How to Perform a Basic Polymerase Chain Reaction (PCR)
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How to Perform a Basic Polymerase Chain Reaction (PCR)

This is just the pure basics to a polymerase chain reaction. It focuses mainly on making the mix for the PCR and why the thermo cycler cycles through the different temperatures. This is a good reference for undergraduate college students learning what a PCR is and how to do one.

Polymerase Chain Reaction (PCR) is a powerful tool for scientists involved in all kinds of research. This reaction allows scientist to take DNA and amplify a specific region to study via cloning/replication of the DNA segment. It is a technique that can be used at almost an everyday basis in genetics labs so it is important to understand what is going on. The general idea of a PCR first involves the DNA you want to amplify. This DNA usually comes from some sort of sample, which can be from anything from a piece of hair to a slab of liver. The important thing is that sample contains DNA. Another important factor concerning the sample is how it is broken down because the sample consists of many cells like all living things. These cells have more than just DNA; they consist of other numerous organelles such as mitochondria and endoplasmic reticulum to function properly. The process of isolating DNA from the cells is another procedure all together.  There are special mixes, solutions, and techniques companies create for this exact purpose. In general the process involves a lysis solution to break up the cells, filter columns to essentially “catch” the DNA, and buffer solutions to wash away all the other material except for the DNA.

Once you finally have your DNA the next step involves primers, deoxynucleotide triphosphates (dNTPs), and polymerase. The primers are small designed fragments of DNA that will help target the region of DNA you want. For example a commonly used primer in population genetic or phylogenetic studies is Cytochrome b, which amplifies mitochondrial DNA of the cell. The primer usually consists of a forward and a reverse complementary strand of the region of DNA to be amplified. These forward and reverse primers can be added one at a time or they can be first mixed together and then added to the DNA for convenience. Next is the dNTPs which is a buffer of nucleotides that are essentially the materials used to build the amplified region of DNA. Finally, the last ingredient is polymerase which is machine that does the actual amplifying. Polymerase is the molecular machine that utilizes the primers to find the region of DNA that is be amplified and uses the dNTPs to build clones of the DNA region. A typical type of polymerase use is taq polymerase.  This polymerase is from a thermophilic bacterium that is easily manipulated with temperature.  Now this is just the basic important steps, there are other buffers and solutions that you can add to this PCR mix such as magnesium chloride and bovine serum albumin to encourage the binding of the polymerase to the DNA. 

Now that you have your mix you can place your mix into the thermo cycler machine. This machine fluctuates the temperature in multiple cycles to help promote the amplification of the DNA region you are targeting. The first temperature the cycler goes to is any temperature that is around 90 Celsius. At this temperature the DNA begins to unravel and separate, allowing access to the primers.  Then the thermo cycler cools down to around 50 Celsius which allows the primers to bind to the corresponding sequence on the DNA. Then the thermo cycler changes the temperature to around 72 Celsius and the polymerase begins replicating the DNA strands. This cycle repeats and at the third cycle you will have two copies of only the DNA region you targeted.  As the cycle continues to repeat, copies of the target region with grow exponentially. By the end you will have billions if not millions of copies of the targeted DNA region in the tube. 

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