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PCR

dna polymerase molecular sequence

PCR (polymerase chain reaction) is a technique in which cycles of denaturation, annealing with primer, and extension with DNA polymerase, are used to amplify the number of copies of a target DNA sequence by more than 100 times in a few hours. American molecular biologist Kary Mullis developed the techniques of PCR in the 1970s. For his ingenious invention, he was awarded the 1993 Nobel Prize in physiology or medicine.

PCR amplification of DNA is like any DNA replication by DNA polymerase in vivo. (in lving cells) The difference is that PCR produces DNA in a test tube. For a PCR reaction to proceed, four components are necessary: template, primer, deoxyribonecleotides (adenine, thymine, cytosine, guanine) and DNA polymerase. In addition, part of the sequence of the targeted DNA has to be known in order to design the according primers. In the first step, the targeted double stranded DNA is heated to over 194°F (90°C) for denaturation. During this process, two strands of the targeted DNA are separated from each other. Each strand is capable of being a template. The second step is carried out around 122° (50°C). At this lowered temperature, the two primers anneal to their complementary sequence on each template. The DNA polymerase then extends the primer using the provided nucleotides. As a result, at the end of each cycle, the numbers of DNA molecules double.

PCR was initially carried out manually in incubators of different temperatures for each step until the extraction of DNA polymerase from thermophilic bacteria. The bacterium Thermus aquaticus was found in Yellow Stone National Park. This bacterium lives in the hot springs at 203°F (95°C). The DNA polymerase from T. aquaticus keeps its activity at above 95°C for many hours. Several additional heat-resistant DNA polymerases have also now been identified.

Genetic engineered heat resistant DNA polymerases, that have proofreading functions and make fewer mutations in the amplified DNA products, are available commercially. PCR reactions are now carried out in different thermocyclers. Thermocyclers are designed to change temperatures automatically. Researchers set the temperatures and the time, and at the end of the procedure take the test tube out of the machine.

The invention of PCR was revolutionary to molecular biology. PCR is valuable to researchers because it allows them to multiply the quantity of a unique DNA sequence to a large—and thus workable—amount in a very short time. Researchers in the Human Genome Project use PCR to look for markers in cloned DNA segments and to order DNA fragments in libraries. Molecular biologists use PCR to cloning DNA. PCR is also used to produce biotin or other chemical-labeled probes. These probes are used in nucleic acid hybridization, in situ hybridization and other molecular biology procedures.

PCR, coupled with fluorescence techniques and computer technology, allows the real time amplification of DNA. This enables quantitative detection of DNA molecules that exist in minute amounts. PCR is also used widely in clinical tests. Today, routine to use PCR in the diagnosis of infectious diseases such AIDS and in a number of forensic tests.

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