The Polymerase Chain Reaction or PCR for short can be used to make many copies of DNA. This allows DNA to be visualized using a dye such as ethidium bromide after gel electrophoresis. The process has been refined over the years, however the basic steps are similar. The first consists of denaturing dsDNA by heating it to ~96 °C. This separates the two strands of DNA. The exact temperature to use can be calculated with Tm = 4oC x (no. of G and C) + 2oC x (no. of A and T). Tm is the melting point of the strands and primer is used to provide the number of G, C, A and T. Annealing of primers is then possible when the temperature cools down to 37-65 °C. Extension from these primers can be made by the use of thermostable DNA polymerase (must be able to resist the denaturation process). Taq polymerase is often used for this purpose. The process is repeated for many cycles to create more copies. This happens in theory with a speed of 2n, with n = number of cycles. This process is performed in a thermocycler, which can be set to different temperatures depending on the organism used. The primer choice to select genes of interest should be at least 18 nucleotides long, so they should be unique in the genome. They should also operate at a similar annealing temperature and be different (otherwise they will anneale each other). However, the final step must be visualization. A DNA ladder is a collection of DNA fragments with known molecular weights. This works in conjunction with the PCR process. When used on the gel, it then provides a comparison to determine the molecular weight of the target sequence after it has been run on an agarose gel. If possible, a positive and a negative control should be used. The first to see the PCR reaction actually working and the bands...... in the center of the paper after electrophoresis. Real-time PCR or qPCR allows you to monitor the process taking place in a cell. This results in quantifiable quantities using fluorescent probes. Now the amount of gene expression can be compared over time. Therefore it is possible to follow the up or down regulation of transcription, for now it is possible to obtain a comparative measure. PCR has become a valuable tool for analysis. As techniques have improved, costs have decreased, making it more accessible to laboratories around the world. It also produces results quickly and requires only small amounts of genetic material. The techniques above are just a small example of what can be done to analyze 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.