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PCR DNA Cloning

The polymerase chain reaction (PCR) is a method for producing a large number of copies of a specific DNA sequence. For example, let's say a dog owner has an albino dog. As you learned in the More About Mutations section of this web site, albinism can be caused by mutations in the tyrosinase gene. Thus, studying that gene in an albino dog could be quite interesting. In order to start the process, however, we must first make enough copies of this tiny piece of DNA to study in the first place. This is where PCR becomes invaluable.

PCR is just a fancy type of DNA replication, so you might want to review that process in our Basic Concepts section before you continue. The starting material for the process is a mixture that contains the target DNA gene (in this case nuclear dog DNA would work very well), DNA polymerase, nucleotides, and DNA primers that are complimentary to the ends of the gene of interest. As you recall, DNA polymerase cannot copy DNA unless it has a primer to start the process. Thus, if we use gene specific primers, only our gene (tyrosinase) will get copied.

The process of PCR is as follows:

1. Denature the DNA by heating it so that it breaks apart to single strands.

2. Lower the temperature to allow gene specific primers to attach to their target on the sequence.

3. Extend the primers using a special DNA polymerase that will not break down at high temperatures. Taq polymerase, the first such heat stable polymerase to be identified, was isolated from the bacterium Thermus aquaticus, which lives in the hot springs of Yellowstone National Park.

4. Repeat this cycle approximately 30 times.

You now have millions of copies of your gene!

There are some problems that must be avoided before PCR data can be used reliably. First, Taq DNA polymerase does not have a proofreading mechanism. This means that it cannot correct any errors it makes while synthesizing DNA. Thus, your PCR product may not be an exact copy of your gene of interest. PCR is also very sensitive, so if you contaminate your DNA sample with even a tiny bit of foreign DNA you will get copies of the contaminant in addition to those of your gene. However, if care is taken when performing PCR, and when interpreting its results, it is an incredibly valuable technique. In fact, Kary Mullis was awarded a Nobel Prize for inventing PCR.





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