with a thin-walled material that facilitates heat transfer between the heating block and the reaction mixture within the reaction vessel when a portion of the outer surface of the vessel is in direct contact with the heating block. Preferably the vessel has a rim about the periphery of its top, which provides a seating surface for contact with the holding means attached to the mechanical arm 24. A flat cap that sits partially inside the top of the vessel seals the top of the tube, providing an air tight seal to prevent evaporation. The walls of the vessel are formed to be vertically rigid up to a temperature of about 110° C. to ensure a tight seal with the vessel slot of the heating block 10,20 during heating. The vessel is formed from a transparent material, preferably a plastic such as a polypropylene derivative or glass. Preferably, the vessel has high transmittance of visible light, low vessel wall gas permeability and sterile inner surface.
As shown in
As shown in
The heated metal lid 46 is connected via wires 52 to a circuit and controller which will be described further below. Alternatively, if a heated metal lid is not included, the reaction can be overlaid with a mineral oil, or a similar substance, which would also minimize or eliminate condensation of the reaction solvent on the lid of the vessel. A substance, such as mineral oil, with a boiling point much greater than the reaction solvent reduces evaporation of the reaction solvent.
As shown in
A circuit board 53 electrically attached by means of wires 54 to the resistive heating elements attached to each block 10,20, the temperature sensors attached to each block, the motor 30 driving the arm movement, and the microcontroller 26. The circuit board consists of:
The current or voltage regulator circuit, Circuit 1, regulates the current and voltage passed to the resistive heating elements. Circuit 1 can be any well-known current and voltage regulator, such as a MOSFET circuit driver or relay driver. The current and voltage passed to the resistive heating elements is regulated by the microcontroller 26.
The temperature sensor circuit, Circuit 2, can be any well known circuit that responds to a change in voltage, current or resistance transmitted by the temperature sensor. In this embodiment, the temperature sensor circuit consists of a linear resistance input varying with temperature from the temperature sensor, National Semiconductor™ model number LM335AZ, connected to an analog to digital integrated circuit, National Semiconductor model number ADC0831CCN. The digital number representation of the temperature sensor is transmitted via wire to the microcontroller.
The motor driver circuit, Circuit 3, can be any well known motor driver circuit, such as Texas Instruments™ motor driver integrated circuit model number L293D. The motor driver transmits current to the arm motor and is capable of forward and reverse current polarity to move the arm mechanism horizontally back and forth. The motor driver is connected via wire to the microcontroller 26.
The microcontroller 26, controls the overall operation of all circuit components and mechanical parts. In the embodiment of the invention shown in
The microcontroller 26 can be programmed to actuate the motor driver circuit, Circuit 2, at predetermined time intervals, directions and durations. This has the effect of activating the DC motor 30, thereby driving the arm gearing system 34,40. This translates into horizontal movement of the arm platform which shuttles the vessel between the slots of the heating blocks 10,20.
An example operation of the thermal cycling device consists of programming the microcontroller 26 via keypad (not shown) and display (not shown) to hold an individual temperature of each heating block 10,20. The microcontroller 26 is also programmed with a set number of cycles of arm movement. The microcontroller is also programmed with a set dwell time of the vessel as it is moved into the slot of each heating block and as it is located between the two heating blocks.
A sample programmed run of the microcontroller 26 could consist of (A) waiting for heating blocks to reach set temperatures, (B) movement of the vessel by the mechanical arm to the slot opening of the first block, (C) holding the cuvette at the first block for a set period of time (D) movement of the vessel by the mechanical arm to the slot opening of the second block, (E) holdings the vessel at the second block for a set period of time, (F) repeating steps B to E for a set number of cycles, (G) moving the vessel to a location between the two heating blocks for removal.
The thermal cycling device component of the present invention, due to the constant temperature heating blocks, is capable of cycling reaction samples in a vessel through significantly shortened temperature versus time profiles compared to prior art. The device depicted
Furthermore, a shortened time (for example, less than 5 seconds) required to bring the temperature of the reaction mixture from one temperature level to the next temperature level corresponding to phases in the amplification process, is facilitated in the system of the present invention. Specifically, the time is shortened in comparison to ramp times in standard PCR, especially standard PCR performed using a single-block device. The decrease in time required to change the temperature of the reaction, decreases the overall time required for to complete nucleic acid amplification.
The simplicity of the horizontal movement of the mechanical arm system between the two heating blocks, significantly decreases the complexity of control and cost of the thermal cycling device compared to those currently in use. Previous device require complicated robotic arm construction and precise microprocessor control to achieve a similar movement of a reaction sample between heating blocks.
Amplification products obtained through the use of the thermal cycling device of the present invention are qualitatively and quantitatively similar to those obtained through the standard Peltier heating block cycling method. However, advantages in specificity and yield are possible with rapid thermal control of the reaction mixture using the device of the present invention. Such a rapid response is not possible with prior art systems.
By reducing the ramping time of the reaction sample, the present invention can markedly decrease the total time required for the polymerase chain reaction. In addition, the vessel can be designed to hold small reaction samples which reduces the amounts of expensive reagents which must be used thus further reducing the cost of carrying out procedures using the present invention.
The thermal cycling apparatus component of the present invention is useful for amplifying DNA from any source. Although particular configurations and arrangements of the present invention have been discussed in connection with the specific embodiments of the thermal cycling device as constructed in accordance with the teachings of the present invention, other arrangements and configurations may be utilized. For example, various cuvette or heating block configurations may alternatively be used in the thermal cycling device.
As will be appreciated by a worker skilled in the art the thermal cycling device of the present invention provides even greater improvement over the prior art in the speed at which thermal cycling can be carried out, e.g., 30 cycles of DNA amplification in 10-30, or fewer, minutes.
It will be appreciated that the apparatus described herein can readily be used for many different applications including; polymerase chain reaction processes; cycle sequencing; and, other amplification protocols such as the ligase chain reaction, The present invention also advantageously provides an apparatus for accurately controlling the temperature of samples located in the reaction vessel and quickly and accurately varies the temperature of samples located in a vessel according to a predetermined temperature versus time profile.
The configuration of the thermal alternator device of the present invention allows it to be readily combined with detection systems, such as the fluorescence detection system described in more detail below. It should be readily appreciated, however, that the device is not limited to combination with a fluorescence detection system. For example, it can be easily adapted for use with systems, including but not limited to, a visible light detection system, a luminescence detection system or a magnetic detection or separation system. The configuration of the present device permits such adaptation to be well within the abilities of the skilled worker.
As shown in
The excitation source 60 is located on one side of the vessel 23 at an optimal distance to focus light into the chamber, thereby illuminating the sample. Preferably, the excitation source has a peak wavelength compatible with fluorescent dyes; for example 480 nm. In this embodiment, the excitation source is a blue Light Emitting Diode (LED) with a 3000 mcd at 30 mA and 15-degree focusing angle. The excitation source 60 is enclosed within an opaque tube to prevent excess leakage of light from the source. The excitation light source wavelength is restricted with an optical low pass filter 64 placed directly in the path of light. An optical filter is needed to differentiate the emission from excitation wavelength. In this embodiment, a 500 nm low pass blue dichronic filter is placed in the path of light from the excitation source.
The emission source consists of a fluorescent entity and a nucleic acid amplification product. When illuminated by the excitation source, the entity (for example, a double-stranded DNA specific dye or a fluorescently labeled probe) and nucleic acid amplification product emits light at a different peak wavelength than the excitation source. Examples of suitable fluorescent dyes include, but are not limited to, thiazole orange, SYBR™ GREEN I, ethidium bromide, pico green, acridine orange, YO-PRO-1, and chromomycin A3. Alternatively the fluorescent entity is a nucleic acid probe that is specific for the amplification product and that is labeled with a fluorescent tag.
The detector 62 is located on one side of the vessel 23, directly opposite the excitation source 60, at an optimal distance to collect light from the emission source. Preferably, the detector is a photosensitive sensor capable of differentiation of visible light at a chosen peak wavelength. In this embodiment, the detector consists of a CDS photodiode. The detector is enclosed within an opaque tube to prevent excess light from being detected by the sensor. The wavelength of light detected by the sensor is restricted with an optical filter placed direction in the path of the emission source. An optical filter is needed to differentiate the emission source from the excitation wavelength, hi this embodiment, a 520 nm band pass green dichronic filter is placed in the path of light from the emission source.
Both the excitation source and the detector are activated by means of the microcontroller 26. In this embodiment, when a fluorescence reading of the sample within the vessel is desired, the microcontroller 26 activates the excitation source 60. The excitation source then illuminates the emission source. The light generated by the emission source strikes the detector. In this embodiment, a relative amount of light from the emission translates into a change in resistance of the photodiode detector. This resistance is monitored via wire by the microcontroller. Any change in fluorescence translates into a change detected by the microcontroller.
In a sample run of the fluorescence detection system, a sample within a reaction vessel is placed into the thermal cycling device. A fluorescence measurement is taken at the ambient temperature of the device. Following this measurement, the PCR reaction takes place over a predetermined number of cycles. Following the completion of the PCR reaction, the vessel is positioned between the fluorescence detection system and another measurement is taken. By comparing the initial and final fluorescence of the sample in the vessel, a corresponding increase in nucleic acid amplification product can be determined. Fluorescence measurements may also be taken at the completion of each PCR cycle, thereby quantifying per cycle the relative amount of nucleic acid amplification product increase.
The fluorescence detection component of the present invention, due to the simplicity of design, is capable of measuring per cycle results of nucleic acid amplification product by means of fluorescence. Compared to prior art fluorescence detection systems implemented in heating block thermal cyclers, this device component offers significant reduction in mechanical complexity and cost, while maintaining similar performance capabilities. DNA amplification can be measured by means of fluorescence at the beginning and end of the PCR reaction, as well as during each step. This same performance measurement using prior art system would be approximately 100 times more expensive. The simplistic optics, excitation source and detector of the present invention, have proven also to produce comparable results to more expensive and complex prior art systems.
Furthermore, the rapid cycling of the thermal cycling component means a quantitative fluorescence measurement of nucleic acid product can be accomplished much faster than prior art systems. This greatly reduces the time required to quantify any nucleic acid product generated by the PCR reaction.
Turning now to
A holder 100 for the reaction vessels 120 was constructed by drilling holes of the appropriate size into a chassis 140 comprising a flat sheet of metal. The reaction vessel holder 100 was bolted onto chassis 140. Underneath the reaction vessel holder 100 proximal heater block 160a, and distal heater block 160b were affixed to a support board 180. Grooves (not seen) were machined into the proximal heater block 160a, and distal heater block 160b, the grooves being shaped precisely to fit the shape of the reaction vessels 120. The proximal heater block 160a, and distal heater block 160b also contained resistive heaters (not seen) which were controllable to maintain a set temperature. The support board 180 was configured and arranged to be able to move in one dimension by sliding along two metal shafts 120. Motion of the support board 180 was driven by a cam shaft 122 which was configured and structured to be rotatable in one direction by a motor (not seen). The cam shaft 122 was thus configured to rotate in between a pair of plastic or metal leaf springs 124. The cam shaft 122 was configured and structured to have three main positions, namely: (1) pointing parallel away from the proximal heater block 160a, and distal heater block 160b to result in a configuration where the distal heater block 160b came into contact with the reaction vessels 120; (2) pointing perpendicular to the proximal heater block 160a, and distal heater block 160b to result in a configuration where neither of the proximal heater block 160a, and distal heater block 160b were in contact with the reaction vessels 120; and (3) pointing parallel towards the proximal heater block 160a, and distal heater block 160b to result in a configuration where the proximal heater block 160a came into contact with the reaction vessels 120.
Thus, when the cam shaft 122 were in the 2nd position, the reaction vessels 120 are not in contact with either the proximal heater block 160a, nor the distal heater block 160b. If the middle section 126 were to be cut out of the support board 180, then this middle position 126 would be convenient for imaging the bottom of the reaction vessels 120. Specifically, a blue LED light source may be shone at the bottom of the reaction vessel to excite the contents of the vessel, e.g. SYBR® Green Dye (Molecular Probes, Inc.). Emitted light from the vessel may be detected by means of a CCD camera. To filter out blue light from the LED source, a bandpass filter may be placed in front of the CCD camera so that only higher wavelengths e.g., green and red are allowed to pass through. This helps improve the signal-to-noise ratio.
The use of a cam shaft 122 with the leaf springs 124 attached to the support board 180 helps ensure good contact between the proximal heater block 160a, and distal heater block 160b and the reaction vessels 120. The reason is because the cam shaft 122 is able to deflect the leaf springs 124 when the cam shaft 122 is parallel to, and facing either towards or away from the proximal heater block 160a, and distal heater block 160b. This enables the cam shaft 122 to exert extra force, thereby to drive the proximal heater block 160a, and distal heater block 160b into contact with the reaction vessels 120, and to correct for dimensional tolerances.
It is important to note that the according to certain aspects of the present invention the reaction vessels 120 only come into partial contact with the proximal heater block 160a, and distal heater block 160b. This means that there is a non-uniform (i.e. nonzero) temperature gradient across the reaction vessel 120. The reason is because, although the proximal heater block 160a, and distal heater block 160b are set at a certain temperature, the top of the reaction vessels 120 experience a different temperature because it is held in place by a material which serves as a passive insulator, and the side walls of the reaction vessels 120 which are not in contact with the proximal heater block 160a, and distal heater block 160b are exposed to the temperature of the ambient air.
The steps through which the microprocessor (μP) shown in
It will be understood by those skilled in the art that the embodiments described herein are merely exemplary and that the person skilled in the art may make many modifications and variations without departing from the scope of the invention. The various embodiments may be practiced in the alternative or in combination as appropriate. All such variations and modifications are intended to be included within the scope of the invention.
This is a continuation-in-part of co-pending U.S. national phase application Ser. No. 11/578,440 entered nationally on Oct. 13, 2006, and filed internationally on Apr. 15, 2005 as PCT International Application No. PCT/CA2005/000576, which claim the benefit of U.S. provisional application Ser. No. 60/563,061 filed on Apr. 16, 2004, and both of which are incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60563061 | Apr 2004 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11578440 | Oct 2006 | US |
| Child | 11768605 | US |
