The polymerase chain reaction or PCR for short can be used to create many copies of DNA. This allows the DNA to then be visualized using a dye like ethidium bromide after gel electrophoresis. The process has been refined over the years, however the basic steps are similar.
The first is to denature dsDNA through heating to ~96 °C. This separates the two strands of DNA. The exact temperature to be used can be calculated with Tm = 4oC x (no. of G & C) + 2oC x (no. of A & T). Tm is the melting point of the strands and to supply the number of G, C, A, & T ‘s the primer is used.
Annealing of primers is then possible when the temperature cools down to 37-65 °C.
Extension from these primers can be done through the use of heat stable (has to be able to withstand the denaturing process) DNA polymerase. Taq polymerase is often used for this.
The process is repeated for many cycles to create more copies. This occurs in theory at 2n rate, with n = number of cycles. This process is done in a thermal cycler, which can be set at different temperatures dependent on the organism used.
The choice of primer to select the genes of interest should at least be 18 nucleotides long, so they should be unique in the genome. Also they should work at a similar annealing temperature and be dissimilar (or they’ll anneal to each other).
The final step must be visualisation however. A DNA ladder is a set of DNA fragments with known molecular weights. This runs alongside the PCR process. When used on the gel, it then provides a comparison to determine the molecular weight of the target sequence after they have been run on an agarose gel. If possible a positive and negative control should be used. The first to see the PCR reaction actually worked, and secondly to exclude contamination of the reagents to rule out false positive results. Ethidium bromide can be used in combination with UV lighting to visualise the bands. However it is a known carcinogen and therefore alternatives have been developed. These, for instance gelred, can be used without the use of UV lighting. However they are often a lot more expensive.
A general issue is that PCR techniques can be very sensitive; any contamination can be amplified. This in combination with the improvements in technique seen over the last few years can mean that for instance in forensics, extensive care needs to be taken when handling samples to prevent contamination. The interpretation of results can also prove tricky for levels of contamination (or DNA that was there due to circumstances unrelated to the crime) that may not have been noticeable with earlier techniques can point the blame to someone who was in fact innocent.
However forensics is not the only area that has been advanced by PCR techniques. Diseases diagnosis can be made much more accurately and quickly. The diagnosis however is not just limited to infectious diseases caused by bacteria, tumours can also be analysed. Therefore it may become apparent if it is...