Polymerase Chain Reaction
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 106 times in a few hours. American molecular biologist Kary Mullis developed the idea of PCR in the 1970s. The idea was conceived while he was cruising in a Honda Civic on Highway 128 from San Francisco to Mendocino, California. For his ingenious invention, he was awarded a Nobel Prize in 1993.
PCR amplification of DNA is like any DNA replication by DNA polymerase in vivo. The difference is that PCR produces DNA in a test tube. For a PCR to happen, 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 90°C for denaturation. During this process, two stands of the targeted DNA are separated from each other and each stand is ready as template. The second step is carried out around 50°C. At this lowered temperature, the two primers annealed 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 is carried out manually in incubators of different temperatures for each step until the discovery of DNA polymerase from thermophilic bacteria. The famous bacterium Thermus aquaticus was found in Yellow Stone National Park. This bacterium lives in the hot springs at 95°C.
The DNA polymerase from T. aquaticus keeps its activity at above 95°C over hours. Several heat-resistant DNA polymerases have been found since.
Genetically engineered heat resistant DNA polymerases, which have proofreading functions and make fewer mutations in the amplified DNA products, are available commercially. PCR reactions are 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 is a revolution to molecular biology. PCR is valuable to researchers because it allows them to multiply a unique DNA sequence to a large amount in a very short time. Researchers in the Human Genome Project are using PCR to look for markers in cloned DNA segments and to order DNA fragments in libraries. Molecular biologists use PCR in cloning DNA. PCR is 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, it has become a routine to use PCR in the diagnosis of infectious diseases such AIDS.
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