Polymerase chain reaction (PCR)

OK, so in the previous step, we extracted our DNA. So we isolated and now we have our extracting 50 microlitres of pure DNA extract, and we’re going to amplify now, because what we want is to obtain thousands of thousands of copies of the target DNA. So it’s going to amplify just the region we want to amplify. For the purpose of this test in criminal cases, we’re using DNA-17, which is a complex mixture of 16 markers called autosomal short tandem repeat, STRs, and one gender marker - so 17 different areas. STRs are regions that are present in the non-coding part of the genome.

The genome is really big, but only 1.5 per cent actually is the one that is producing proteins that are useful for the cell functioning. So all the rest of DNA contain information that are also useful that can identify who you are. So those STR regions are regions that are maternally and paternally inherited, and they can be really useful in forensic science to link to the identity of an individual. So what we are going to use at the beginning is now a primer mix, so all this complex mixture, which is produced already together, and it is fluorescently labelled in order to be detected on the following step we are going to do.

And a master mix: a master mix that contains the enzyme that will put all the bases one after the other, the water, the salts that have the reaction.

We’re going to use small tubes, 0.2 tubes, and I have labelled them, so it will be one tube per the samples that we’re going to have. And we’re going to have an extraction, a PCR negative and a PCR positive as well. So, for the sake of this, I already prepared the master mix into the tubes. I’m just going to add the samples in.

Again, we need to be very careful on the labelling. So I’m going to add the sample one, into the tube labelled as number one, and the sample two into number two.

And finally, the extraction negative. So as we said before, the extraction negative is to check that the reagents that we used to extract were actually clean from any contamination. For the same issues, we’re going to use a PCR negative and a PCR positive. So for PCR negative, we’re going to use just normal PCR-grade water, and with this sample, we want to see if there’s any possible contamination

in the reagents we’re using: so either in the primer mix or in the mass mix. We’re going to use a positive control as well, which is a DNA sample where all the locations of all the STRs are really well-known, so that will help us in the analysis. So, whether if we have a failed reaction and the positive control failed as well, that probably might mean that there is a problem with the enzyme functioning or the PCR itself - the method itself - and so the run needs to be repeated.

Whereas if the sample is not working but the positive is working, then you can tell that the reaction is working but just the sample didn’t work, and then we will have to proceed with re-extraction or re-evaluation of another method. So now that we prepared our tube, we’re going to transfer them.

One of the bigger concerns of a forensics lab is contamination and being able not to carry over any possible contamination. So, we’re going to transfer into another rack and try to keep the pre-PCR area and post-PCR area the most separate as possible. So Natsumi, would you mind following me to the post-PCR area please? Sure.

OK. So we’re going to transfer the samples from the transfer rack into the thermocycler, and we’re going to keep the positive control separate, because we don’t want it to contaminate any of the other samples by any chance. Oh, yes. I’m going to close. So PCR is a technique developed in the mid-80s that really revolutionised the way we’re looking at molecular biology right now, and if you come closer, you can have a look at the stage, because it’s actually very explanatory on how it happens. OK. So the first stage is where we’re activating the enzymes, so the DNA polymerase in action. Then, we’re going to have, as you can see on stage two, a repetition of 28 times the cycles.

So the first step is the step where we are denaturating the double strands of the DNA. So we are denaturating. Then, the step in the middle is a polymering step, and we’re going to have the primer linking to the targeted DNA area that we select with the primers. In the third step of the cycle is an elongation step. So 72 degrees is an optimal temperature for the DNA preliminaries to start polymerasing base after base. This is repeating 28 times.

So the DNA is exponentially amplified 1,000 of million of times, and at the end of this process, we’re going to have our targeted area - only those ones, so not the whole genome that we started with - amplified million times, and this is exactly what we need to detect the DNA onto the capillary electrophoresing in our following step.