Positive controls
The present invention relates to the use of one or more amplicons as temperature calibrators. In some embodiments, the calibrators may be used to calibrate the temperature of a microfluidic channel in which amplification and/or melt analysis is performed. In some embodiments, the amplicons may be genomic, ultra conserved elements and/or synthetic. The amplicon(s) may have a known or expected melt temperature(s). The calibrators may be added to primers of study or may follow or lead the primers of study in the channel. The amplicon(s) may be amplified and melted, and the temperature(s) at which the amplicon(s) melted may be determined. The measured temperature(s) may be compared to the known temperature(s) at which the amplicon(s) was expected to melt. The difference(s) between the measured and expected temperatures may be used to calibrate/adjust one or more temperature control elements used to control and/or detect the temperature of the channel. In other embodiments, the UCE primers may function as a positive control to validate amplification has occurred.
1. A method for providing a positive control in a nucleic acid amplification reaction followed by a nucleic acid melting using ultra-conserved elements (UCE), the method comprising:
providing a control sample, wherein the control sample comprises human genomic DNA, the human genomic DNA including a UCE sequence;
providing a device configured to receive the control sample, wherein the device comprises a system controller configured to control fluid flow, control temperature for the PCR reaction and control temperature for a thermal melt;
adding one or more UCE specific primers to the control sample, wherein the one or more UCE specific primers have the sequence shown in SEQ ID NOs 2 and 3;
loading the control sample into the device performing PCR to amplify the UCE sequence;
performing PCR on the control sample;
following the PCR, performing a thermal melt analysis on the control sample to obtain melting curves, wherein the system controller analyzes the melting curve and determines a melting temperature for the control sample;
wherein the system controller analyzes a reaction result using the control sample as a positive control, wherein
(i) if the control sample produces an expected UCE melting temperature, the amplification reaction followed by the thermal melt analysis is determined to have been successful; and
(ii) if the control sample produces an unexpected UCE melting temperature, the amplification reaction followed by the thermal melt analysis is determined to have been unsuccessful.
2. The method of claim 1 , wherein the device is a microfluidic device comprising multiple microchannels and both performing the PCR and the thermal melt analysis for the control sample occurs in a single channel.
3. The method of claim 1 , further comprising adjusting operating conditions and repeating the PCR followed by the thermal melt analysis if the reaction is determined to be unsuccessful.