The last of General Microbiology, looking at PCR… (Day 50)

Microbiology zones of inhibition antimicrobial resistance

Today was my day of Microbiology, one of my favourite days of the week as the practicals are always interesting. And as the rest of the week is bank holiday and rector days the last day of school this week. Last week we looked at testing for antimicrobial resistance and efficiency and I promised that I would so you the results so here they are…

Microbiology zones of inhibition antimicrobial resistanceThe bacteria I cultured on this plate was Staphylococcus aureus, which is more commonly known as MRSA. The effectiveness of the antimicrobial is determined by the size of the circle around it, you can see that pennicillin (PEN) and Erythromycin (ERY) have very little effect. Kanamycin (KAN) has limited effect, and Streptomycin (STR) has the biggest effeciency. The resistance of bacteria to antibiotics is something I will write a bit more on another day as its a large topic area to cover and PCR has already taken me over my word limit.

Today was kind of sad as this was our last practical session in the Microbiology lab as next week we start working in the Immunology lab downstairs. The topic today was the use of PCR and wasn’t as practical as usual, instead it was the theory and use of PCR, then a walk through the PCR laboratories (which actually had work in progress which kinda explains why we didn’t anything ourselves). PCR stands for Polymerase Chain Reaction which is a technique developed in 1983 allowing the rapid multiplication of a single (or few) pieces of DNA into millions of copies of a particular DNA sequence.

This is based both on the chemical and physical properties of DNA. Now without getting to complicated DNA is in a double helix shape with the structure given by a backbone composed of alternating sugar (2-deoxyribose) and phosphate molecules to which the molecules coding the gene are attached. Now there are just four different molecules that are used for coding in DNA; Adenine, Thymine, Cytosine, and Guanine. Now these are then broken down into two groups; the purines (Adenine and Guanine) and the pyrimidines (Cytosine and Thymine). Now at each position on the DNA backbone a purine will bind with a pyrimidine to form a base pair. It gets even simplier here as Adenine will only bind to Thymine and Guanine will only bind to Cytosine. Basically this means that if you know what one strand of the DNA backbone is you can simply work out what the second strand should be just by looking at the bindings.

Now the molecules forming the base pairs are joined by hydrogen bonds (which are noncovalent) so DNA can be unzipped and the two strands seperated which is pretty essential in living organisms. In the PCR process this is done by raising the temperature to between 94 – 98 degrees celcius which breaks these bonds and seperates the two strands of DNA. This process is called thermocycling as it is done repeatedly to multiply the strands of DNA.

Now you understand how DNA is split its important to look at how the copy is made. First of all once the DNA splits we need to know which part of the DNA we want to copy, this is done using using a forward and reverse primer which are specifically manufactured to attach to the molecule pattern at each end of the region we want to copy. We then use something called a DNA Polymerase which is a enzyme that can add molecules to the end of a newly forming strand of DNA (this new strand starts at the primer position). Finally we cook this all together in a soup containing a mix of unbound molecules (Adenine, Thymine, Cytosine, and Guanine) to be used to create the new strands. This heating and splitting cycle is repeated 20 – 30 times each time splitting and replicating every DNA chain within the solution (aka doubling the amount of DNA each time).

Just repeating this for 30 cycles will produce over 1,073,741,824 identical DNA strands which can then be used for further analysis whether that is looking at identifying DNA in forensics or looking for a disease causing gene. One of the most exciting applications is the identification of viral infections before the onset of clinical disease allowing the treatment to start earlier and potentially have a better success rate.

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