Top picture shows the uniformity of PCR products which is what matters, below is given some insights and data analysis of the temperature performance of the MiniCube PCR machine to illustrate that this machine is a different kind of PCR machine due to its precision, accuracy and stability.
In general it takes around 26-30 min to complete a 2 stage PCR protocol at the default precision settings where the thermal controller only accepts a temperature as achieved when a successive number of recordings fall with ±0.020 °C of the target temperature
Let us look at the stability of the device. Many PCR machines are not capable of sustaining the same temperature over one day operation and vary between 0.5-2.0°C degrees. The electronic design governing each well is a high quality low drift sigma-delta ADC design coupled to a high quality low-drift PT1000 sensor mounted on each well – that gives unprecendented stability.
The data belows shows the stability of well number 2 on a minicube PCR measured with a Fluke 1586A instrument and a NIST calibrated temperature probe. The recording is carried out for 45 hours and the recorded standard deviation of the temperature is 3 milli-kelvin – that is precision.
The recorded mean temperature is 54.999 °C which is 1 milli-kelvin from the target temperature of 55.000 °C – that is accuracy
The lower trace is 256 cycles from 6 wells superimposed on each other and represents 1518 temperature traces. Data was recorded with an external precision temperature recorder Fluke 1586A with a calibrated NIST probe.
The well-to-well variation on these 1518 traces is less than 3 milli-kelvin– that is stability
The recording below shows traces from 16 wells running the same protocol where the temperature is plotted on the left Y-axis and the derivative of the temperature dT/dt is plotted on the right Y-axis.
From the derivative curve we can see that is possible to get a heating rate of 9.3 C/sec and a cooling rate of 2.3 C/sec
The date tables show row statistics for the 16 traces with hold time, meanvalue of 16 traces, standard deviation and number of wells.
The first row statistics at annealing of 65C at 177.150 seconds has a standard deviation of 16 milli-kelvin between the wells.
The second row statistics at extension 72 C at 188.550 seconds has a standard deviation of 9 milli-kelvin between the wells.
The third row statistics at melting 95 C at 210.250 seconds has a standard deviation of 13 milli-kelvin between the wells.
The larger variation seen in this trace compared to previous figure is due to that we have fewer data points in the statistics, but still standard deviations between wells in the order of 9-16 milli-kelvin gives possibilities for some detailed primer performance analysis which is not possible on a standard block cycler PCR machine
Accuracy takes time and sometimes we just want the PCR to finish as fast as possible. It is easy to change the firmware in the device so that the temperature controllers run with less accuracy which makes the transition between the different stages to go faster. With less accuracy the controller will over- and undershoot the temperature but because we have a rather large volume of liquid there is a large heat capacity in a volume of liquid that evens out the over and undershoots of the temperature. Below we tried to run a 40 cycles PCR between 67C and 87C (the melting point of our product) to see how fast we can complete them. We have loaded a “relaxed mode” firmware which has an accuracy of 2C vs the standard 0.02C.
We can see that the run with no tubes inserted in the wells completes in 12 min and 19 sec wheras the wells with tubes with 20 ul H2O completes in 12 min and 39 sec. So the extra mass did not slow our device down too much. However, let us see how that works out when we actually run a PCR reaction.
Here is our results from our rapid PCR (relaxed mode, with 2 C accuracy), and with a two-stage protocol of annealing at 67C and melting at 95C. We chose 95C and not 87C because we wanted to make sure we got a product and in next picture below you can see that when we approach the annealing temp of the product we get weaker bands.
Anyway, here is the good news:
The 2-stage PCR completed in 15 minutes and 30 seconds. That is impressive in a 0.2 ml PCR tube with a volume of 20 ul with a almost perfectly spheric geometry (a lumped system – as it is called in thermal engineering)
Below you can see why we ran the melting temperature at 95C in the rapid PCR experiment above. We tried to lower the melting temperature towards the melting temperature of the product which is 87C and below 91C we did not get that good performance, but maybe if we had run the assay slower we could get better products at 87 C but our purpose was to reduce time. Anyway, these data shows that if we repeat the experiment above with a melting temperature of 91C then we might be able to reduce the time with another 2 minutes and still get fairly strong bands – this would take the time down to 13 minutes and 30 seconds.
HIV2 – LTR gene
In collaboration with AAU University Hospital, Department of Clinical Molecular Diagnostics in Denmark we used to minicube PCR to amplify the LTR gene from HIV2 with only 35 cycles and detected as little as 10 copies.
Conclusion: Minicube PCR is an accurate and precise thermal cycler that can be used to:
1. Reproducible and rapid detection of presence of a gene in a sample with amplification efficiencies of 2 and with detection limits down to 10 copies or below.
2. Due to its accuracy and precision it and asynchroneous mode it offers a better alternative to gradient PCR in terms of primer optimization
3. Protocols can easily be ported between different devices because of the high accuracy and precision
4. Asynchron control allows optimization of many parameters and assays at the same time and with a significant time saving. A weeks work can be completed in a hour.