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Polymerase Chain Reaction Technology
Several of the detectors used in our project use a polymerase chain reaction in order to amplify and identify pathogens. This reaction, which is now widely used in research laboratories and doctor's offices, relies on the ability of DNA-copying enzymes to remain stable at high temperatures. The secret for this reaction to work is the use of the DNA polymerase from thermophilic bacterium, Thermus aquaticus. Kary Mullis, who says this idea came to him in a 1983 moonlight drive in the California mountains, won the Nobel prize for this reaction in 1993. (For information about the historic papers surrounding PCR, readers may want to visit Berkeley's excellent site on the topic.) In nature, all organisms rely on the DNA molecule for replication of genetic material. The PCR mimics this process; only it does it in vitro (in test tubes). When a cell divides, DNA polymerase makes a copy of the entire DNA in each chromosome. To copy the DNA, polymerase requires a supply of the four nucleotide bases (adenine, guanine, thymine, and cytosine) and primers. The primers are required to initiate the copying of the sequence of nucleotides. The PCR contains the same components for DNA duplication: a piece of DNA, a supply of the four nucleotides, a supply of the primer sequence, the DNA polymerase. However, in PCRs, the polymerase used is the Taq polymerase, isolated from the thermos aquaticus. |
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The PCR involves three steps that are carried out at different temperatures. The first process separates the two DNA chains in the double helix, which merely requires heating the vial to 90-95 degrees centigrade. The primers cannot bind to the DNA strands at such a high temperature, so the vial is then cooled to 55 degrees centigrade. The primers then bind to the ends of the DNA strands. The final step is to make complete copies of the template. The Taq polymerase works best at 75 degrees centigrade (the temperature of the hot springs where the bacteria were discovered), so the temperature is raided. The Taq polymerase begins adding nucleotides to the primer, by adding the complement of each of the nucleotide bases. Adenine always bonds with guanine, and thyme always bonds with cytosine. When the polymerase is finished, one copy of the DNA sequence is completed. This cycle can be repeated many, many times. Each new piece of DNA can act as a new template, so after 50 cycles, over one billion copies can be produced.
The amount of time it takes for one cycle to occur, and the amount of DNA needed for identification are variable and scientists are constantly trying to decrease the amount of time needed to identify a piece of DNA. Works Cited The
1993 Nobel Prize in Chemistry -- The PCR Method -- Nobel e.Museum |
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