Patent Application
20130157376
Thermal cyclers are known in the art. Thermal cyclers can be field calibrated by using software to adjust well-to-well variation in measured temperature. Hardware calibration often requires a skilled service technician.
The present disclosure relates generally to a method for calibrating an apparatus for thermal cycling. Certain embodiments relate more specifically to a container that fits within a thermal cycling apparatus, which is configured to provide information related to the functional, experimental, or actual parameters of the thermal cycling apparatus into which the container is placed, in order to determine when apparatus calibration is required or recommended or to calibrate the apparatus manually or via software or automation.
Thus, in one embodiment a method for calibrating a thermal cycling apparatus having at least one heat source is provided, the method comprising the steps of providing a container comprising at least one sample vessel, wherein vessel comprises a calibration mixture comprising nucleic acids having at least one nucleic acid melting domain, the melting domain having a true temperature, and a reagent that produces a signal to differentiate between single-stranded and double-stranded nucleic acids, introducing the container into the thermal cycling apparatus, heating the contents of the container, monitoring the reagent to calculate a measured melting temperature, and adjusting the heat source to correct discrepancies between the true melting temperature and the measured melting temperature of melting domains. In one illustrative embodiment, the reagent is a dsDNA binding dye, while in another illustrative embodiment, the reagent is a fluorescent dye that is bound to one of the nucleic acids. Optionally, the calibration mixture comprises a second melting domain, the second melting domain having a true temperature that is different from the true temperature of the melting domain, and both melting domains are used in the adjusting step, wherein the melting domain is a low temperature melting domain, the second melting domain is a high temperature melting domain, and the adjustment is an adjustment across temperatures using a LowCalibrationTemperature point and HighCalibrationTemperature point, wherein the two points are calculated as follows:
LowCalibrationTemperature=(PreviousLowCalibrationTemperature*A)+B
HighCalibrationTemperature=(PreviousHighCalibrationTemperature*A)+B
where A and B are:
where PreviousLowCalibrationTemperature and PreviousHighCalibrationTemperature were determined in a previous round of calibration. Optionally, the adjustment across temperatures is a linear adjustment across temperatures, and a separate adjustment is made for each of a plurality of heat sources.
In another aspect of the invention, a device for use in calibrating a thermal cycling apparatus is provided, the device comprising a container comprising a plurality of sample vessels, each sample vessel comprising a calibration mixture comprising nucleic acids having at least one nucleic acid melting domain, the melting domain having a true temperature, and a reagent that produces a signal to differentiate between single-stranded and double-stranded nucleic acids and configured to generate a measured temperature for the melting domain, wherein the calibration mixture does not contain sufficient components for amplification and the sample vessel is provided sealed to prevent addition of components for amplification
In yet another aspect of the invention, a method for calibrating a thermal cycling apparatus having at least one heat source is provided, the method comprising the steps of providing a container comprising at least one sample vessel, wherein vessel comprises a calibration mixture comprising temperature-indicating reagent that provides a measurable signal a temperature, the reagent having a true temperature, introducing the container into the thermal cycling apparatus, heating the contents of the container, monitoring the reagent to calculate a measured temperature, and adjusting the heat source to correct discrepancies between the true temperature and the measured temperature.
In yet another aspect of the invention a system for calibration is provided comprising a container comprising at least one sample vessel, wherein vessel comprises a calibration mixture comprising nucleic acids having at least one nucleic acid melting domain, the melting domain having a true temperature, and a reagent that is configured to produce a measured melting temperature, a thermal cycler system comprising at least one heat source, and computing device configured to calculate desired adjustment of heat output from the heat source using the true temperature and the measured temperature.
Additional features of the present invention will become apparent to those skilled in the art upon consideration of the following detailed description of preferred embodiments exemplifying the best mode of carrying out the invention as presently perceived.
The written disclosure herein describes illustrative embodiments that are non-limiting and non-exhaustive. Reference is made to certain of such illustrative embodiments that are depicted in the figures, in which:
As used herein, “nucleic acid,” “nucleotide,” “oligonucleotide,” “DNA,” and similar terms also include RNA, nucleic acid analogs, and nucleic acid substitutes, i.e. naturally occurring or synthetic analogs or substitutes having other than a phosphodiester backbone. Non-limiting examples, including the so called “peptide nucleic acids” (PNAs) and the so called “locked nucleic acids” (LNAs) are considered within the scope of this invention. Non-analogous nucleic acid substitutes are also considered within the scope of this invention.
As used herein, “base pair,” “base pairing,” and similar terms refer to the association of complementary nucleotides or nucleic acids as previously defined and are not limited to canonical Watson-Crick base pairing or association via hydrogen bonding.
As used herein, “double-stranded” refers to the base pairing of at least one pair of nucleotides and is not limited to oligonucleotides or nucleic acids of any particular length or base pairs from separate nucleic acid strands.
As used herein, “melting domain,” “nucleic acid melting domain,” and similar terms refer to portion, unit, or segment of double-stranded nucleic acid that remains in a double-stranded configuration at certain temperatures and separates or melts into single-stranded nucleic acid at other temperatures.
As used herein, “calibrator plate,” “sample plate,” “sample container,” and similar terms refer to a container comprising a plurality of sample vessels or compartments, and does not necessarily imply the presence of a sample, known or unknown, within the sample vessels, or a rigid, plate-like configuration. Non-limiting examples of illustrative configurations include 8-tube strips, 12-tube strips, 48-well plates, 96-well plates, 384-well plates, and 1536-well plates. Furthermore, use of a multi-well sample plate or a multi-tube strip herein is illustrative only. Other configurations of sample vessel-comprising containers are known in the art. Additional non-limiting embodiments of sample plate containers, including sample tubes, capillaries, and flexible pouches, are also considered within the scope of this invention.
As used herein, “sample vessel,” “sample compartment,” “sample well,” and similar terms refer to a portion or partition of a sample container that is configured to provide a barrier that limits fluid communication between adjacent portions or partitions, and does not imply the presence of a sample, known or unknown, within the sample vessel, compartment, or well. Non-limiting, illustrative examples include wells of a sample plate or tube strip and blisters formed in a flexible or non-flexible sample pouch.
As used herein, “Temperature-indicating reagents” and similar terms refer to molecules, components, chemicals, compounds, or other materials that are capable of demonstrating, suggesting, or revealing an actual, experimental, or approximate temperature and may include nucleic acids illustratively with associated indicators such as dyes, nucleic acid binding dyes, covalently-bound dyes, probes, fluorescent probes, and other temperature indicators such as fluorescent moieties, fluorescent units, temperature-sensitive liquid crystals, or other thermochromic, temperature-sensitive, or temperature-responsive substances.
More detail regarding the plurality of wells 120 of well block 110 can be seen in
The layers of adhesive 150 and 170 may be the same material. The adhesive is ductile and flexible, has relatively high thermal conductivity and low viscosity. Illustratively, the adhesive enhances the uniformity of heat transfer between peltier 160 and wells 120. In one embodiment, the adhesive permits apparatus 100 to be assembled without the use of conventional clamps used to clamp a well block to a heat sink. When an adhesive is used in an embodiments such as apparatus 100, the adhesive is capable of retaining the peltier device 160 adjacent to the structure contacted by the adhesive such as the wells 120 of well block 110 and/or heat sink 180 even when apparatus 100 is turned upside down without clamping well block 110 to heat sink 180.
Various embodiments of a suitable adhesive are capable of cycling between a temperature at least as high as 95° C. and at least as low as 60° C. at least about 5,000 times, at least about 10,000 times, at least about 100,000 times, or at least about 200,000 times and still be capable of retaining peltier device 160. Various embodiments of a suitable adhesive may have an elongation, as defined below in the Examples, of at least about 15%, 20%, 22%, 35%, 40%, 50%, 55%, 60%, 70%, 90%, 110%, 120%, 180%, 200%, 400% or ranges within combinations of these values such as about 15% to about 1,000%, about 35% to about 700%, about 70% to about 500%, or between 100% to about 200%.
Suitable adhesives may also have an unprimed adhesion lap shear of between about 1 kgf/cm2 and about 75 kgf/cm2, over about 10 kgf/cm2, between about 10 kgf/cm2 and about 45 kgf/cm2. The viscosity of the adhesive may range between about 1,000 centipoise and about 200,000 centipoise, between about 10,000 centipoise and about 150,000 centipoise, between about 20,000 centipoise and about 80,000 centipoise, or between about 30,000 centipoise and about 40,000 centipoise.
Various embodiments may also have a thermal conductivity, as defined below in the Examples, of at least about 0.39, 0.40, 0.74, 0.77, 0.84, 0.85, 0.9, 0.92, 0.95, 1.1, 1.4, 1.53, 1.8, 1.9, 1.97, 2.2, 2.5 or ranges within combinations of these values such as about 0.74 to about 2.5 or about 0.9 to about 1.8. In one embodiment, the adhesive has a thermal conductivity at 25° C./77° F. of between about 0.7 Watt/meter-K and about 2.5 Watt/meter-K. In another embodiment, the adhesive has a thermal conductivity at 25° C./77° F. of between about 0.8 Watt/meter-K and about 2.0 Watt/meter-K. In one embodiment, the adhesive has a thermal conductivity at 25° C./77° F. of between about 0.9 Watt/meter-K and about 1.5 Watt/meter-K. In yet another embodiment, the adhesive has a thermal conductivity at 25° C./77° F. of over about 1.0 Watt/meter-K. In a further embodiment, the polymer has a thermal conductivity at 25° C./77° F. of about 1.1 Watt/meter-K.
Examples of suitable adhesives include thermally conductive silicone pastes, which are non-curing. Specific trade names of suitable thermally conductive silicone pastes, which are non-curing, are provided by those listed in the Examples.
The embodiment depicted in
More detailed information about the configuration of well 120 can be appreciated with reference to
An advantage of flat bottom 128 relative to prior art configurations is that the shape can be manufactured with greater uniformity, and provides additional surface area that enables heat to be transferred with greater uniformity and at a more rapid rate. However, it is understood that flat bottom 128 may have rounded edges near sidewall 126 or otherwise may not be completely flat from one side of cylindrical sidewall 126 to the other. Moreover, because lower cylindrical sidewall 126 does not interfere with insertion of the sample well 82 into well 120, the shape of the well 120 allows sample well 82 to have maximal contact with the sidewall 122 of the wells in each well block.
An average well 120′ of well block 110′, as shown in
As shown in
The system includes a computing device 104, which may comprise one or more processors, memories, computer-readable media, one or more HMI devices 103 (e.g., input-output devices, displays, printers, and the like), one or more communications interfaces (e.g., network interfaces, Universal Serial Bus (USB) interfaces, etc.), and the like. Computing device 104 may be provided within housing 101, or may be provided separately, such as a laptop or desktop computer, or portions of computing device 104 may be resident within housing 101, while other portions are located separately and may be coupled through wiring or wirelessly. Computing device 104 may be configured to load computer-readable program code for controlling thermal cycler apparatus 100 and optics block 109. In one illustrative embodiment, thermal cycler apparatus 100 in housing 101 may be provided in an automated system with a robotics unit 105. The robotics unit 105 may be programmed to load the samples into sample wells 82 and then load sample plate 80 into housing 101 through opening 102. Optionally, robotics unit 105 may also prepare the samples prior to loading into sample wells 82. Teach points may be used by robotics unit 105 for orienting plate 80 into well block 110. Teach points 134a-c are best seen in
It will be understood that reference to PCR is illustrative only and the devices of this disclosure may be compatible with other methods of amplification. Such suitable procedures include strand displacement amplification (SDA); nucleic acid sequence-based amplification (NASBA); cascade rolling circle amplification (CRCA), Q beta replicase mediated amplification; isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN); transcription-mediated amplification (TMA), and the like. Asymmetric PCR may also be used. Therefore, when the term PCR is used herein, it should be understood to include variations on PCR as well as other alternative amplification methods, as well as post-PCR processing, such as melt curve analysis. Illustrative examples of suitable melt curve analysis can be found in U.S. Pat. No. 7,387,887, herein incorporated by reference. Furthermore, the devices of this disclosure may be suitable for a variety of other biological and non-biological reactions that require temperature control.
As peltiers 160 may be individually driven, an illustrative embodiment for determining when each peltier requires calibrating, adjusting, or replacing in order to achieve uniformity among peltiers or to correct for differences between the actual, measured temperature produced by a peltier and the apparatus temperature set point that attained the actual temperature, is provided. Methods for calibrating peltiers may involve adjusting, controlling, or resetting the energy output of each peltier individually to allow for proper and adequate control of sample temperature in wells corresponding to each pettier-controlled temperature zone. Methods for calibrating peltiers may also involve replacing the peltier entirely.
In one illustrative embodiment of the present invention, a calibrator sample plate comprising a plurality of sample wells is provided. Illustratively, as in
It is noted, and one skilled in the art would be aware, that a wide variety of thermal cyclers are known in the art and the embodiment represented in
One or more peltiers are then gradually heated to a point beyond the known temperature-indicating range of the reagent. The temperature-indicating reagents in each sample well are monitored by optics block 109 (shown in
In one illustrative embodiment, each sample well of a 96-well calibrator plate comprises a calibration mixture comprising one or more nucleic acids that anneal to form at least one intramolecular or intermolecular nucleic acid melting domain, wherein each melting domain melts at a distinct melting temperature (Tm), and a reagent that differentiates between single-stranded and double-stranded nucleic acids. Illustratively, each melting domain may have a Tm within the normal thermal cycling or melting range for PCR or post-PCR melting, or one or more of the melting domains may have a Tm that brackets the thermal cycling or melting range. The calibrator plate is inserted into a thermal cycling apparatus and the peltiers are gradually heated to a point beyond the known melting temperatures for nucleic acid melting domains in the calibration mixture. As the temperature of calibration mixture in each well increases, nucleic acid melting domains in each sample begin to transition from a double-stranded to a single-stranded configuration and the signal from the reagent is monitored by optics to determine the change in relative amounts of single-stranded or double-stranded nucleic acids. Change in signal can be plotted, illustratively on a computer monitor, to generate a melting curve, from which an experimental melting temperature may be calculated. Peltiers are then adjusted, calibrated, or replaced to ensure that the temperatures displayed or recorded by the thermal cycling apparatus during nucleic acid melting or transition is comparable and within acceptable limits, deviation, or error from the known melting temperature of each nucleic acid melting domain.
A thermal cycler apparatus similar to device 180 of
It is noted, and one skilled in the art would be aware, that a wide variety of other temperature indicators are known in the art and contemplated within the scope of this invention, such as temperature sensitive optical materials that undergo a color change at a specific temperature. It will be appreciated that the goal is to provide a detectable signal at a predetermined known or calculated temperature such that the measured temperature can be compared to the predetermined known or calculated temperature.
Because the illustrative calibrator plate 80 is to be used for instrument calibration, other components for PCR or other amplification methods (e.g. polymerase, dNTPs) need not be provided. Additionally, the plate 80 may be provided with the calibration mixture sealed in each well, thus preventing addition of sample materials and allowing for multiple calibration uses while minimizing risk of spilling the contents. However, it is understood that the calibration mixture may be configured for PCR, in which case the calibration plate would be thermal cycled prior to generation of the calibration melt curve.
The calibrator plate of this illustrative example was placed in the thermal cycler apparatus. A melting program comprising parameters designed specifically to execute a series of steps within the thermal cycler apparatus to melt the double-stranded nucleic acids within an acceptable temperature range was initiated. The signal produced by the Oregon Green dye was monitored throughout the method and calibration process. Upon completion of the melting program, signal data were processed by computer software designed to generate and produce melting curve data and calculate the experimental Tm for each double-stranded oligonucleotide.
It is noted that melting parameters and signal data processing are variable and often specific to the reagents comprising the experiment or calibration procedures. The scope of this invention is not limited to a single or set of melting parameters or signal data processing procedures. However, various melting data processing procedures, including such for high resolution melting, are known in the art and are within the scope of the present invention.
The melting temperatures of one such calibration are shown below in Tables 1-4.
Table 1 shows the average low calibration temperature for each of the 24 temperature zones prior to calibration, wherein the individual temperatures from each well is averaged to produce the zone temperature. In this example, zone C1 is the warmest, with an average temperature of 42.43° C., while zone D6 is the coolest, with an average temperature of 41.69° C. The difference between the warmest and coolest zones is 0.74° C. Table 2 is similar to Table 1, except showing the average high calibration temperature for each of the 24 temperature zones. At the high calibration temperature, zone A1 is the warmest, with an average temperature of 79.28° C., while zone D5 is the coolest, with an average temperature of 77.28° C. The difference between the warmest and coolest zones is 2.00° C. It is noted that different zones were the hottest and coolest for the two different melts.
Discrepancies or differences between the theoretical, known, or pre-determined Tm and the mean experimental Tm may be corrected by adjusting, resetting, or calibrating the output of each peltier to bring the experimental Tm into agreement with the pre-determined Tm, thereby allowing adjustment of each temperature zone to provide a measured temperature output approximately equal to the known temperature output at the pre-determined Tm. Such adjusting, resetting, or calibrating of each peltier used for a temperature zone may include adjusting the value of the electrical input delivered thereto for a desired temperature output, or adjusting the pre-programmed temperature level for a particular value of the electrical input made to the peltier. As such, the correction of each peltier used for a temperature zone against the pre-determined Tm establishes a significant increase in the consistency of the measured temperatures across each sample vessel across the calibrator plate, thereby significantly increasing the consistency of any measured results in subsequent thermal cycling activities.
Because peltiers may vary to a different degree at different voltages, it may be desirable to use multiple predetermined known or calculated Tm's to measure the temperatures when adjusting the setting for each peltier. As discussed above, in the illustrative example, two different double-stranded oligonucleotides are used, generating two distinct Tms, a measured high calibration temperature and a measured low calibration temperature.
For a two-point calibration, for each temperature zone a low calibration temperature and a high calibration temperature are calculated, as follows:
LowCalibrationTemperature=(PreviousLowCalibrationTemperature*A)+B
HighCalibrationTemperature=(PreviousHighCalibrationTemperature*A)+B
Where A and B are:
A=(TrueHighTM−TrueLowTM)/(MeasuredHighTM−MeasuredLowTM)
B=((TrueLowTM*MeasuredHighTM)−(TrueHighTM*MeasuredLowTM))/(MeasuredHighTM−MeasuredLowTM),
and where the TrueHighTM and TrueLowTM are predetermined temperatures, and the Previous LowCalibrationTemperature and PreviousHighCalibrationTemperature were determined in a previous round of calibration. This may have occurred when a service technician or end-user performs a calibration using calibration plate 80, or it may have occurred during initial factory calibration.
A linear adjustment using the LowCalibrationTemperature and the HighCalibrationTemperature is then calculated for each peltier. Because each peltier 160 is controlled independently, the input to each peltier, such as voltage or resistance, is optionally adjusted across the temperature range according to the calculated linear adjustment. Referring back to Tables 1-4 in this exemplary embodiment, after calibration, the difference between the warmest low temperature zone (D4 at 42.08° C.) and the coolest low temperature zone (A6 at 41.97° C.) is only 0.11° C. Similarly, after calibration, the difference between the warmest high temperature zone (C1 at 78.84° C.) and the coolest high temperature zone (B1 at 78.72° C.) is only 0.12° C. It is noted that both before and after calibration in the melts represented in Tables 1-4, different zones had the warmest melt at the low and high temperatures, and different zones had the coolest melt at both the high and low calibration temperatures, thus demonstrating that each individual temperature zone is adjusted based on its own linear adjustment across temperatures. If a single calibration melt had been used for each zone, the extent of calibration needed at the other end of the melt range would have been missed, resulting in a better adjustment at the measured calibration temperature and potentially decreasing the calibration at the other end of the temperature range. Calibration may be repeated, if desired, and may be done in an iterative process, until sufficient temperature uniformity is achieved. If sufficient uniformity cannot be achieved, such failure may be due to one or more peltiers that are failing to perform satisfactorily. If a particular temperature zone continually provides results that are not sufficiently uniform (either run-to-run uniformity or uniformity between temperature zones), it may be desirable to replace the peltier for that temperature zone. It is understood that ultimate block uniformity may be limited by the worst performing peltier.
It is understood that the instrument, either internally or through an external computing device 104, may be programmed to initiate calibration subsequent to the generation of the melt curves. Alternatively, the calibration may be performed manually using the true and experimental Tms.
While 2 calibration temperatures are used in this illustrative example, it is understood that other numbers of calibration temperatures may be used, such as 3, 4, 5, or n calibration temperatures. Furthermore, while the illustrative adjustment is linear across the temperature range, depending on the heat source used, it is understood that the adjustment may be non-linear (e.g. exponential), or may be a best fit curve if more than two calibration temperatures are used. Also, while the illustrative example provides calibration for 24 temperature zones, it is understood that the methods described herein can be used for adjusting the heat source for any number of temperature zones, depending on the configuration of the instrument, including an instrument having one temperature zone. It is also understood that the calibration methods described herein may be used in combination with software that adjusts melt curve data subsequent to melting, as is known in the art.
It will be understood by those having skill in the art that changes may be made to the details of the above-described embodiments without departing from the underlying principles presented herein. For example, any suitable combination of various embodiments, or the features thereof, is contemplated.
Any methods disclosed herein comprise one or more steps or actions for performing the described method. The method steps and/or actions may be interchanged with one another. In other words, unless a specific order of steps or actions is required for proper operation of the embodiment, the order and/or use of specific steps and/or actions may be modified.
Throughout this specification, any reference to “one embodiment,” “an embodiment,” or “the embodiment” means that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. Thus, the quoted phrases, or variations thereof, as recited throughout this specification are not necessarily all referring to the same embodiment.
Similarly, it should be appreciated that in the above description of embodiments, various features are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure. This method of disclosure, however, is not to be interpreted as reflecting an intention that any claim require more features than those expressly recited in that claim. Rather, inventive aspects lie in a combination of fewer than all features of any single foregoing disclosed embodiment. It will be apparent to those having skill in the art that changes may be made to the details of the above-described embodiments without departing from the underlying principles set forth herein.
The claims following this Detailed Description are hereby expressly incorporated into this Detailed Description, with each claim standing on its own as a separate embodiment. This disclosure includes all permutations of the independent claims with their dependent claims. Recitation in the claims of the term “first” with respect to a feature or element does not necessarily imply the existence of a second or additional such feature or element. Embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows.
