Source: University of Nebraska, Institute of Agriculture and Natural Resources
In this interactive activity adapted from the University of Nebraska's Library of Crop Technologies, learn the basic steps of polymerase chain reaction (PCR), a technique used to produce short sections of DNA for analysis. In this technique, various components are combined in a microfuge tube and heated in a thermal cycler. The activity details the process, which results in thousands of identical copies of a DNA fragment. Note: The activity provides a simulation of the process. The characterizations of depicted components and their behaviors should not be taken literally.
Scientists have been able to amplify, or reproduce, DNA fragments in the lab since the 1970s. In the first decade, they exclusively used a cell-based cloning process, which was time-consuming, labor-intensive, and produced uncertain results. Researchers had to manually remove a fragment of DNA from a complete strand, insert the fragment into a host cell using bacteria, yeast, or some other vector organism, and allow the fragment to replicate as the host cell and vector reproduced.
In the 1980s, a completely automated and far more efficient technique was introduced. Using the polymerase chain reaction, or PCR, scientists can amplify DNA fragments from much smaller samples of DNA. Further, they can make millions of copies in just a few hours. Whereas cell-based cloning experiments took two to four days to complete, using PCR, amplification and analysis rarely takes more than four to five hours. Not only is PCR more efficient than cell-based cloning, it's much cheaper as well.
The major breakthrough in PCR technology was the discovery of a bacteria species (Thermus aquaticus) that lives in hot springs and produces a heat-resistant DNA polymerase enzyme. Because "Taq" polymerase can withstand the high heat required to separate DNA strands, it is the only enzyme needed to complete PCR. PCR bypasses the need for costly vectors, restriction enzymes (enzymes that cut double-stranded DNA at specific nucleotide sequences), and ligases (enzymes used to insert a DNA fragment into a vector). Instead, Taq polymerase binds to the separated DNA strand to be copied, beginning where the primer sequence leaves off. It synthesizes a new strand of DNA by matching nucleotide bases in the chain with their counterparts in solution (e.g., "A"s pair with "T"s).
PCR soon became automated, which provided a reliable and quick means of temperature and time control during each amplification cycle. PCR has been a significant breakthrough in genetic research. It has allowed labs that were previously underequipped, underfunded, or understaffed to amplify DNA exponentially in a short time, which in turn allows for DNA analysis.
Apart from its essential role in the Human Genome Project—the international research project whose goals were to sequence the 3 billion or so chemical bases that make up human DNA and to identify and map the 20,000 to 25,000 genes in the human genome—PCR is used to detect infections, diagnose genetic disease both in the womb and in living patients, and perform DNA "fingerprinting." Applications of DNA fingerprinting include paternity testing, crime scene analysis, and victim identification from incomplete bodily remains. PCR has also revealed some important details concerning evolution. For example, PCR was used to trace human origins to Africa.
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