What is the Polymerase Chain Reaction (PCR)

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 s...

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...ent bands following electrophoresis.

Real-time PCR or qPCR allows the on going process in a cell to be monitored. This results in quantifiable amounts using fluorescent probes. Now the amount of gene expression can be compared over time. Therefore up or down regulation of transcription can be followed, for now a comparative measure can be obtained.

PCR has become a valuable tool for analysis. As techniques have improved, the costs have gone down, making it more accessible for labs across the globe. Next to this it produces results quickly and it only needs small amounts of genetic material to be present. The above techniques are only a small sample of those that are possible to analyse DNA.

Works Cited

Reed R., Holmes D., Weyers J. and Jones A. (2007). Practical skills in Biomolecular Sciences. Chapter 61:Molecular genetics II – PCR and related applications.