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Polymerase Chain Reaction - From Simple Ideas


The Polymerase Chain Reaction (PCR) is a powerful technique, which results in the rapid production of multiple copies of a target DNA sequence. The PCR technique has made it possible to analyze DNA fragments in samples that contain amounts of DNA that are either too small, or too degraded, to permit other types of nucleic acid analysis. As a result, it now becomes possible to analyze and characterize DNA fragments found in minute quantities in places like a drop of blood at a crime scene or a cell from an extinct dinosaur.

The PCR method is a cycling reaction in which template DNA is denatured by heating to separate the strands of the molecule. Primer (20-30 base fragment of DNA complementary to a region of the template) is annealed to the single-stranded templates. The cycle ends as the primer molecules are elongated by the action of DNA polymerase to produce molecules that are identical copies of the original template.

Because the products of one PCR cycle can act as templates for the next PCR cycle, the number of new identical molecules produce doubles with each repetition of the cycle.

When first developed, multiple cycles of the PCR process were cumbersome for two reasons. First, the DNA polymerases (Klenow fragment) available at the time were inactivated each time the temperature was raised to denature the template strand. This meant that polymerase had to be replenished with every repetition of the PCR cycle. Second, three water baths at three different temperatures were necessary, which meant that constant human attention was required: A technician had to keep moving the reaction vessel to the next water bath at one-minute intervals. This minute-by-minute change had to be repeated a minimum of thirty times!

Two developments were instrumental in the maturation of the PCR process. First was the purification of a heat-stable DNA polymerase (Taq DNA polymerase). This enzyme was originally isolated from a thermophilic bacterium, Thermus aquaticus, found in the hot springs of Yellowstone National Park. Because Taq DNA polymerase is not inactivated by the higher temperatures of PCR, it only needs to be added to the sample once, rather than with every repeat of the cycle.

The second development was the invention of a thermal cycler. The first automated PCR machine was invented by Cetus Instrument Systems and was originally dubbed "Mr. Cycle." It was fabricated by modifying a multi-channel automated liquid handler and two aluminum blocks. Currently, the market is flooded with more sophisticated thermal cyclers, which can meet any researchers' needs.

PCR technology is unique in its ability to locate and exponentially amplify a small quantity of a specific nucleotide sequence which is "lost" against a large background of total nucleic acid. This feature of PCR has made possible the development of a vast number of experimental and diagnostic molecular biology techniques, which were previously extremely time consuming or, in many cases, impossible to perform.


Go to next story: Polymerase Chain Reaction - Xeroxing DNA

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