Patent Publication Number: US-11022573-B2

Title: Positive controls

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     The present application is a continuation-in-part of the U.S. application Ser. No. 13/223,258 claiming the benefit of priority to U.S. Provisional Application Ser. No. 61/378,927, filed on Aug. 31, 2010, the entire disclosure of which is incorporated herein by reference. In addition, the present application claims the benefit of priority to U.S. Provisional Application Ser. No. 61/378,591, filed on Aug. 31, 2010, the entire disclosure of which is incorporated herein by reference. 
     SEQUENCE SUBMISSION 
     The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is entitled 3400233SequenceListing.txt, was created on Aug. 30, 2011 and is 11 kb in size. The information in the electronic format of the Sequence Listing is part of the present application and is incorporated herein by reference in its entirety. 
     BACKGROUND 
     Field of the Invention 
     The present invention relates to methods, devices, and systems for calibrating a temperature of one or more microfluidic channels in a microfluidic device. More particularly, aspects of the present invention relate to methods, devices, and systems for using an amplicon having a known or expected thermal melting temperature to calibrate a thermal sensor and/or thermal control element used to control the temperature of a microfluidic channel in a microfluidic device. The present invention also relates to methods, devices and systems for calibrating the temperature of a thermal melt in a microfluidic channel of a microfluidic device. More particularly, aspects of the present invention relate to methods, devices, and systems for using a calibrant present in a test sample and amplifying the calibrant to produce a calibrant amplicon in which the calibrant amplicon has a known or expected thermal melting temperature to calibrate the melting temperature of an amplicon of a nucleic acid of interest. The present invention also relates to the use of a calibrant present in a test sample as a control for determining whether amplification of a sample has occurred. 
     Description of the Background 
     Devices for performing chemical, biological, or other reactions (e.g., a microfluidic device for performing polymerase chain reaction (PCR) amplification of DNA molecules, or a microfluidic molecular diagnostic platform that performs PCR on a patient sample and then uses the PCR product for genotyping by performing a high resolution melt analysis) often feature one or more thermal control elements that are used to subject reactants to a desired thermal profile. A description of PCR amplification, and an example of one possible microfluidic device including thermal control elements for PCR amplification and thermal melt analysis, are provided in U.S. Patent Application Publication Nos. 2009/0248349 and 2011/0056926, the entire disclosures of which are incorporated herein by reference. 
     In many applications of such microfluidic devices (e.g., PCR and/or thermal melt analysis), the thermal control elements of those devices must be precisely calibrated. That is, the correspondence between the temperature of the thermal control element and an electrical characteristic of the thermal control element must be precisely determined. For example, in the case of a resistance temperature detector and/or heater, the correspondence between temperature and resistance must be precisely determined. Additional types of thermal control elements can include platinum resistive heaters, thermistors, diode temperature sensors, thermocouples, or any other suitable temperature measuring devices. Additional electrical characteristics of thermal control elements that correspond to temperature can include capacitance or inductance of an element, frequency, pulse width, or amplitude of a signal, or other sensor characteristics known in the art. 
     Among many variations that can occur in microfluidic devices, temperature variation between channels is particularly relevant. Securing the right and uniform temperature among channels in a microfluidic device or chip leads to a reliable and consistent PCR and thermal melt analysis. In turn, having a successful amplification and melt of the target sequence may lead to a correct sample genotyping, which may lead to the characterization of a patient with a genetic disorder or a genetic predisposition or the efficiency of drug metabolism. 
     As noted above, assuring that microfluidic channels in a chip present the same temperature during a PCR and thermal melt analysis reaction will result in improved reliability and consistency. However, with such small elements and such small volumes of reagents, it is very challenging to test and prove that channels at the same time are performing a PCR plus thermal melting under the same optimal temperature conditions. 
     Methods of calibrating thermal control elements of a microfluidic device often include generating a lookup table or a series of coefficients that define a calibration equation, i.e., a lookup table or an equation relating the temperature of the thermal control element with the electrical characteristic. 
     Calibration can be performed by sending the device to a third party laboratory for taking accurate measurements and generating the lookup table or series of coefficients; however, this procedure is generally expensive and time consuming. Furthermore, for many devices (e.g., many common microfluidic devices) there may be many thermal control elements (e.g. dozens or even hundreds of heaters and sensors), each of which requires its own precise calibration, making third-party calibration impractical. 
     Accordingly, there is a need in the art for a reliable calibrant that will be useful in calibrating the temperature of one or more microfluidic channels in a microfluidic device or chip. Similarly, there is a need for robust calibration of thermal sensors and/or heaters that can be accurate, reduce downtime and maintain high throughput. 
     SUMMARY 
     In one aspect, the present invention provides a method of calibrating the temperature of a microfluidic channel present in a microfluidic system comprising a controller. The method may comprise: (a) introducing into the microfluidic channel reagents including one or more amplicons; (b) melting the one or more amplicons and determining a melting temperature of each of the one or more amplicons; (c) comparing the determined melting temperature of each of the one or more amplicons with a respective expected melting temperature of each of the one or more amplicons; and (d) adjusting the controller based on the comparison of the determined melting temperature of each of the one or more amplicons with the respective expected melting temperature of each of the one or more amplicons. 
     In some embodiments, one or more of the one or more amplicons are prepared synthetically and then introduced into the microfluidic channel. In some embodiments, the one or more amplicons may be prepared from genomic DNA. In some embodiments, the amplicons may be prepared from Ultra Conserved Elements (UCEs). In other embodiments, the amplicons may be prepared from any coding sequences, such as sickle cell coding sequence. In other embodiments, the one or more amplicons may be prepared from any synthetic DNA. In additional embodiments, the one or more amplicons may be prepared from both genomic DNA or synthetic DNA. In some embodiments, one of the one or more amplicons may be prepared from RFCAL-100. In other embodiments, one of the one or more amplicons may be prepared from RFCAL-200. In additional embodiments, the amplicons may be prepared from both RFCAL-100 and RFCAL-200. The controller may control and/or detect the temperature of the microfluidic channel. 
     In one embodiment, the microfluidic channel reagents may include at least two amplicons; the melting and determining may comprise melting the two or more amplicons and determining a melting temperature of each of the two more amplicons; the comparing may comprise comparing the determined melting temperature of each of the two or more amplicons with a respective expected melting temperature of each of the two or more amplicons; and the adjusting may comprise adjusting the controller based on the comparison of the determined melting temperature of each of the two or more amplicons with the respective expected melting temperature of each of the two or more amplicons. The two or more amplicons may have known melting temperatures more than 5 degrees Celsius apart. The two or more amplicons may have known melting temperatures more than 10 degrees Celsius apart. The two or more amplicons may have known melting temperatures more than 15 degrees Celsius apart. The two or more amplicons have known melting temperatures more than 20 degrees Celsius apart. 
     In some embodiments, the determining the melting temperature of each of the two or more amplicons may comprise: obtaining a derivative of a melt curve of the melting of the two or more amplicons using a Savitzky-Golay filter, and finding the temperatures at the maximum negative derivatives of the derivative of the melt curve. The determining the melting temperature of each of the two more amplicons may comprise: cross-correlating peaks of a derivative of a reference melt curve against peaks of a derivative of a melt curve of the melting of the two or more amplicons, and selecting the temperatures having the highest correlation as the melting temperatures of the two more amplicons. The comparing the determined melting temperature of each of the two or more amplicons with the respective expected melting temperature of each of the two or more amplicons may comprise calculating a slope correction factor and an intercept correction factor using the respective expected melting temperature of each of the two or more amplicons. The adjusting the controller may comprise calculating an adjusted temperature using the calculated slope correction factor and the calculated intercept correction factor. 
     In one embodiment, a derivative of a melt curve of the melting of the one or more amplicons may exhibit a single peak, for instance where only one amplicon is used. The melting of the one or more amplicons may comprise a first melting of the one or more amplicons and a second melting of the one or more amplicons. The determining the melting temperature of each of the one or more amplicons may comprise (i) determining a first temperature corresponding to a peak of the derivative of the melt curve of the first melting of the one or more amplicons and (ii) determining a second temperature corresponding to a peak of the derivative of the melt curve of the second melting of the one or more amplicons. The adjusting the controller may comprise calculating a temperature adjustment using a linear interpolation of the first and second temperatures. 
     In another aspect, the present invention provides a method of calibrating the temperature of a thermal melt in a microfluidic channel present in a microfluidic system. The method may comprise: (a) introducing into the microfluidic channel reagents comprising a test sample containing human genomic DNA, at least a pair of primers for one or more nucleic acids of interest and a pair of primers for a calibrant, wherein the calibrant is found in genomic DNA; (b) amplifying the genomic DNA to produce a calibrant amplicon and an amplicon of the at least one nucleic acid of interest; (c) melting the amplicons and determining a melting temperature of each of the amplicons; (d) comparing the determined melting temperature of the calibrant amplicon with an expected melting temperature of the calibrant amplicon; and (e) calibrating the melting temperature of the amplicon of the at least one nucleic acid of interest based on the melting temperature of the calibrant amplicon. 
     It is within the scope of the invention that segments of fluid may be added to the microfluidic channel sequentially. Each segment of fluid may be of different composition than a segments directly adjacent to it. In one embodiment, the pair of primers for the calibrant may be added to the microfluidic channel in a separate fluid segment (“calibrant fluid segment”) than the at least one pair of primers for the one or more nucleic acids of interest (“assay fluid segment”). In some embodiments, one or more calibrant fluid segments may be added to the microfluidic channel prior to multiple assay fluid segments being added to the microfluidic channel. In other embodiments, one or more calibrant fluid segments may be added to the microfluidic channel before multiple assay fluid segments are added to the microfluidic channel, and one or more calibrant fluid segments may be added after the assay fluid segments. In other embodiments, calibrant fluid segments may alternate with assay fluid segments such that each assay fluid segment has a calibrant fluid segment adjacent on either side. It is therefore noted that throughout the specification, the introduction of at least one pair of primers for the one or more nucleic acids of interest and the introduction of the pair of primers for the calibrant into the microfluidic channel can occur in the same or in separate fluid segments, wherein the separate fluid segments may be introduced in any order, alternately, sequentially, or at the start and end of multiple fluid segments. 
     In some embodiments, the microfluidic channel reagents include a pair of primers for one nucleic acid of interest. As used herein, nucleic acid of interest refers to a nucleic acid whose presence or absence is to be determined in a test sample. In other embodiments, the microfluidic channel reagents include a pair of primers for each of two or more nucleic acids of interest. In some embodiments, the calibrant found in genomic DNA is a DNA segment that is known to be present in all genomic DNA of interest, such as human genomic DNA. In other embodiments, the calibrant is a UCE. In some embodiments, the microfluidic channel reagents may contain a pair of primers for the calibrant. In certain embodiments, the amplification of the microfluidic reagents produce a calibrant amplicon. In certain embodiments, the amplification of the calibrant dos not affect amplification of the one or more nuclei acids of interest. 
     In some embodiments, the determining the melting temperature of each of the amplicons may comprise: obtaining a derivative of a melt curve of the melting of the amplicons using a Savitzky-Golay filter, and finding the temperatures at the maximum negative derivatives of the derivative of the melt curve. The determining the melting temperature of each of the amplicons may comprise: cross-correlating peaks of a derivative of a reference melt curve against peaks of a derivative of a melt curve of the melting of the amplicons, and selecting the temperatures having the highest correlation as the melting temperatures of the amplicons. The comparing the determined melting temperature of the calibrant amplicon with the respective expected melting temperature of the calibrant amplicon may comprise calculating a slope correction factor and an intercept correction factor using the respective expected melting temperature of the calibrant amplicon. Calibrating the melting temperature of each of the amplicons of the nucleic acids of interest may comprise calculating an adjusted temperature using the calculated slope correction factor and the calculated intercept correction factor of the calibrant amplicon. 
     The method may comprise using the comparison of the determined melting temperature of the calibrant amplicon with the respective expected melting temperature of the calibrant amplicon to validate amplification of the one or more amplicons of the one or more nucleic acids of interest. The validation may comprise determining the corrected melting temperatures of the amplicons of the nucleic acids of interest. 
     The method may also comprise using the melt curve of the calibrant amplicon as a control to validate amplification has occurred. Obtaining a melt curve for the calibrant can serve as a positive control to demonstrate that no contamination of the reagents or other anomalies have occurred that would prevent amplification of the genomic DNA. 
     In another aspect, the present invention provides a method of calibrating the temperature of a microfluidic channel. The method may comprise: performing in the microfluidic channel a thermal melt analysis of at least one amplicon having a known thermal melting temperature and adjusting a thermal control element based on a deviation of the thermal melting temperature from the known value. 
     In still another aspect, the present invention may provide a calibrant comprising RFCAL-100. 
     In yet another aspect, the present invention may provide a calibrant comprising RFCAL-200. The calibrant may further comprise RFCAL-100. 
     In another aspect, the present invention provides a calibrant (a) which is present in human genomic DNA, (b) amplification of which produces an amplicon having a known melting temperature that does not interfere with the amplification of one or more nucleic acids of interest that may be present in the human genomic DNA. 
     In one aspect of the invention, a system and a method for a positive control of a biological reaction are provided. Specifically, the positive control may be based on the usage of ultra-conserved elements (UCE) in the biological reaction. A device is configured to receive a biological sample to perform a biological reaction. A control sample and a test sample, each of which comprises reagents for the biological reaction, are provided. Both the control and the test sample comprise biological material from the sample to be tested. The control sample will contain reagents specific to the UCE, such as primers. The control and the test samples are loaded into the desired device and the biological reaction is run on the test sample and on the control sample. Furthermore, a controller is configured to analyze a reaction result for the test sample using the control sample as a positive control. Specifically, if the control sample produces an expected result the biological reaction on the test sample is determined to have been successful. If the control sample produces an unexpected result, the biological reaction on the test sample is determined to have been unsuccessful. 
     In one embodiment, the biological reaction is performed separately on each of the test and control samples. In yet another embodiment, the control sample is combined with the test sample and a single biological reaction is run on the sample including both the test and control material. In one embodiment, the biological reaction may be any amplification, thermal melt, or other nucleic acid processing reaction. In the case of a thermal melt or nucleic acid processing reaction, the control sample comprising UCE is used to validate the test sample, which may include the products of a previous amplification of the test nucleic acids. In the case of an amplification reaction, the test sample may comprise test sample and test primers while the control sample may comprise test sample (necessarily containing the UCE) and UCE primers. An expected result from the control sample resulting in UCE amplification indicates a successful sample preparation and subsequent amplification of the test sample. 
     In another embodiment, reaction components specific to UCEs may be used as a positive control in combination with any biological reaction on any biological sample. An expected result from the positive control (i.e., the UCE) will confirm that the reaction components and equipment are functioning properly, regardless of the outcome of the test sample. For instance, such an embodiment would be useful when testing for the presence of a particular organism in a sample. Running a control using UCE specific reagents will allow for a determination of whether the test was successful, regardless of whether the organism of interest was present in the test sample. 
     The above and other aspects and features of the present invention, as well as the structure and application of various embodiments of the present invention, are described below with reference to the accompanying drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The accompanying drawings, which are incorporated herein and form part of the specification, illustrate various embodiments of the present invention. In the drawings, like reference numbers indicate identical or functionally similar elements. Additionally, the left-most digit(s) of the reference number identifies the drawing in which the reference number first appears. 
         FIG. 1  illustrates a microfluidic device embodying aspects of the present invention. 
         FIG. 2  is a functional block diagram of a system for using a microfluidic device embodying aspects of the present invention. 
         FIG. 3A  is a flow chart illustrating a process for calibrating the temperature of a microfluidic channel of a microfluidic device in accordance with an embodiment of the present invention. 
         FIG. 3B  is a flow chart illustrating a process for calibrating the temperature of a thermal melt in a microfluidic channel present in a microfluidic system in accordance with an embodiment of the present invention. 
         FIG. 4  illustrates thermal melt curves of an exemplary Ultra Conserved Element (UCE) calibrant in accordance with an embodiment of the invention on a microfluidic instrument showing three different genomic DNA. 
         FIG. 5  illustrates thermal melt curves of an exemplary UCE calibrant in accordance with an embodiment of the invention on a Roche LightCycler® 480 (LC480) cycler platform showing seven different genomic DNA. 
         FIG. 6  illustrates thermal melt curves showing three different genotypes of a primer of study. 
         FIG. 7  illustrates thermal melt curves showing UCE products in accordance with an embodiment of the invention. 
         FIG. 8  illustrates a thermal melt curve showing three different genotypes. 
         FIG. 9  illustrates a thermal melt curve showing a UCE amplification product in accordance with an embodiment of the invention. 
         FIG. 10  illustrates a thermal melt curve showing an amplification product of a UCE primer and a primer of study in accordance with an embodiment of the invention. 
         FIG. 11  illustrates a thermal melt curve showing the amplification product of the primer of study only. 
         FIG. 12  illustrates a diagram depicting a pGOv4 vector map in relation to a synthetic sequence in accordance with an embodiment of the invention. 
         FIG. 13  illustrates a graph depicting the calibrators&#39; product amplification on a LC480 cycler platform in accordance with an embodiment of the invention. 
         FIG. 14  illustrates a graph depicting the calibrators&#39; product melting on a LC480 cycler platform in accordance with an embodiment of the invention. 
         FIG. 15  illustrates a graph depicting the results of RFCAL-100 on a microfluidics-based analysis platform in accordance with an embodiment of the invention. 
         FIG. 16  illustrates a graph depicting the results of RFCAL-200 on a microfluidics-based analysis platform in accordance with an embodiment of the invention. 
         FIG. 17  illustrates a graph depicting the RFCAL-melt profile on Idaho Technologies HR-1 high resolution melter in accordance with an embodiment of the invention. 
         FIG. 18A  illustrates fluorescence versus temperature melt curves for Warfarin CyP2C9*2. 
         FIG. 18B  illustrates derivative of fluorescence with respect to temperature melt curves for Warfarin CyP2C9*2. 
         FIG. 19A  illustrates fluorescence versus temperature melt curves for a calibrant according to an embodiment of the invention on a well plate based system, and  FIG. 19B  illustrates that the derivative of fluorescence with respect to temperature melt curves for the UCE calibrant exhibits a single peak. 
         FIG. 20A  illustrates fluorescence versus temperature melt curves for a calibrant according to an embodiment of the invention on a microfluidic channel based system, and  FIG. 20B  illustrates that the derivative of fluorescence with respect to temperature melt curves for the calibrant exhibits two peaks. 
         FIG. 21A  illustrates calculation of the relative temperature shift of an experimental calibrant relative to reference calibrant from a calibrated instrument in accordance with an embodiment of the invention. 
         FIG. 21B  illustrates calculation of the temperature shift on the experimental calibrant as the shift required to maximize the correlation with the reference calibrant in accordance with an embodiment of the invention. 
         FIG. 22A  is a plot illustrating melting temperatures of assays of the same genotype and interlaced UCEs collected from 7 different channels. 
         FIG. 22B  is a plot illustrating the standard deviation of the assays melted at the same time across the 7 channels in  FIG. 22A . 
         FIG. 22C  is a plot illustrating the changes in the melt temperatures of the assays relative to the interpolated UCE melt temperature using the immediately preceding and immediately following UCE. 
         FIG. 22D  is a plot illustrating the corresponding standard deviation of the changes in the melt temperatures shown in  FIG. 22C . 
         FIG. 22E  is a plot illustrating the changes in the melt temperatures of the assays relative to the interpolated UCE Tm using only the first and last UCE. 
         FIG. 22F  is a plot illustrating the corresponding standard deviation of changes in the melt temperatures of the assays shown in  FIG. 22E . 
         FIG. 23A  is a plot illustrating a reference dual peak thermal melt curve from a calibrated instrument. 
         FIG. 23B  is a plot illustrating an out of calibration dual peak experimental thermal melt curve. 
         FIG. 23C  is a plot illustrating the out of calibration dual peak experimental thermal melt curve shown in  FIG. 23B  lined up with the reference dual peak thermal melt curve shown in  FIG. 23A . 
         FIG. 23D  is a plot illustrating a subsequent melt curve of an independent calibration check melt. 
         FIG. 23E  is a schematic diagram illustrating the functioning of the automatic calibration procedure in accordance with an embodiment of the invention. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       FIG. 1  illustrates a microfluidic device  100  embodying aspects of the present invention. As described herein, the microfluidic device  100  may also be referred to interchangeably as the reaction chip or as the U-chip. In the illustrated embodiment, the microfluidic device  100  includes several microfluidic channels  102  extending across a substrate  101 . Each channel  102  includes one or more inlet ports  103  (the illustrated embodiment shows two inlet ports  103  per channel  102 ) and one or more outlet ports  105  (the illustrated embodiment shows one outlet port  105  per channel  102 ). In exemplary embodiments, each channel may be subdivided into a first portion extending through a PCR thermal zone  104  (as described below) and a second portion extending through a thermal melt zone  106  (as described below). 
     In an embodiment, the microfluidic device  100  further includes thermal control elements in the form of thin film resistive heaters  112  associated with the microfluidic channels  102 . In one non-limiting embodiment, the thin film resistive heaters  112  may be platinum resistive heaters whose resistances are measured in order to control their respective temperatures. In the embodiment illustrated in  FIG. 1 , each heater element  112  comprises two heater sections: a PCR heater  112   a  section in the PCR zone  104 , and a thermal melt heater section  112   b  in the thermal melt zone  106 . 
     In one embodiment, the microfluidic device  100  includes a plurality of heater electrodes  110  connected to the various thin-film heaters  112   a  and  112   b . In non-limiting embodiments, heater electrodes  110  may include PCR section leads  118 , one or more PCR section common lead  116   a , thermal melt section leads  120 , and one or more thermal melt section common lead  116   b . According to one embodiment of the present invention, a separate PCR section lead  118  is connected to each of the thin-film PCR heaters  112   a , and a separate thermal melt section common lead  1120  is connected to each of the thin-film thermal melt heaters  112   b.    
       FIG. 2  illustrates a functional block diagram of a system  200  for using a microfluidic device  100 , in accordance with one embodiment. The DNA sample is input in the microfluidic chip  100  from a preparation stage  202 . As described herein, the preparation stage  202  may also be referred to interchangeably as the pipettor system or as the pre-K stage. The preparation stage  202  may comprise appropriate devices for preparing the sample  204  and for adding one or more reagents  206  to the sample. Once the sample is input into the microfluidic chip  100 , e.g., at an input port  103 , the sample flows through a channel  102  into the PCR zone  104  where PCR takes place. That is, as explained in more detail below, as the sample flows within a channel  102  through the PCR zone  104 , the sample is exposed to the PCR temperature cycle a plurality of times to effect PCR amplification. Next, the sample flows into the thermal melt zone  106  where a high resolution thermal melt process occurs. Flow of sample into the microfluidic chip  100  can be controlled by a flow controller  208 . The flow controller may be part of a control system  250  of the system  200 . The control system  250  may comprise the flow controller  208 , a PCR zone temperature controller  210 , a PCR flow monitor  218 , a thermal melt zone temperature controller  224 , and/or a zone fluorescence measurement system  232 . 
     The temperature in the PCR zone  104  can be controlled by the PCR zone temperature controller  210 . The PCR zone temperature controller  210 , which may be a programmed computer or other microprocessor, sends signals to the heater device  212  (e.g., a PCR heater  112   a ) based on the temperature determined by a temperature sensor  214  (such as, for example, an RTD or thin-film thermistor, or a thin-film thermocouple thermometer). In this way, the temperature of the PCR zone  104  can be maintained at the desired level. According to some embodiments of the present invention, the PCR zone  104  may also be cooled by a cooling device  216  (for example, to quickly bring the channel temperature from 95° C. down to 55° C.), which may also be controlled by the PCR zone temperature controller  210 . In one embodiment, the cooling device  216  could be a peltier device, heat sink or forced convection air cooled device, for example. 
     The flow of sample through the microfluidic channels  102  can be measured by a PCR zone flow monitoring system  218 . In one embodiment, the flow monitoring system can be a fluorescent dye diffusion imaging and tracking system illustrated in U.S. Pat. No. 7,629,124, which is incorporated herein by reference in its entirety. According to one embodiment of the present invention, the channels in the PCR zone can be excited by an excitation device  220  and light fluoresced from the sample can be detected by a detection device  222 . An example of one possible excitation device and detection device forming part of an imaging system is illustrated in U.S. Patent Application Publication No. 2008/0003593 and U.S. Pat. No. 7,629,124, which are incorporated herein by reference in their entirety. 
     The thermal melt zone temperature controller  224 , e.g. a programmed computer or other microprocessor, can be used to control the temperature of the thermal melt zone  106 . As with the PCR zone temperature controller  210 , the thermal melt zone temperature controller  224  sends signals to the heating component  226  (e.g., a thermal melt heater  112   b ) based on the temperature measured by a temperature sensor  228  which can be, for example, an RTD or thin-film thermocouple. Additionally, the thermal melt zone  106  may be independently cooled by cooling device  230 . The fluorescent signature of the sample can be measured by the thermal melt zone fluorescence measurement system  232 . The fluorescence measurement system  232  excites the sample with an excitation device  234 , and the fluorescence of the sample can be detected by a detection device  236 . An example of one possible fluorescence measurement system is illustrated in U.S. Patent Application Publication No. 2008/0003593 and U.S. Pat. No. 7,629,124, which are incorporated herein by reference in their entirety. 
     In accordance with aspects of the present invention, the thin film heaters  112  may function as both heaters and temperature detectors. Thus, in one embodiment of the present invention, the functionality of heating element  212  and  226  and temperature sensors  214  and  228  can be accomplished by the thin film heaters  112 . 
     In one embodiment, the system  200  sends power to the thin-film heaters  112   a  and/or  112   b , thereby causing them to heat up, based on a control signal sent by the PCR zone temperature controller  210  or the thermal melt zone temperature controller  224 . The control signal can be, for example, a pulse width modulation (PWM) control signal. An advantage of using a PWM signal to control the heaters  212  is that with a PWM control signal, the same voltage potential across the heaters may be used for all of the various temperatures required. In another embodiment, the control signal could utilize amplitude modulation or alternating current. It may be advantageous to use a control signal that is amplitude modulated to control the heaters  212  because a continuous modest change in voltage, rather than large voltage steps, avoids slew rate limits and improves settling time. Further discussion of amplitude modulation can be found in U.S. Patent Application Publication No. 2011/0048547, which is incorporated herein by reference in its entirety. In some embodiments, the desired temperature for the heaters is reached by changing the duty cycle of the control signal. For example, in one non-limiting embodiment, the duty cycle of the control signal for achieving 95° C. in a PCR heater might be about 50%, the duty cycle of the control signal for achieving 72° C. in a PCR heater might be about 25%, and the duty cycle of the control signal for achieving 55° C. in a PCR heater might be about 10%. In other embodiments, other duty cycles may be used as would be apparent to persons skilled in the art. 
     The microfluidic device  100  and the system  200  can be used in conjunction with aspects of the present invention. For example, the system  200  may calibrate the thermal control elements  112  of the microfluidic device  100  using an amplicon having a known or expected thermal melting temperature, in accordance with aspects of the invention. However, an amplicon having a known or expected thermal melting temperature may be used to calibrate thermal control elements in microfluidic devices and systems other than the microfluidic device  100  and the system  200  illustrated in the  FIGS. 1 and 2 . 
     In high resolution thermal melting instruments, such as system  200 , temperature precision is of extreme importance. For example, a small bias or shift in temperature may cause a genotype misclassification when analyzing melt curves. Whether analyzing fluorescence versus temperature or derivative of fluorescence with respect to temperature, a homozygous DNA sample containing a single nucleotide polymorphism and a wildtype DNA sample have the same shape and only differ by a small temperature shift. For example,  FIG. 18A  illustrates fluorescence versus temperature melt curves for Warfarin CyP2C9*2, and  FIG. 18B  illustrates the derivative of fluorescence with respect to temperature melt curves for Warfarin CyP2C9*2. As shown in  FIG. 18B , the temperature difference between the two profiles, or average difference in true melting temperature, is only 0.4° C. For such genotypes that exhibit the same shape and only differ by a shift, the melting temperature of the DNA, T m , estimated by the peak temperature of the derivative is often used to classify the genotypes. A small drift or bias in measured temperature may increase the misclassification of these genotypes. If there is a positive temperature shift or drift, a homozygous mutant can be misclassified as a wildtype. Conversely, if there is a negative temperature shift or drift, a wildtype can be misclassified as homozygous mutant. 
     A temperature shift may be due to spatial differences in temperature such as in a well plate (gradient), or a shift in time (drift). In an ideal system, where there are no temperature shifts, or biases in measured temperature in space or time, there would be a much lower variance in melting temperatures for a particular genotype. However, in real instruments, temperature gradients and drift are real phenomena. 
     One aspect of the present invention is the calibration of the temperature of a microfluidic channel (e.g., channel  102 ) of a microfluidic device (e.g., device  100 ). In some embodiments, the calibration may be performed by calibrating one or more thermal control elements (e.g., thin film resistive heaters  112 ) that control and/or detect the temperature of a microfluidic channel. In some embodiments, the temperature of a plurality of microfluidic channels (e.g., channel  102 ) may be calibrated. In some embodiments, the calibration may be performed using a single amplicon having a known or expected thermal melting temperature as a calibrant. The amplicon is introduced into the thermal melting zone of the microfluidic channels and the amplicon is melted. In one embodiment, the amplicon may be derived from genomic DNA. In one particular embodiment, the amplicon may be produced from an Ultra Conserved Element (UCE). In an alternative embodiment, the amplicon may be produced from a synthetic DNA. In a further embodiment, the amplicon may be produced from coding sequences, such as a sickle cell coding sequence. 
     In some embodiments, the calibration may be performed using a two or more amplicons each having a known or expected thermal melting temperature. In particular embodiments, the two or more amplicons may be produced from genomic DNA, synthetic DNA or a combination of one or more amplicon derived from genomic DNA and one or more synthetic amplicons. In addition, one or more of the two or more amplicons may be UCEs. 
       FIG. 3A  illustrates a process  300   a  for calibrating the temperature of a microfluidic channel of a microfluidic device in accordance with an embodiment of the present invention. The process  300   a  may begin at step  302  at which amplicons are introduced into the microfluidic channel. In a preferred embodiment, each one of the one or more amplicons has a known thermal melting temperature. In other words, in a preferred embodiment, the temperature at which each of the one or more amplicons is expected to melt is known. In one embodiment, the one or more amplicons comprise any synthetic sequence. 
     At step  304 , a thermal melt analysis may be performed on the amplification product. In other words, the amplicons may be melted, and the temperature at which the amplicons melt may be measured. In this way, the process  300   a  may determine the temperature at which each one of the one or more amplicons in a microfluidic channel melted. In one embodiment, the thermal melt analysis may be performed by exposing the amplicons to a temperature ramp. The amplicons may be exposed to the temperature ramp (e.g., heating the thermal control element at, for example, 0.5 degrees Celsius per second over a range of temperatures that includes temperatures corresponding to the features of the thermal response profiles of the amplicons) in a thermal melt zone of a microfluidic device (e.g., thermal melt zone  106  of microfluidic device  100 ). During the thermal ramp, a dependent variable (e.g., fluorescence intensity) of the amplicons and the measurement value of the thermal control element is monitored to generate a thermal response profile. The temperature may be measured by a thermal melt zone fluorescence measurement system (e.g., thermal melt zone fluorescence measurement system  232 ). However, any known method and/or device for performing thermal melt analysis may be used. 
     In one embodiment, thermal melt analysis may be an analysis of a dependent variable related to a solution (e.g., reagent solution) that is subject to a thermal variation, i.e., the relationship between a solution&#39;s temperature and the dependent variable. In some embodiments, the thermal response profile may be a melt curve, i.e. the fluorescent melt analysis of a solution to determine the relationship between the amount of fluorescence and the solution&#39;s temperature. In some embodiments, generating such a thermal response profile can include loading a microfluidic chip (e.g., the microfluidic chip  100 ) into a system for controlling reactions in the microfluidic chip (e.g., system  200 ), loading a droplet, plug, slug, or continuous flow of the reagents including the one or more amplicons into the chip (e.g., into a microfluidic channel  102 ), and controlling a thermal control element (e.g., heater  112 ) that is in thermal communication with the amplicons to heat the amplicons while monitoring the temperature of the thermal control element and while monitoring the dependent variable (e.g., fluorescence in the case in which a melt curve is obtained) of the amplification product. 
     In one embodiment, the apparent (i.e., measured) temperatures of each feature (e.g., the apparent melt temperature corresponding to each amplicon) may be identified from a thermal response profile. In some embodiments, the apparent temperatures may be determined from the derivative plot using peak-picking. In other embodiments, the apparent temperatures may be determined by cross-correlating the derivative plot with a known template (i.e., an expected thermal response profile) for each amplicon, or shifting and stretching the thermal response profile to match a predefined template. 
     At step  306 , for each of the one or more amplicons, the measured temperature at which the amplicon melted is compared to the known (i.e., expected) thermal melt temperature for the amplicon. The comparison may be performed by, for example, a digital or analog comparator. The comparator may be part of a control system of a microfluidic system (e.g., control system  250  of system  200 ). However, any known method and/or device for performing the comparison may be used. 
     At step  308 , the temperature of the microfluidic channel is calibrated based on the results of the comparison(s) of the measured melt temperature(s) to the respective known thermal melt temperature(s). The calibration may be performed by adjusting a controller (e.g., control system  250 ) and/or one or more thermal control elements (e.g., heater  112 ) that control and/or detect the temperature of the microfluidic channel (e.g., channel  102 ). 
     In some embodiments, calibration of a thermal control element can include determining the coefficients of a calibration equation, i.e., an equation that models the relationship between the temperature of the thermal control element and a measurement value. In an embodiment, that measurement value may be an electrical characteristic of the thermal control element. For example, a calibration equation for a resistive heater  112  may model the relationship between the temperature of the heater  112  and the resistance of the heater  112 . In this embodiment, the calibration equation may model a linear relationship as shown in Equation 1:
 
 T=k   0   +k   1   R   Equation 1
 
     In this case, T is temperature of the thermal control element, R is the resistance of the thermal control element, and k 0  and k 1  are constants (i.e., the coefficients) to be determined by calibration. Two calibration coefficients can be determined by, for example, measuring the resistance R at two known temperatures (T 1  and T 2 ). In some aspects of the present invention, these measurements can be obtained by heating the thermal control element (e.g., a heater  112 ) in thermal contact with a droplet, plug, slug, or continuous flow of the reagents including one or more ampliocns (e.g., while the reagents are in a microfluidic channel  102  corresponding with the heater  112 ) and measuring the resistance of the thermal control element (e.g., measuring the resistance of the heater  112 ) at temperatures that coincide with two or more features (e.g., melting points) of the thermal response profile of the amplicons. 
     In other embodiments, the calibration equation may model the relation between other electrical factors, such as the current, electric potential, applied power, resistivity, conductivity, or other related quantities. In some aspects, the measurement value may be an independently controlled aspect of the thermal control element that is related to the temperature of the thermal control element. In other aspects, the measurement value could be any factor related to the temperature of the thermal control element. 
     In some embodiments, the calibration equation may contain more coefficients to be determined. For example, in the case where the measurement value is resistance, some calibration equations can model a quadratic relationship as shown in Equation 2:
 
 T=k   0   +k   1   R+k   2   R   2   Equation 2
 
In this case, an amplification product resulting from reagents having three or more features in its thermal response profile may be preferable to more accurately determine all of the coefficients. Further, one of ordinary skill will comprehend that this approach may be expanded for a compound calibrator having n or more features, using Equation 3:
 
 T=k   0   +k   1   R+k   2   R   2   + . . . k   n-1   R   n-1   Equation 3
 
     Furthermore, in some embodiments, more accurate values for the coefficients may be obtained by utilizing reagents having more features than there are coefficients to be determined (i.e., determine more correspondences between temperature and resistance of the thermal control element than there are coefficients). The resulting over-determined system can be solved, for example, using the least squares method. 
     In some embodiments, the thermal control element can be calibrated using an ambient temperature (i.e., room temperature) in addition to one or more temperatures determined based on features (e.g., melting points) of thermal response profiles. For example, calibration of the temperature of the microfluidic channel may include determining the thermal control element&#39;s output (e.g., the resistance of the heater  112 ) while the thermal control element is at a known, ambient temperature. In some embodiments, the ambient temperature can be measured with a separate temperature measurement device such as, for example, a precision RTD or thermocouple. However, any other suitable temperature measurement device including non-contact methods are appropriate as will be understood by those skilled in the art. 
     In other embodiments, the one or more amplicons used to calibrate temperature in a microfluidic channel of a microfluidic device may be synthetic amplicons, i.e. amplicons produced from synthetic sequences. In a preferred embodiment, the amplicon(s) derived from synthetic sequences will not interfere with clinical samples and may be produced from a vector. In a preferred embodiment, the one or more calibrants are designed from a synthetic sequence will not interfere with clinical samples and may easily be produced from a vector. In some embodiments, the one or more sequences are chosen such that they are not present in human genomic DNA. In other embodiments, the one or more sequences are chosen such that they are not present in mammalian genomic DNA. In additional embodiments, the one or more sequences are chosen such that they are not present in vertebrate DNA. In one embodiment, one of the one or more amplicons is named RFCAL-100. In another embodiment, one of the one or more amplicons is named RFCAL-200. In certain embodiments, the one or more amplicons are cloned in a vector. Any suitable vector well known to the skilled artisan may be used, such as a plasmid, a virus, and the like, into which a synthetic sequence has been inserted. 
     In some embodiments of the present invention, one or more of the one or more amplicons used to calibrate temperature in a microfluidic channel of a microfluidic device may be produced from UCEs. UCEs are involved in transcription regulation and in early development, and scientists believe UCEs were functional introns common to all life forms in the evolutionary time. In the human genome, there are a large number of UCEs, which may be sequences longer than 200 base pairs (bp). UCEs are also conserved among vertebrates&#39; genomes as well. UCEs are present in all human chromosomes, except for Y and 21 chromosomes. UCEs have a very low mutation rate and have not changed at all during the last 300 millions years or more. 
     Aspects of the present invention can be used to calibrate resistive sensors, thermistors, diode temperature sensors, thermocouples, or any other suitable temperature measuring devices. The present invention can further be used to calibrate resistive sensors that are also used for heating, such as thin-film platinum elements (or nickel or copper or any other material as would be understood by those skilled in the art). 
     Another aspect of the present invention is, for temperature shifts in space and/or time that follow an observable pattern or trend, the application of a temperature correction to regenerate and/or replot temperature corrected melt curves and decrease the variance in melting temperatures. In other words, aspects of the present invention may recalibrate the temperature profile such that the measured temperatures are closer to their true values. Accordingly, aspects of the present invention may be used to decrease the misclassification rate when genotyping. In some embodiments, neighboring positive control DNA samples (in space or time) of known melting temperatures may be used to calculate and apply a temperature correction to DNA melt curves of unknown genotypes. In some embodiments, positive control samples (i.e., calibrants or amplicons having a known or expected melt temperature) may be melted before and/or after and/or simultaneously with unknown samples, and the derivative of their melt curves may exhibit one or two or more peaks. 
     For example,  FIG. 19A  illustrates fluorescence versus temperature melt curves for a calibrant (e.g., a 207 bp UCE sequence described in more detail below) according to an embodiment of the invention on a well plate based system, and  FIG. 19B  illustrates that the derivative of fluorescence with respect to temperature melt curves for the UCE calibrant exhibits a single peak. For another example,  FIG. 20A  illustrates fluorescence versus temperature melt curves for a calibrant (e.g., a synthetic calibrant comprising RFCAL-100 and RFCAL-200 described in more detail below) according to another embodiment of the invention on a microfluidic channel based system, and  FIG. 20B  illustrates that the derivative of fluorescence with respect to temperature melt curves for the calibrant exhibits two peaks (i.e., one peak around 70° C. and a second peak around 90° C.). Aspects of the present invention may use a calibrant producing a melt curve having a derivate that exhibits one or two peaks, such as those shown in  FIGS. 19B and 20B , to calibrate and apply a temperature correction to DNA melt curves of unknown genotypes. 
     One aspect of the present invention is a method of calibrating the temperature of a thermal melt in a microfluidic channel present in a microfluidic system. The method may comprise: (a) introducing into the microfluidic channel reagents comprising a test sample containing human genomic DNA, at least a pair of primers for one or more nucleic acids of interest and a pair of primers for a calibrant, wherein the calibrant is found in genomic DNA; (b) amplifying the genomic DNA to produce a calibrant amplicon and an amplicon of the at least one nucleic acid of interest; (c) melting the amplicons and determining a melting temperature of each of the amplicons; (d) comparing the determined melting temperature of the calibrant amplicon with an expected melting temperature of the calibrant amplicon; and (e) calibrating the melting temperature of the amplicon of the at least one nucleic acid of interest based on the melting temperature of the calibrant amplicon. 
       FIG. 3B  illustrates a process  300   b  for calibrating the temperature of a thermal melt in a microfluidic channel of a microfluidic device in accordance with an embodiment of the present invention. The process  300  may begin at step  312  at which reagents are introduced into the microfluidic channel. The reagents may include a test sample, a pair of primers for a nucleic acid of interest and/or a pair of primers for a calibrant. In some embodiments, the reagents may also include one or more additional pairs of primers for one or more additional nucleic acids of interest. In one embodiment, the test sample may contain human genomic DNA. However, the test sample may contain other types of DNA in addition to or as an alternative to human genomic DNA. In some embodiments, the calibrant is found in genomic DNA, such as, for example, human genomic DNA. In one non-limiting embodiment, the calibrant is a UCE. However, in other embodiments, the calibrant may be a synthetic calibrant. In some embodiments, such as embodiments where the calibrant is a UCE, the calibrant may be included in the test sample. In other embodiments, such as embodiments where the calibrant is synthetic, the reagents additionally include the calibrant. In some embodiments, the reagents may include one or more additional pairs of primers for one or more additional calibrants, which may also be included in the reagents. In a preferred embodiment, each one of the one or more calibrants has a known thermal melting temperature. In other words, in a preferred embodiment, the temperature at which each of the one or more amplicons is expected to melt is known. Step  312  may include the reagents being introduced in separate fluid segments, such that the primers for the nucleic acids of interest may be in a separate fluid segment than the primers for the calibrant. A fluid segment containing the primers for the calibrant may be introduced before and after multiple fluid segments containing primers for the nucleic acid of interest, or the fluid segment containing the primers for the calibrant may be alternately introduced with fluid segments containing the primers for the nucleic acids of interest, such that a fluid segment containing the primers for the calibrant may be present before and after each fluid segment containing primers for the nucleic acids of interest. 
     At step  314 , the reagents, which may include the test sample, at least a pair of primers for one or more nucleic acids of interest and at least of pair of primers for one or more calibrants are amplified. Amplification of the reagents in step  314  may produce an amplification product comprising one or more calibrant amplicons and an amplicon of the one or more nucleic acids of interest. In one embodiment, the amplification may be performed by exposing the reagents to a PCR temperature cycle a plurality of times to effect PCR amplification. The reagents may be exposed to the PCR temperature cycle in a PCR zone of a microfluidic device (e.g., PCR zone  104  of microfluidic device  100 ). However, any known method and/or device for amplication of reagents may be used. 
     At step  316 , a thermal melt analysis may be performed on the amplification product. In other words, the one or more calibrant amplicons and an amplicon of the one or more nucleic acids of interest may be melted, and the temperature at which the amplicons melt may be measured. In this way, the process  300   b  may determine the temperature at which each of the amplicons in the amplification product in a microfluidic channel melted. In one embodiment, the thermal melt analysis may be performed by exposing the amplicons to a temperature ramp. The amplicons may be exposed to the temperature ramp (e.g., heating the thermal control element at, for example, 0.5 degrees Celsius per second over a range of temperatures that includes temperatures corresponding to the features of the thermal response profiles of the amplicons) in a thermal melt zone of a microfluidic device (e.g., thermal melt zone  106  of microfluidic device  100 ). During the thermal ramp, a dependent variable (e.g., fluorescence intensity) of the amplicons and the measurement value of the thermal control element is monitored to generate a thermal response profile. The temperature may be measured by a thermal melt zone fluorescence measurement system (e.g., thermal melt zone fluorescence measurement system  232 ). However, any known method and/or device for performing thermal melt analysis may be used. 
     In one embodiment, thermal melt analysis may be an analysis of a dependent variable related to a solution (e.g., reagent solution) that is subject to a thermal variation, i.e., the relationship between a solution&#39;s temperature and the dependent variable. In some embodiments, the thermal response profile may be a melt curve, i.e. the fluorescent melt analysis of a solution to determine the relationship between the amount of fluorescence and the solution&#39;s temperature. In some embodiments, generating such a thermal response profile can include loading a microfluidic chip (e.g., the microfluidic chip  100 ) into a system for controlling reactions in the microfluidic chip (e.g., system  200 ), loading a droplet, plug, slug, or continuous flow of the reagents including the one or more amplicons into the chip (e.g., into a microfluidic channel  102 ), and controlling a thermal control element (e.g., heater  112 ) that is in thermal communication with the amplicons to heat the amplicons while monitoring the temperature of the thermal control element and while monitoring the dependent variable (e.g., fluorescence in the case in which a melt curve is obtained) of the amplification product. 
     In one embodiment, the apparent (i.e., measured) temperatures of each feature (e.g., the apparent melt temperature corresponding to each of the one or more calibrant amplicons and/or each of the amplicon of the one or more nucleic acids of interest) may be identified from a thermal response profile. In some embodiments, the apparent temperatures may be determined from the derivative plot using peak-picking. In other embodiments, the apparent temperatures may be determined by cross-correlating the derivative plot with a known template (i.e., an expected thermal response profile) for each amplicon, or shifting and stretching the thermal response profile to match a predefined template. 
     At step  318 , for each of the one or more calibrant amplicons, the measured temperature at which the calibrant amplicon melted is compared to the known (i.e., expected) thermal melt temperature for the calibrant amplicon. The comparison may be performed by, for example, a digital or analog comparator. The comparator may be part of a control system of a microfluidic system (e.g., control system  250  of system  200 ). However, any known method and/or device for performing the comparison may be used. 
     At step  320 , the melting temperature(s) of the amplicon of the one or more nucleic acids of interest of the microfluidic channel is calibrated based on the measured melt temperature(s) of the one or more calibrant amplicons. For example, the melting temperature(s) of the amplicon of the one or more nucleic acids of interest of the microfluidic channel is calibrated based on the results of the comparison(s) of the measured melt temperature(s) of the one or more calibrant amplicons to the respective known thermal melt temperature(s). In one non-limiting embodiment, the calibration is performed by shifting the melt temperature(s) of the amplicon of the one or more nucleic acids of interest in accordance with the results of the comparison(s) of the measured melt temperature(s) of the one or more calibrant amplicons to the respective known thermal melt temperature(s). 
     As noted above, in some embodiments, the microfluidic channel reagents include a pair of primers for one nucleic acid of interest. As used herein, nucleic acid of interest refers to a nucleic acid whose presence or absence is to be determined in a test sample. In other embodiments, the microfluidic channel reagents include a pair of primers for each of two or more nucleic acids of interest. In some embodiments, the calibrant found in genomic DNA is a DNA segment that is known to be present in all genomic DNA of interest, such as human genomic DNA. In other embodiments, the calibrant is a UCE. In some embodiments, the microfluidic channel reagents may contain a pair of primers for the calibrant. In certain embodiments, the amplification of the microfluidic reagents produce a calibrant amplicon. In certain embodiments, the amplification of the calibrant does not affect amplification of the one or more nucleic acids of interest. 
     In some embodiments, the determining the melting temperature of each of the amplicons may comprise: obtaining a derivative of a melt curve of the melting of the amplicons using a Savitzky-Golay filter, and finding the temperatures at the maximum negative derivatives of the derivative of the melt curve. The determining the melting temperature of each of the amplicons may comprise: cross-correlating peaks of a derivative of a reference melt curve against peaks of a derivative of a melt curve of the melting of the amplicons, and selecting the temperatures having the highest correlation as the melting temperatures of the amplicons. The comparing the determined melting temperature of the calibrant amplicon with the respective expected melting temperature of the calibrant amplicon may comprise calculating a slope correction factor and an intercept correction factor using the respective expected melting temperature of the calibrant amplicon. Calibrating the melting temperature of each of the amplicons of the nucleic acids of interest may comprise calculating an adjusted temperature using the calculated slope correction factor and the calculated intercept correction factor of the calibrant amplicon. 
     The method may comprise using the comparison of the determined melting temperature of the calibrant amplicon with the respective expected melting temperature of the calibrant amplicon to validate amplification of the one or more amplicons of the one or more nucleic acids of interest. The validation may comprise determining the corrected melting temperatures of the amplicons of the nucleic acids of interest. 
     In one embodiment, thermal melt analysis may be an analysis of a dependent variable related to a solution (e.g., reagent solution) that is subject to a thermal variation, i.e., the relationship between a solution&#39;s temperature and the dependent variable. In some embodiments, the thermal response profile may be a melt curve, i.e. the fluorescent melt analysis of a solution to determine the relationship between the amount of fluorescence and the solution&#39;s temperature. In some embodiments, generating such a thermal response profile can include loading a microfluidic chip (e.g., the microfluidic chip  100 ) into a system for controlling reactions in the microfluidic chip (e.g., system  200 ), loading a droplet, plug, slug, or continuous flow of the reagents including test sample and primers into the chip (e.g., into a microfluidic channel  102 ) amplifying the calibrant and nucleic acid of interest, and controlling a thermal control element (e.g., heater  112 ) that is in thermal communication with the amplification product to heat the amplification product while monitoring the temperature of the thermal control element and while monitoring the dependent variable (e.g., fluorescence in the case in which a melt curve is obtained) of the amplification product. 
     In one embodiment, the apparent (i.e., measured) temperatures of each feature (e.g., the apparent melt temperature corresponding to each amplicon) may be identified from a thermal response profile. In some embodiments, the apparent temperatures may be determined from the derivative plot using peak-picking. In other embodiments, the apparent temperatures may be determined by cross-correlating the derivative plot with a known template (i.e., an expected thermal response profile) for each calibrant, or shifting and stretching the thermal response profile to match a predefined template. 
     In some embodiments of the present invention, one or more of the one or more calibrants used to calibrate temperature of a thermal melt in a microfluidic channel of a microfluidic device may be UCEs. Because UCEs are present in all genomic DNA, every time that an amplification is done using a genomic DNA, one or more specific Ultra Conserved Element (UCE) DNA sequences may also be amplified. 
     In some embodiments of the present invention, a pair of primers may be designed to amplify a specific UCE sequence that is used as a calibrant. Using an amplification process (e.g., step ***), such as PCR, the pair of primers may be used to make the same UCE amplicon over and over. The UCE amplicon is will always shows the same melt temperature (T M ). Based on the characteristic of the UCE amplicons always showing the same T M , the UCE smplicon can be used, for example, for calibrating melting temperature in a microfluidic system having one or more microfluidic channels. 
     Accordingly, in one aspect, the present invention provides methods, devices, and systems for using a pair of primers for amplifying a calibrant found in human genomic DNA in PCR and thermal melt analysis devices such that the calibrant amplicon is used to calibrate the melting temperature of an amplicon of a nucleic acid of interest. In one embodiment, the UCE primers are used at the same time with at least a pair of primers for one or more nucleic acids of interest, during PCR amplification and melting, the melt temperature (T M ) of an amplicon generated from a nucleic acid of interest is correct. This may be accomplished because the UCE primer product may have a known T M . 
     Embodiments of the present invention can be used in a variety of instruments but are particularly useful in PCR and thermal melt systems that perform in vitro diagnostics. Embodiments of the present invention may be used to calibrate sensors and heaters that are intended for thermal melt of samples (diagnostics) as well as other heaters and sensors within the instrument that perform entirely different functions (e.g., sample prep or PCR). 
     In another aspect, the present invention provides a multiple amplicon calibrator system. In other words, reagents containing two or more amplicons may be used to calibrate the temperature of a microfluidic channel. For instance, a dual amplicon calibrator may be used to generate accurate high resolution melting on certain microfluidic chip based systems. In a non-limiting embodiment, one or more of the multiple amplicons in the multiple amplicon calibrator system are Ultra Conserved Elements (UCEs). In another non-limiting embodiment, one or more of the multiple amplicons in the multiple amplicon calibrator system are synthetic. In another non-limiting embodiment, one or more of the multiple amplicons are UCEs, and one or more of the multiple amplicons are synthetic. 
     As described above, one embodiment of the present invention may utilize two amplicons. In one non-limiting example, one amplicon may be from a desired DNA sequence, and the other amplicon may be a UCE amplicon. In one embodiment, the DNA sequence may be derived from the sequence of any human gene or other sequence. One example of a gene that could be used is a sickle cell gene. One example of a sequence for the sickle cell gene is a 444 bp fragment. In another embodiment, the UCE amplicon is a 415 bp UCE amplicon. The melt temperature Tm for the sickle sequence is approximately 84° C., and the melt temperature Tm for the UCE sequence is 74° C. In this non-limiting example, both amplicons were derived by amplifying human genomic DNA. However, it is not necessary that both amplicons in a dual amplicon calibrator system be derived by amplifying human genomic DNA. 
     In certain embodiments, certain limitations may exist with the dual calibrant system using the amplicon derived from the 444 bp fragment of the sickle gene and the 415 bp fragment of the UCE described above. First, the span covered by the two calibrators may not cover the full range of anticipated melt temperatures. Second, the amplicons were derived from genomic DNA and thus potentially could serve as a source of contamination for subsequent amplification reactions. Finally, manufacture of these calibrators may require amplification from human DNA, which was obtained from DNA banks, such as the Coriell DNA banks. Thus, these calibrators may not represent material that would be easy to manufacture or guarantee as a source. 
     Another aspect of the present invention is a dual amplicon calibrator system with a wide temperature span and two or more amplicons derived from synthetic sequences. In a preferred embodiment, the amplicons are designed from synthetic sequences so that they may be readily produced. In some embodiments, the sequences are chosen such that they are not present in human genomic DNA. In other embodiments, the sequences are chosen such that they are not present in mammalian genomic DNA. In additional embodiments, such sequences are chosen such that they are not present in vertebrate DNA. In some embodiments, the sequences are chosen to produce amplicons with a T M  span greater than 5 degrees Celsius, preferably greater than 10 degrees Celsius, and more preferably greater than 20 degrees Celsius. In one embodiment, a first amplicon is named RFCAL-100. In another embodiment, a second amplicon is named RFCAL-200. In certain embodiments, the two or more amplicons are cloned in a vector. Any suitable vector well known to the skilled artisan may be used, such as a virus, and the like. 
     Non-Limiting Calibrant Examples 
     Genomic UCE Sequence 
     In a non-limiting example of a calibrant that may be used to calibrate the temperature of a microchannel of a microfluidic device is a UCE sequence with 207 base pairs. In development, the 207 bp UCE sequence was found using the Bioscience World web site www.bioscienceworld.ca. The sequence was then checked against the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) database. The 207 bp sequence (SEQ ID NO:1) was shown to be part of the human chromosome 17 sequence (GenBank accession number AF287967.1). Using Primer 3 software, a set of two primers was designed (SEQ ID NO:2 and SEQ ID NO:3) which amplified a 71 bp product (SEQ ID NO:4). 
     Experiments using the UCE primers and different genomic DNA samples showed that all samples were amplified, and all samples amplified show the same TMs. This experiment was done several times using the Roche LightCycler® 480 (LC480) cycler platform and a microfluidic instrument. The results of the experiments are shown in  FIGS. 4 and 5 .  FIGS. 4 and 5  illustrate a so-called “derivative plot” which describes the derivative of fluorescence with respect to temperature as a function of temperature (e.g., dF/dT vs. T).  FIG. 4  illustrates thermal melt curves of an amplification product generated using the UCE primers on the microfluidic instrument and shows three different genomic DNA.  FIG. 5  illustrates thermal melt curves of an amplification product generated using the UCE primers on the LC480 cycler platform and shows seven different genomic DNA. The results of the experiments demonstrate that there were not variations on the melt temperatures T M s. Thus, the experiments show that the UCE primers can be used to amplify UCE amplicons that act as temperature calibrators. 
     A second experiment was also performed. In the second experiment, a pair of primers for amplifying a nucleic acid of interest was loaded into the device and then subsequently the UCE primers were loaded into the device. The experiment was performed using a microfluidic instrument, and the results are shown in  FIG. 6  and  FIG. 7 .  FIG. 6  illustrates thermal melt curves showing three different genotypes of the primer of study (i.e., a wildtype genotype (Wt), a homozygous genotype (Hm), and a heterozygous genotype (Ht)).  FIG. 7  illustrates thermal melt curves showing the UCE amplicon of the UCE primers. 
     In the event that the amplicon of the UCE primers show variations in melt temperatures T M s, the amplification product of the primers of study also may also show variation on the melt temperatures T M s. This may indicate an impossible genotype differentiation between DNA samples. An example of results indicating an impossible genotype differentiation between DNA samples are shown in  FIG. 8  and  FIG. 9 .  FIG. 8  illustrates a thermal melt curve showing three different genotypes, i.e. a wildtype genotype (Wt), a homozygous genotype (Hm), and a heterozygous genotype (Ht).  FIG. 9  illustrates a thermal melt curve showing the UCE amplicon of the UCE primers. As illustrated in  FIG. 9 , the melt temperatures T M s of the UCE amplicon of the UCE primers showed variation, and, as illustrated in  FIG. 8 , the melt temperatures of amplicon of the nucleic acid of interest showed the same variation. 
     To demonstrate that the UCE primers would not interfere with the amplification of a nucleic acid of interest and also help as a temperature calibrator, the following experiment was performed. The UCE primers and primers for a nucleic acid of interest were added together with the reaction mix components (i.e., reagents) and loaded into wells of a 96-well plate. PCR amplification and melting analysis were performed using a LC480 cycler platform. The results are shown in  FIGS. 10 and 11 .  FIG. 10  illustrates a thermal melt curve showing the amplicon of the UCE primers added to the primers of study.  FIG. 11  illustrates a thermal melt curve showing the amplicon of the nucleic acid of interest. The results illustrated in  FIGS. 10 and 11  confirm that the UCE primers do not affect the amplification of the nucleic acid of interest. In other words, the same melt temperatures were obtained for the amplicon of the nucleic acid of interest regardless of whether the UCE primers were added with the primers of the nucleic acid of interest. The results also show that the UCE amplicon can be used as temperature calibrator. 
     As shown by the experiments discussed above, in some embodiments, two or more UCE primers may be used to provide a simple, fast and reliable procedure to check temperature inside a microfluidic channel in a microfluidic device or chip. Also, the experiments show that, in some embodiments, the two or more UCE primers may work as temperature calibrator when added following or ahead of the primers of study in a chip. In this case, the UCE amplicons may be used to determine whether the microfluidic channels temperature did or did not reach the ideal temperature set, and, therefore, may be used to validate a PCR run. The experiments further show that the two or more UCE primers works in the same fashion when used in a standard PCR instrument such as a LC480 cycler platform. In some embodiments, the UCE primers may be added with two or more primers of study and may not interfere with the amplification product of the primers of study. 
     In other embodiments, the UCE primers may function as a positive control to validate amplification has occurred. Using positive controls in biological experiments is well known in the art. Positive controls are defined as biological reactions where a specific phenomenon is expected. Specifically, the positive controls ensure that there is an effect when there should be an effect, by using an experimental treatment that is already known to produce that effect. Positive controls are often used to assess a success of a biological reaction. If a positive control does not produce an expected result, there may be something wrong with the experimental procedure, the reagents or the equipment used, and the experiment should be repeated. Accordingly, suitable control samples include samples of a like or similar nature to a test sample but which unlike the test sample have a known or expected characteristic. 
     As UCEs are present in all genomic DNA, every time that an amplification is done using a genomic DNA, the UCE sequence is also amplified. Specifically, UCE primers are used to amplify UCE sequences to make the same product repeatedly. Accordingly, amplification reactions performed on the UCE sequences can be used as a positive control in biological reactions. UCE presence in a biological test sample or in a separate control sample will allow a researcher to determine whether a biological reaction has been successful based upon an outcome of the biological reaction performed on the control sample and/or a UCE related portion of a reaction outcome for the test sample containing the UCE. Specifically, if the control sample containing UCE and UCE primers produces an expected result, the biological reaction on the test sample is determined to have been successful. If the control sample produces an unexpected result, the biological reaction performed on the test sample is determined to have been unsuccessful. 
     In one embodiment of the present invention, a testing device is configured to receive a biological sample and to run a biological reaction on the biological sample. A control sample and a test sample, each comprising reagents for the biological reaction, are provided to be tested in the testing device. The test sample comprising a biological material and the control sample comprising the biological material and reagents specific to one or more UCE are loaded into the testing device and the biological reaction is run separately on the test sample and on the control sample. In yet another embodiment of the invention, the one or more UCE reagents are added to the test sample and the biological reaction is run on the combined sample. 
     The test sample may comprise one or more test samples and test primers while the control sample may comprise test sample and UCE primers. Alternatively, the test sample is combined with the control sample and the biological reaction is performed on the combined sample. Specifically, one or more UCE primers may work as a positive control when added together with the test primers to the test sample. By way of a non-limiting example, the biological reaction may be an amplification reaction and/or a DNA analysis reaction. In this case, the UCE may be used to validate an amplification of the test amplicons. The amplification of UCE indicates a successful sample preparation and subsequent amplification. The one or more UCE primers added to the test sample in addition to the test primers do not interfere with the amplification product of the test primers. Accordingly, two amplifications can be achieved simultaneously in one amplification reaction. In this case, results of the amplification reactions for the test amplicons and for the UCE may be separately monitored and evaluated. An expected amplification result of the UCE indicates a successful sample preparation and subsequent amplification. By way of non-limiting example, the amplification reaction may be a PCR reaction and/or polymerase-endonuclease amplification reaction. The nucleic acid analysis reaction includes, but is not limited to a thermal melt analysis, sequencing, separation methods including slab gel and capillary electrophoresis (CE), fluorescence detection methods, short tandem repeat analysis, and other analysis methodologies known to those of skill in the art. In one embodiment the control sample comprises all of the components of the test sample and UCE specific reagents. 
     In one embodiment of the invention, a controller is configured in communication with the testing device to analyze a reaction result for the test sample using the control sample as a positive control. Specifically, if amplification of UCE produces an expected result, the amplification of the test sample is determined to have been successful. To make a determination with respect to the UCE amplification result, a detection method may be used. In one embodiment, a thermal response profile, such as a melt curve, may be obtained. In such an instance, the UCE would have an expected melting temperature, and a melt curve showing the expected melting temperature would be indicative of a positive result. An expected thermal response profile can serve as a positive control to demonstrate that no contamination of the reagents or other anomalies have occurred that would prevent amplification of the genomic nucleic acid. The control sample and the test sample can undergo the biological reaction in series or in parallel. The device used to run the biological reaction can be a microfluidic device or a non-fluidic device such as a well plate loaded into a PCR machine. 
     In the illustrated embodiment shown in  FIG. 1 , the microfluidic device  100  includes several microfluidic channels  102 . The test sample and the control sample may be loaded into the same channel or into different channels  102  of the microfluidic device  100 . Each channel may be subdivided into a first portion extending through a PCR thermal zone  104  and a second portion extending through a thermal melt zone  106 . In one embodiment, the test sample and the control sample may undergo an amplification reaction separately. In yet another embodiment, the biological sample and UCE primers that would make up a separate control sample may instead be added to the test sample to undergo an amplification reaction and a melt analysis in a single reaction. If the UCE amplification reaction exhibits expected results, the amplification reaction performed on the test sample is determined to be successful, regardless of the actual presence of amplicons in that test sample. In this case, the reaction result may be reported and/or used for further processing. In contrast, if the UCE amplification shows unexpected results, the reaction performed on the test sample is determined to be unsuccessful. In this case, the experimental procedure associated with the PCR and melting reaction may be readjusted and repeated. In yet another embodiment of the present invention, there is provided a positive control formulation comprising one or more primers specific to a UCE. In one non-limiting embodiment, the positive control formulation comprises one or more primers which have one or more of the sequences shown in SEQ ID NOs 2 and 3. 
     Synthetic Calibrator 
     In one non-limiting embodiment of one or more amplicons that may be used to calibrate the temperature of a microchannel of a microfluidic device, one or more of the one more amplicons may be synthetic amplicons. In an exemplary embodiment, the invention provides a dual amplicon calibrator system. The dual amplicon calibrator system may be used to improve calibrator performance and production. In one non-limiting example, the dual amplicon calibrator system may be used in a high resolution DNA melting instrument to improve calibrator performance and production. However, this is not required, and the dual amplicon calibrator system may be used in other systems. In some embodiments, the dual amplicon calibrator system may have individual calibrators designed for the dual system. In one embodiment, testing of the dual amplicon calibrator system was performed on a LC480 cycling platform, and non-limiting examples of possible formulations of the dual calibrator system were developed. 
     In one non-limiting example, two amplicons that may be used calibrate the temperature of a microfluidic channel in a microfluidic device are referred to herein as “sequence A” and “sequence B.” Sequence A is 100 base pairs in length (SEQ ID NO:5), and sequence B is 200 base pairs in length (SEQ ID NO:6). Although a particular example is described, different sequences and sequences having different lengths may also be used in embodiments of the present invention. 
     In development of the non-limiting example of a dual calibrator in accordance with embodiments of the present, synthetic DNA constructs for the two calibration points were used to allow specific targeting of melting temperatures TMs. Two DNA (i.e., sequence A and sequence B) sequences were created to work as a DNA template. The sequences were generated using a web-based program called ‘Random DNA Sequence Generator”. 
     The two sequences were checked for similarity using BLAST program from NCBI, After no similarities were found with any sequence on the database, primers were targeted to the last 20 or 22 base pairs of the calibrator sequences. Primers for Sequence A are those set forth in SEQ ID NO:7 and SEQ ID NO:8. Primers or Sequence B are those set forth in SEQ ID NO:9 and SEQ ID NO:10. To check for primer specificity, the University of California, Santa Cruz, (UCSC) Genome Browser was used. The primers were verified to amplify only sequence A and sequence B. 
     After this preliminary work was done, a sequence was constructed and ordered for both sequences using Gene Oracle clone services. Each construct was separately cloned into a pGOv4 vector. The pGOv4 vector is a pUC based vector which is Ampicillin and Kanamycin resistant. The pGOv4 map is shown in  FIG. 12 . 
     After the sequences were incorporated into a vector, the vectors were RFCAL-100 which contains Sequence A and RFCAL-200 which contains Sequence B. The sequence for RFCAL-100 is set forth in SEQ ID NO:11. The sequence for RFCAL-200 is set forth in SEQ ID NO:12. 
     All primers were ordered high-performance liquid chromatography (HPLC) purified at 200 nM from Invitrogen. However, primers of different purifications and/or from different providers may be used. The primers were then resuspended in PCR clean water at 100 uM. Genomic DNA was used as a control DNA. For example, VKORC1 wild type from Paragon Dx may be used as a control DNA. Tables below provide additional details regarding the reagents mixes and reagents catalog numbers. 
     
       
         
           
               
             
               
                 TABLE 01 
               
             
            
               
                   
               
               
                 PCR reagents 
               
            
           
           
               
               
               
               
            
               
                   
                 Reagent 
                 Manufacture 
                 Catalog # 
               
               
                   
                   
               
               
                   
                 Taq polymerase 
                 Clontech Takara 
                 RR006L 
               
               
                   
                 dNTPs 
                 Fisher Scientific 
                 AB-1124 
               
               
                   
                 PCR water 
                 Mo Bio 
                 17000-10 
               
               
                   
                 MgCl2 
                 Ambion 
                 AM9530G 
               
               
                   
                 LC Green Plus 
                 Idaho Technology 
                 BCHM-ASY-000 
               
               
                   
                 2× CULS Buffer 
                 CULS 
                 CULS0017 
               
               
                   
                 Genomic DNA control 
                 Paragon Dx 
                 004-GGCCGG 
               
               
                   
                   
               
            
           
         
       
     
     The reagents described above were used in the following concentrations depicted on tables below to create a DNA Reagent Mix, a Taq Polymerase Reagent Mix and a Primer Reagent Mix. 
     
       
         
           
               
             
               
                 TABLE 02 
               
             
            
               
                   
               
               
                 DNA mix 
               
               
                 DNA Reagent Mix 
               
            
           
           
               
               
               
               
            
               
                   
                 Volume 1 Rx 
                 Channel conc. 
                 Stock conc. 
               
               
                   
               
               
                 DNA 
                  1.8 uL 
                 1 cp/nL 
                 50 ng/uL 
               
               
                 2× CULS Buffer 
                  7.5 uL 
                 1× 
                 2× 
               
               
                 Alexa 
                   3 uL 
                 10 uM 
                 50 uM 
               
               
                 Water 
                 2.69 uL 
                 — 
                 — 
               
               
                 Total 
                   15 uL 
                 — 
                 — 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 03 
               
             
            
               
                   
               
               
                 CR mix 
               
               
                 Taq Polymerase Reagent Mix 
               
            
           
           
               
               
               
               
            
               
                   
                 Volume 1 Rx 
                 Channel conc. 
                 Stock conc. 
               
               
                   
               
               
                 Enzyme 
                 3.3 uL 
                 0.25 U/uL 
                 5 U/uL 
               
               
                 dNTPs 
                   1 uL 
                 0.37 mM 
                 25 mM 
               
               
                 2× CULS Buffer 
                   5 uL 
                 1× 
                 2× 
               
               
                 Water 
                 0.7 uL 
                 — 
                 — 
               
               
                 Total 
                  10 uL 
                 — 
                 — 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 04 
               
             
            
               
                   
               
               
                 Primer mix 
               
               
                 Primer Reagent Mix 
               
            
           
           
               
               
               
               
            
               
                   
                 Volume 1 Rx 
                 Channel conc. 
                 Stock conc. 
               
               
                   
               
               
                 Forward primer 
                 0.4 uL 
                 1 uM 
                 100 uM 
               
               
                 Reverse primer 
                 0.4 uL 
                 1 uM 
                 100 uM 
               
               
                 2× CULS Buffer 
                   5 uL 
                 1× 
                  2× 
               
               
                 MgCl2 
                 0.1 uL 
                 3 mM 
                 1000 mM 
               
               
                 LC Green Plus 
                   4 uL 
                 1× 
                 10× 
               
               
                 Water 
                 0.1 uL 
                 — 
                 — 
               
               
                 Total 
                  10 uL 
                 — 
                 — 
               
               
                   
               
            
           
         
       
     
     In this example, a total of 20 uL of a sum of all three reagent mixes were added in a well of a 96-well plate. Each primer was added in duplicate. The volume and percentage of each reagent mix is shown in the table below. 
     
       
         
           
               
             
               
                 TABLE 05 
               
             
            
               
                   
               
               
                 Volume and proportion of reagents per well 
               
               
                 Reagents per well 
               
            
           
           
               
               
               
               
            
               
                   
                 Reagent Mix 
                 Volume 
                 Percentage 
               
               
                   
                   
               
               
                   
                 DNA 
                 12 uL 
                  60% 
               
               
                   
                 Polymerase 
                  3 uL 
                  15% 
               
               
                   
                 Primer 
                  5 uL 
                  25% 
               
               
                   
                 Total 
                 20 uL 
                 100% 
               
               
                   
                   
               
            
           
         
       
     
     All three groups of reagents were ordered from the laboratory&#39;s in-house stock supply. DNA and primers were added subsequently on their respective mixes. 
     In this example, primers were added in duplicates. Positive control was added (e.g., VKORC1 primers plus control DNA (VKORC1)) as well as a Negative control (e.g., VKORC1 primers plus 2×CULS buffer). 
     Reactions were amplified on the Roche LC480 cycling platform. The PCR protocol used is shown on table below. 
     
       
         
           
               
             
               
                 TABLE 07 
               
               
                   
               
               
                 PCR protocol 
               
               
                 PCR Protocol 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Pre Amplification 
                 95° C./1 min 
                  1× 
               
               
                   
                 Amplification 
                 95° C./5 sec 
                 40× 
               
               
                   
                   
                 38° C./5 sec 
                   
               
               
                   
                   
                 72° C./5 sec 
                   
               
               
                   
                 Pos Amplification 
                 37° C./10 sec 
                  1× 
               
               
                   
                   
                 95° C./10 sec 
                   
               
               
                   
                 Melt 
                 55° C.-95° C./15 sec 
                  1× 
               
               
                   
                   
               
            
           
         
       
     
     Annealing temperature at 38° C. worked well for both primers set. 
       FIG. 13  illustrates a graph depicting the calibrators&#39; product amplification on a LC480 cycler platform. As illustrated in  FIG. 13 , the samples were successfully amplified. RFCAL-200 DNA showed very strong fluorescence values. RFCAL-100 showed lower fluorescence values compared with RFCAL-200 as expected due to the sequence size and GC content. VKORC1 was used as a PCR positive control. 
     Melting peaks for both DNA samples also were successfully obtained. Both presented a single and defined peak that was far apart from each other. RFCAL-100 has a melt temperature T M  around 70° C., and RFCAL-200 has a melt temperature T M  around 90° C.  FIG. 14  illustrates thermal melt curves depicting the amplification product of the calibrators melting on a LC480 cycler platform. 
     An aliquot of the PCR product was then checked for target size confirmation using an Agilent Bioanalyzer 2100 microfluidics-based analysis platform.  FIG. 15  illustrates the RFCAL-100 analysis results, and  FIG. 16  illustrates the RFCAL-200 results. 
     Expected and observed band size for RFCAL 100 and RFCAL-200 PCR product is shown below. 
     
       
         
           
               
               
               
             
               
                   
               
               
                 Seq. product 
                 Bp expected 
                 Bp observed 
               
               
                   
               
             
            
               
                 RFCAL-100 
                 100 bp 
                 113 bp 
               
               
                 RFCAL-200 
                 200 bp 
                 204 bp 
               
               
                   
               
            
           
         
       
     
     In a preferred embodiment, RFCAL-100 and RFCAL-200 are added on a 2:1 ratio because RFCAL-100 presents a lower fluorescent values than RFCAL-200. By doing so, RFCAL-100 and RFCAL-200 achieve rough fluorescence equivalence in the final formulation.  FIG. 17  illustrates an RFCAL-melt profile for RFCAL-100 and RFCAL-200 on Idaho Technologies HR-1 high-resolution melter with the calibrator formulated in a 2:1 ratio of RFCAL100 to RFCAL-200. As shown in  FIG. 17 , the fluorescent values of RFCAL-100 and RFCAL-200 are substantially the same at these ratios. However, it is not necessary that RFCAL-100 and RFCAL-200 achieve rough fluorescence equivalence, and other ratios may be used. 
     In the exemplary embodiment, the two constructs and respective primers worked as desired. The primers were able to demonstrate a successful amplification of the expected product. Each of their amplification products showed a single peak with high fluorescent values upon melt. Product specificity was confirmed by demonstration of expected fragment sizes on the Bioanalyzer microfluidics-based analysis platform. RFCAL-100 and RFCAL-200 are thus useful for temperature calibration purposes given their performance. 
     Although the specific examples of UCE and synthetic calibrants have been described above, other calibrants may be used in accordance with aspects of the present invention. 
     Embodiments of the present invention have been fully described above with reference to the drawing figures. Although the invention has been described based upon these preferred embodiments, it would be apparent to those of skill in the art that certain modifications, variations, and alternative constructions could be made to the described embodiments within the spirit and scope of the invention. 
     Non-Limiting Temperature Calibration/Correction Examples 
     As noted above, the computation from a resistance measurement, R, of a heating sensing element to measured temperature, T mes , may take the form of the linear equation:
 
 T   mes   =k   1   R+k   0   Equation 4
 
     An error in terms k 1 , or k 0  would lead to a bias or shift in the measured temperature relative to the true temperature. Calibrants (i.e., positive control DNA having a known or expected melt temperature) may be used to calculate the temperature compensation to shift the measured temperature closer to the true value. 
     Single Peak Temperature Calibration with Interpolation 
     When a single peak amplicon (e.g., derived from the 207 bp UCE sequence) is used as a calibrant (i.e., positive control), a single temperature shift (correction in intercept k 0 ) may be applied to the span of all measured temperatures of a particular melt. In one embodiment, this shift correction value k 0 ′ may be applied to an unknown DNA sample as follows. First, the peak temperatures of a pre UCE melt (T m   pre UCE ) and a post UCE melt (T m   post UCE ) are obtained. Second, the adjusted temperature of an assay melt that is run in between the two UCEs (i.e., calibrants) is calculated by linear interpolation. 
     The peak temperatures of the pre UCE amplicon melt (T m   pre UCE ) and the post UCE amplicon melt (T m   post UCE ) may be obtained, for example, in step  304  of the process  300   a  for calibrating the temperature of a microfluidic channel of a microfluidic device shown in  FIG. 3A  or in step  316  of the process  300   b  for calibrating the temperature of a thermal melt shown in  FIG. 3B . In other embodiments, the peak temperatures of the pre UCE amplicon melt (T m   pre UCE ) and the post UCE amplicon melt (T m   post UCE ) may be obtained using any suitable approach. For example, one suitable approach is a peak picking approach that obtains T m   pre UCE  and T m   post UCE  by (i) using a Savitzky-Golay filter with a predefined window size, polynomial and temperature resolution (e.g., 2° C., 2 nd  order, and 0.01° C. respectively) to obtain the negative derivatives of the pre and post UCE amplicon melts and (ii) finding the temperatures at the maximum negative derivatives of the pre and post UCE amplicon melts. Here, the temperatures at the maximum negative derivatives of the pre and post UCE amplicon melts are T m   pre UCE  and T m   post UCE . 
     In another embodiment, the peak temperatures of the pre UCE amplicon melt (T m   pre UCE ) and the post UCE amplicon melt (T m   post UCE ) may be obtained using a maximal correlation approach. Under the maximal correlation approach, T m   pre UCE  and T m   post UCE  are obtained by using the derivative of the melt curve of a calibrant (i.e., positive control) (e.g., shown as a solid line in  FIG. 21A ) from a calibrated instrument as a reference to which a derivative of a melt curve of the calibrant from one or more subsequent experiments (e.g., shown as a dotted line in  FIG. 21A ) are shifted. Accordingly, y(x) for the experimental calibrant may be shifted by various dx:
 
 y ′( x )= y ( x−dx )  Equation 5
 
     In this way, the negative derivative of the reference calibrant (e.g., shown as a solid line in  FIG. 21A ) may be correlated against the shifted calibrant of a subsequent experiment, y′(x), as shown in  FIG. 21A . The optimal shift may be given by the dx that maximizes the correlation coefficient as shown in  FIG. 21B . In  FIG. 21A , the optimal shift results in the dashed line. T m   pre UCE  and T m   post UCE  may then be calculated by subtracting the optimal dx from the T m  of the reference UCE. This maximal correlation approach to calculating shifts in T m  may be more robust to noise than the picking the peak alternative, but either method may be used. 
     Once T m   pre UCE  and T m   post UCE  are obtained, the adjusted temperature of an assay melt that is run in between the two UCEs may be calculated by linear interpolation as follows: 
     
       
         
           
             
               
                 
                   
                     
                       Δ 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       
                         T 
                         m 
                         
                           Assay 
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           minus 
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           interpolated 
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           UCE 
                         
                       
                     
                     = 
                     
                       
                         T 
                         Assay 
                       
                       - 
                       
                         T 
                         m 
                         preUCE 
                       
                       + 
                       
                         
                           
                             
                               
                                 ( 
                                 
                                   
                                     melt_index 
                                     Assay 
                                   
                                   - 
                                   
                                     melt_index 
                                     preUCE 
                                   
                                 
                                 ) 
                               
                             
                           
                           
                             
                               
                                 ( 
                                 
                                   
                                     T 
                                     m 
                                     postUCE 
                                   
                                   - 
                                   
                                     T 
                                     m 
                                     preUCE 
                                   
                                 
                                 ) 
                               
                             
                           
                         
                         
                           ( 
                           
                             
                               
                                 melt_index 
                                 ⁢ 
                                 
                                     
                                 
                               
                               postUCE 
                             
                             - 
                             
                               melt_index 
                               
                                 
                                     
                                 
                                 ⁢ 
                                 preUCE 
                               
                             
                           
                           ) 
                         
                       
                     
                   
                   ⁢ 
                   
                     
 
                   
                   ⁢ 
                   
                     
                       T 
                       
                         Assay 
                         ⁢ 
                         
                             
                         
                         , 
                         adjusted 
                       
                     
                     = 
                     
                       
                         T 
                         m 
                         
                           reference 
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           UCE 
                         
                       
                       + 
                       
                         Δ 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         
                           T 
                           m 
                           
                             Assay 
                             ⁢ 
                             
                                 
                             
                             ⁢ 
                             minus 
                             ⁢ 
                             
                                 
                             
                             ⁢ 
                             interpolated 
                             ⁢ 
                             
                                 
                             
                             ⁢ 
                             UCE 
                           
                         
                       
                     
                   
                 
               
               
                 
                   Equation 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   7 
                 
               
             
           
         
       
     
     Thus, the calculation for the shift correction value k 0 ′ is: 
     
       
         
           
             
               
                 
                   
                     k 
                     0 
                     ′ 
                   
                   = 
                   
                     
                       T 
                       m 
                       
                         reference 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         UCE 
                       
                     
                     - 
                     
                       T 
                       m 
                       
                         pre 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         UCE 
                       
                     
                     + 
                     
                       
                         
                           
                             
                               ( 
                               
                                 
                                   melt_index 
                                   Assay 
                                 
                                 - 
                                 
                                   melt_index 
                                   
                                     pre 
                                     ⁢ 
                                     
                                         
                                     
                                     ⁢ 
                                     UCE 
                                   
                                 
                               
                               ) 
                             
                           
                         
                         
                           
                             
                               ( 
                               
                                 
                                   T 
                                   m 
                                   
                                     post 
                                     ⁢ 
                                     
                                         
                                     
                                     ⁢ 
                                     UCE 
                                   
                                 
                                 - 
                                 
                                   T 
                                   m 
                                   
                                     pre 
                                     ⁢ 
                                     
                                         
                                     
                                     ⁢ 
                                     UCE 
                                   
                                 
                               
                               ) 
                             
                           
                         
                       
                       
                         ( 
                         
                           
                             melt_index 
                             
                               
                                   
                               
                               ⁢ 
                               
                                 post 
                                 ⁢ 
                                 
                                     
                                 
                                 ⁢ 
                                 UCE 
                               
                             
                           
                           - 
                           
                             melt_index 
                             
                               
                                   
                               
                               ⁢ 
                               
                                 pre 
                                 ⁢ 
                                 
                                     
                                 
                                 ⁢ 
                                 UCE 
                               
                             
                           
                         
                         ) 
                       
                     
                   
                 
               
               
                 
                   Equation 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   8 
                 
               
             
             
               
                 
                   
                       
                   
                   ⁢ 
                   and 
                   ⁢ 
                   
                       
                   
                 
               
               
                 
                     
                 
               
             
             
               
                 
                   
                       
                   
                   ⁢ 
                   
                     
                       T 
                       
                         Assay 
                         , 
                         adjusted 
                       
                     
                     = 
                     
                       
                         T 
                         assay 
                       
                       + 
                       
                         k 
                         0 
                         ′ 
                       
                     
                   
                 
               
               
                 
                   Equation 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   9 
                 
               
             
           
         
       
     
     Note that in the equations above, T m   reference UCE  is the true, known T m  of the UCE taken from the literature or measured from a calibrated instrument, and T Assay  is the assay temperature vector or T m  of the assay measured by the experimental instrument. The calculation of the shift correction value using Equation 8 may occur, for example, in step  306  of the process  300   a  for calibrating the temperature of a microfluidic channel of a microfluidic device shown in  FIG. 3A  or in step  318  of the process  300   b  for calibrating the temperature of a thermal melt shown in  FIG. 3B . The calculation of the adjusted temperature shown in Equation 9 may occur, for example, in step  308  of the process  300   a  for calibrating the temperature of a microfluidic channel of a microfluidic device shown in  FIG. 3A  or in step  320  of the process  300   b  for calibrating the temperature of a thermal melt shown in  FIG. 3B . 
       FIG. 22A  is a plot illustrating melting temperatures (Tms) of assays of the same genotype (open circles) and interlaced UCEs (solid points connected by lines) collected from 7 different channels.  FIG. 22B  is a plot illustrating the standard deviation of the assays melted at the same time across the 7 channels.  FIG. 22C  is a plot illustrating the changes in the melt temperatures (ΔTms) of the assays relative to the interpolated UCE melt temperature (Tm) using the immediately preceding and immediately following UCE.  FIG. 22D  is a plot illustrating the corresponding standard deviation of the changes in the melt temperatures (ΔTms) shown in  FIG. 22C .  FIG. 22E  is a plot illustrating the changes in the melt temperatures (ΔTms) of the assays relative to the interpolated UCE Tm using only the first and last UCE.  FIG. 22F  is a plot illustrating the corresponding standard deviation of changes in the melt temperatures (ΔTms) of the assays shown in  FIG. 22E . 
     As shown in  FIGS. 22A-22F , when there is a drift in temperature in a particular channel (as shown by the drifting T m  of the UCEs) or if there is a shift in measured temperature from channel to channel, calculating the adjusted measured temperature of the assay relative to the interpolated Tm of the pre and post UCEs may be used to help reduce the standard deviations of T m s of the assays (of the same genotype) across the channels. In this way, calculating the adjusted measured temperature of the assay relative to the interpolated Tm of the pre and post UCEs may be used to improve the accuracy of a genotype call. 
     In the example method using a single calibrant/peak set forth above, use of a genomic UCE (e.g., the 207 bp UCE sequence described above) is not necessary. A synthetic sequence (e.g., RFCAL-100 or RFCAL-200) may alternatively be used as the calibrant. 
     Dual Peak Temperature Calibration 
     When a dual peak calibrant (e.g., a calibrant comprising RFCAL-100 and RFCAL-200) is used as a positive control, a temperature scaling as well as shift (correction in slope k 1  as well as intercept k 0 ) may be applied to the span of all measured temperatures of a particular melt. For this reason, using a dual peak positive control may lead to more accurate compensation of measured temperature values. In one embodiment, this scale correction k 1 ′, and shift correction value k 0 ′ may be applied to an unknown DNA sample as follows. First, the low and high peak temperatures of an experimental dual peak calibrant (e.g., UCE and/orgenomic and/or synthetic) melt T m low   UCE  and T m low   UCE  are obtained. Second, the adjusted temperature of an assay melt that is run following the experimental positive control is obtained. 
     The low and high peak temperatures of an experimental dual peak calibrant (e.g., UCE) melt may be obtained, for example, in step  304  of the process  300   a  for calibrating the temperature of a microfluidic channel of a microfluidic device shown in  FIG. 3A  or in step  316  of the process  300   b  for calibrating the temperature of a thermal melt shown in  FIG. 3B . The low and high peak temperatures of an experimental dual peak calibrant (e.g., UCE) melt T m low   UCE  and T m low   UCE  may be obtained using any suitable approach. For example, one suitable approach (i) obtains the derivative of the melt curve of the experimental calibrant by using a Savitzky-Golay filter with a predefined window size, polynomial and temperature resolution (e.g., 2° C., 2 nd  order, and 0.01° C. respectively) and (ii) finds the temperatures at the maximum negative derivatives (low and high) of the dual peak calibrant. Here, the temperatures at the maximum negative derivatives (low and high) of the dual peak calibrant are T m   pre UCE  and T m   post UCE . 
     Another suitable approach for obtaining the low and high peak temperatures of an experimental dual peak calibrant melt T m low   UCE  and T m high   UCE  may use the derivative of a melt curve for a dual peak calibrant (i.e., positive control) obtained from a calibrated instrument as a reference. The low temperature and high temperature peaks may be found (T m low   UCE reference  and T m high   UCE reference ) from the reference derivative of the melt curve, and a window (e.g., ±1.5° C.) may be placed around each peak (shown by the dashed line segments of  FIG. 23A ). 
     The negative derivative of the left peak (dashed line portion of  FIG. 23A  centered around 70° C.) of the reference derivative may be cross-correlated against the negative derivative of the left peak of the experimental calibrant shown in  FIG. 23B . The two highest cross-correlation points may be obtained, and the temperature of the first (left) highest cross-correlation point may be taken as T m low   UCE . The negative derivative of the right peak (dashed line portion of  FIG. 23A  centered around 90° C.) of the reference derivative may be cross-correlated against the negative derivative of the right peak of the experimental calibrant shown in  FIG. 23B . The two highest cross-correlation points may be obtained, and the temperature of the second (right) highest cross-correlation point may be taken as T m high   UCE . 
     Once T m low   UCE  and T m high   UCE  have been obtained, the adjusted temperature of an assay melt that is run before or following the experimental positive control may be obtained by calculating a slope correction factor and an intercept correction factor as follows: 
     
       
         
           
             
               
                 
                   
                     slope_correction 
                     ⁢ 
                     _factor 
                     ⁢ 
                     
                       : 
                     
                     ⁢ 
                     
                         
                     
                     ⁢ 
                     
                       k 
                       1 
                       ′ 
                     
                   
                   = 
                   
                     
                       ( 
                       
                         
                           T 
                           mhigh 
                           UCEreference 
                         
                         - 
                         
                           T 
                           mlow 
                           UCEreference 
                         
                       
                       ) 
                     
                     
                       ( 
                       
                         
                           T 
                           mhigh 
                           UCE 
                         
                         - 
                         
                           T 
                           mlow 
                           UCE 
                         
                       
                       ) 
                     
                   
                 
               
               
                 
                   Equation 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   10 
                 
               
             
             
               
                 
                   
                     intercept_correction 
                     ⁢ 
                     _factor 
                     ⁢ 
                     
                       : 
                     
                     ⁢ 
                     
                         
                     
                     ⁢ 
                     
                       k 
                       0 
                       ′ 
                     
                   
                   = 
                   
                     
                       T 
                       mhigh 
                       UCEreference 
                     
                     - 
                     
                       
                         k 
                         1 
                         ′ 
                       
                       ⁡ 
                       
                         ( 
                         
                           T 
                           mhigh 
                           UCE 
                         
                         ) 
                       
                     
                   
                 
               
               
                 
                   Equation 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   11 
                 
               
             
           
         
       
     
     The slope correction factor and the intercept correction factor may be calculated, for example, in step  306  of the process  300   a  for calibrating the temperature of a microfluidic channel of a microfluidic device shown in  FIG. 3A  or in step  318  of the process  300   b  for calibrating the temperature of a thermal melt shown in  FIG. 3B . 
     Then, the adjusted temperature may be calculated from the slope correction factor and the intercept correction factor as follows:
 
 T   Assay,adjusted   =k   1 ′( T   Assay )+ k   0 ′  Equation 12
 
     Alternatively, these correction factors may be applied to the resistance values in real time to obtain the recalibrated temperature reading as follows:
 
 T   Assay,adjusted   =k   1   ′k   1   R+k   1   ′k   0   +k   0 ′  Equation 13
 
     The calculation of the adjusted temperature shown in either Equation 12 or Equation 13 may occur, for example, in step  308  of the process  300   a  for calibrating the temperature of a microfluidic channel of a microfluidic device shown in  FIG. 3A  or in step  320  of the process  300   b  for calibrating the temperature of a thermal melt shown in  FIG. 3B . 
     As shown in  FIG. 23C , using an out of calibration dual peak calibrant (e.g., a calibrant producing the dual peak melt curve shown in  FIG. 23B ), the recalibration procedure described above may line up the low and high peaks with the low and high peaks from the reference melt of the calibrator (e.g., the reference melt shown in  FIG. 23A ). As illustrated in  FIG. 23D , the subsequent melt of an independent calibration check melt shows that this recalibration procedure works on subsequent melts.  FIG. 23E  is a schematic diagram illustrating the functioning of the automatic calibration procedure using a dual peak calibrant. 
     As shown in the temperature calibration examples set forth above, calibrants (i.e., positive control DNA) having known or expected melt temperatures and producing single or dual peak melt curves may be used to intermittently account for calibration drift in the instrument.