Restriction-Modification Systems of Bacteria
Southern and Northern Analysis
Polymerase Chain Reaction (or PCR)
Polymerase Chain Reaction (or PCR)
The polymerase chain reaction (PCR) is the most powerful technique that has been developed recently in the area of recombinant DNA research and is having an impact on many areas of molecular cloning and genetics. With this technique a target sequence of DNA can be amplified a billion fold in several hours. This procedure has been applied to forensic analysis where minute samples of DNA was isolated from blood at a crime scene to determine if an individual was actually at the location of the crime. Most recently DNA from mummies and fossil tissue has been analyzed to determine ancient evolutionary relationships.PCR is a DNA polymerase reaction and as with any polymerase reaction it requires a DNA template and a free 3'-OH. The template is provided by the DNA sample to be amplified and the free 3'-OH are provided by site-specific oligonucleotide primers. The primers are complementary to each of the ends of the sequence that is to be amplified. The three steps of the reaction are:
1. Denaturation - the DNA is heated usually to 95oC to render it single-stranded
2. Annealing - the two primers bind the appropriate complementary strand; the temperature for this step varies depending on the of size of the primer and its homology to the target DNA
3. Primer Extension - DNA polymerase extends the primer by its polymerase activity; this is done at a temperature optimal for the particular polymerase that is used; currently the most popular enzyme for this step is Taq polymerase, the DNA polymerase from the thermophilic ("heat-loving) bacteria Thermus aquaticus; the extension is performed at 72oC
These steps are repeated from 28-35 times. Since the reaction is essentially exponential and since each cycle is about 5 minutes, a large quantity of DNA can be produced for analysis in as little as several hours.
The first description of the PCR reaction used the DNA polymerase I enzyme from E. coli, and the reaction was moved manually between the different temperatures. Because the E. coli enzyme is heat sensitive, its activity was killed during the denaturation step at 95oC. Therefore a new aliquot of the enzyme had to be added with each cycle. The purification, and ultimately the cloning, of the DNA polymerase from T. aquaticus made the reaction much simpler. This organism lives in hot springs that can be near boiling and since it can complete its life cycle at these temperatures, its DNA polymerase would need to be able to maintain its functions after being exposed to these temperatures. This property makes it highly favorable for the PCR reactions because the denaturation step is performed at 95oC. Thus a new enzyme aliquot is not required for each cycle. More recently, DNA polymerases have been isolated from organisms that reside in the vicinity of the thermal vents that are present in the oceans at the junction of two tectonic plates. These enzymes are even more heat stable and may eventually replace the T. aquaticus enzyme for the PCR reaction.
Finally, this procedure has been automated by the development of thermal cyclers. These instruments have the capability of rapidly switching between the different temperatures that are required for the PCR reaction. Thus the reactions can be set up, placed in the thermal cycler and the technician can return several hours later and obtain the product and proceed from that point.
Copyright © 1997. Phillip McClean