Last week – we talked about PCR and how important it is in the forensic setting. We will finish off talking about PCR, and then we will discuss how it can be used.
If we go back to the slide of the double stranded DNA, and if we take that to a high temperature, the two strands separate, you then add the primers, which interact with ? On the strand, synthesis takes place in the 5-3 direction, then you end up with 2 molecules identical to the DNA, and then you do another round, so it's an exponential increase.
There are different enzymes and polymerases, which come from bacteria and hot springs. The original polymerase is the taq polymerase. In the forensic setting the copying is extremely important. Now there are vent? Polymerases, which are much more efficient and effective, known for its proof reading, when bases are incorporates you can get errors, with the incorrect base being put in. It will happen 1 in every thousand. If you feed in an enzyme that will release the process to reach the enzyme going in from the 3-5 strand, to take away the base; and put in the correct base.
Some enzymes do less better than others. You get your standard taq, then add a small amount of higher fidelity polymerases, so you can remove the incorrect base and insert the correct one.
Visualising the process of PCR (See Moodle)
In laboratories without a high through point of PCR, reactions will usually happen using a gel 'agrose' (See Moodle) it looks like jelly. You can make slabs of this gel. You boil up the agrose which melts, then you cast it in a square cast, so you have a slab of jelly. You can then put in wells and holes into the slabs. To put in your samples, place gel on piece of equipment, and then you can run electrical currents through. When you put electric through you have a complete current.
DNA is it positive or negative? It is negative. If you put a current through it, it will move towards the positive, so you can then separate your DNA based on the size. Larger fragments won't travel as far, and smaller bits will travel further. You also have a lane that has markers of known DNA size. The size markers determine the size of DNA fragments.
Agrose gel electrophoresis of DNA (See Moodle)
You can incorporate a fluorescent marker within the DNA, so you can see where the bands are. You put it into a chemical known as ? (something beginning with E, See Moodle) If you shine UV light upon it, you can see it under this dye, which makes the bands fluorescent. (See diagram on Moodle)
Gradient PCR is used to optimise annealing temperature (See Moodle)
It is extremely important to have a clean laboratory, as you want to get the best results you can get. When you are looking for a band, you want to see a single band in the right place, so it needs to be as clean as possible. You need to optimise the contamination. You need enzyme regression, when carrying out the experiment, do a series of optimisation reactions. To optimise PCR conditions, change the temperature. You can run the PCR at different temperatures,
What is the optimum temperature for the annealing of primers? 64-66 degrees is your optimum temperature.
Aim for one very clean band, another way is by changing the magnesium concentration, 1mM is the optimal. Mgcl2 concentration. (See Moodle) For a positive result you need standard DNA and standard primers. If you do that and it is clean, what does it tell you? You are doing it right.
Why bother using positive control? If you didn't use it and you got nothing what does it tell you? It tells you you got it right, that you haven't got contamination, so positive control is really important.
Negative control – this is also really important. If you get a bond that has contamination, if you carry out negative reaction and leave out the DNA, if you get a band, you know you've got contamination, as it is a reaction from somewhere else. It isn't...