What is PCR?

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PCR (polymerase chain reaction) is a process (GIF animation) used to make many copies of select regions of DNA.  The process involves three repeated steps.

  1. The template (original) DNA is first denatured or broken apart.  The double stranded helix is separated so that single stranded DNA exists.  This is done by heating the DNA to 94oC.
  2. The temperature is then reduced in order to allow the primer to bind to the single stranded template (original) DNA.
  3. Then the temperature is raised to the temperature where optimum extension (or replication) of the DNA takes place.
In order for the process to take place a mix of the original/template DNA, primers, individual nucleotides (dNTPs - A [adenine], T [thymine], G [guanine], C [cytosine]), MgCl2, a buffer solution, and Taq DNA polymerase (the enzyme responsible for replicating the DNA, originally extracted from bacteria discovered in hot springs - Thermus aquaticus) must be made up.  Primers are simply small segments of DNA that are synthesized to be complimentary to a specific region(s) of the template DNA.  An example of a primer is viewable by clicking on this sentence.  It is characterized by having A's matched with T's (or T's with A's) and G's matched with C's (or C's matched with G's).

A diagram of the process of replication or an animated GIF may clarify what is being described.  The following is an explanation of what is depicted:

  1. Isolated (extracted) double stranded DNA is shown.
  2. Separated single strands of the original DNA template are shown with the primers (looking like small bugs) bound to them.
  3. Extending from the point where the primers were first bound the Taq DNA polymerase replicates the DNA strands.  (The wavy lines indicate that DNA extension has progressed passed the viewable region.  NOTICE - that the DNA is replicated beyond the area that is desired to be copied [which is the region depicted as a straight line.])
  4. Once again the double stranded DNA is denatured to provide single stranded DNA for the primers to bind to.
  5. Replication of the DNA again takes place.  (NOTICE - that half of the copies of newly replicated DNA are now limited in their extension to the point where a primer can bind.)
  6. Again the double stranded DNA is denatured and the primers bind (anneal).
  7. DNA replication takes place once again.  (NOTICE - that the more the replication process is performed the higher the percentage of DNA fragments that are limited in their length to the distance between where the two primers bind will be.  This region is depicted as a straight line.)
  8. Typically the number of times the PCR process is repeated is between 25-35.  A machine called a thermocycler is normally used to automate the process.  This machine also allows the time spent at each temperature and the temperatures themselves to be modified in order to optimize the process.
PCR is used for many genetic analyses ranging from paternity testing and criminal forensic analyses to speciation and phylogenetic tests to examinations of variation between species' subpopulations.  Depending upon the specificity of the primers that are used the number of locations within the genome that are copied and amplified varies.  This means that for very specific primers either one or two sizes of DNA fragments will be produced while for other less specific primers many different sizes of DNA fragments are produced.  The various sizes of DNA originate from multiple sites within the genome where the primers can bind and Taq DNA polymerase can replicate the fragments.

Here are two animated examples of the electrophoresis process (animated GIF) where pieces of DNA are "run" on a gel in order to separate and identify pieces of different lengths (Flash animation), or for a simple descriptive diagram of gel attributes [click here].  Or for a personal explanation and pictures visit this page (electrophoresis pictures).
 

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