15 Experiment continued
- On the ‘PCR program’ tab select the correct PCR program for the test you want to perform and then click on ‘Start’
Look at the ‘Temperature profile’ window – why does the temperature change? What do the different temperatures do?
Answer
The temperature profile is showing the process of thermal cycling. Each PCR cycle consists of three steps and three different temperatures:
- Heating to 94 °C – The DNA strands are separated (denatured).
- Cooling to 64 °C or 60 °C – The primers bond to their target DNA sequences (the required temperature depends on the primer)
- Heating to 72 °C – The Taq polymerase adds nucleotides to the ends of each primer building new DNA strands.
Note that this process is significantly speeded up – a PCR run would take about an hour in real-time.
Look at the ‘Fluorescence output’ window – why does the fluorescence increase?
Answer
As the DNA is amplified then more light is emitted by the dye which is incorporated into its structure.
This emitted light (fluorescence) is measured by the PCR machine. It provides a direct measure of how much DNA is in each tube, which increases with each PCR cycle. (This increase tails off as the supply of nucleotides to build new DNA strands runs out.)
- When the program is complete the PCR machine automatically calculates the Ct values for each sample and deposits these data in the ‘Experimental Record’ window, alongside the record of each control and sample number.
- Click on ‘Clear All’ then click on Well F1 – the negative control.
What is the negative control in Well F1? Look at the ‘Fluorescence Output’ window and the ‘Experimental Record’ window. Is this the outcome you expect?
Answer
As this sample contains no genomic DNA, there should be no PCR amplification present. As amplification of DNA is detected by measuring fluorescence, you would predict that you should see no fluorescence.
- Click on ‘Clear All’ then click on Wells A1, B1, C1, D1 and E1 – the other controls.
Is the outcome as you’d expect for the other five controls? How do the Ct values for these compare? Table 1 may help.
Answer
For the CYP2D6 *4 primer pair you would predict that you would only see amplification in control DNAs 1 and 2. Control DNA 2 has a higher number of copies of the CYP2D6 *4 variant so you would expect the fluorescence to increase more quickly corresponding to the left-most curve. You would also predict that the Ct values for these would differ, with control DNA 2 having a lower Ct value.
- Click on ‘Clear All’ then click on Wells F5, G5 and H5 (Samples 32, 33 and 34)
Which curve in the ‘Fluorescence Output’ window corresponds to which of the three samples and why? Which sample has more copies of the CYP2D6 *4 variant?
Answer
Sample 34 has a lower Ct value than Sample 33. This means that the fluorescence in Sample 34 has reached the threshold value more quickly, corresponding to the left-most curve. Sample 34 must have started with more copies of the CYP2D6 *4 variant than Sample 33.
There is no fluorescence detected in Sample 32 so this corresponds to the horizontal line in the graph.
- Click on ‘Show All’, then highlight the data (headed ‘PCR experiment details’) and copy (Ctrl-C) then paste (Ctrl-V) into the spreadsheet template in the appropriate column, at row 4. (The application doesn’t save your data so this is an important step)
- Return to the ‘Welcome’ tab to repeat the program or choose a different population. Click on ‘Reset’.
- If you are doing this experiment in your classroom at school, your teacher will tell you which other population(s) you should study as well as which test(s) and how many replicates you should do.
- If you are doing this experiment on your own, do both tests for one of the populations A, B or C, performing two replicates for each.
14 So, let’s do the experiment!