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The Polymerase Chain Reaction
It is possible to increase the amount of a given DNA many times over without cloning that DNA. The method that makes this amplification possible is the polymerase chain reaction (PCR). Any chosen DNA can be amplified, and it neednot be separated from the rest of the DNA in a sample before the procedure is applied. PCR copies both complementary strands of the desired DNA sequence. Scientists had long wished for a cell-free, automated way of synthesizing DNA, but any system that could be automated would need to function at high temperatures so that the DNA strands could be separated physically without the need for the many enzymes found in DNA replication, such as topoisomerases and helicases. Unfortunately, such temperatures (around 90°C) would denature and inactivate the DNA polymerases that would be making the DNA strands. What made the process possible was the discovery of bacteria that live around deep-sea hydrothermal vents, under extreme pressures, and at temperatures higher than 100°C. If bacteria can live under those conditions, then their enzymes must be able to function at those temperatures. The bacterium, Thermus aquaticus, from which a heat resistant polymerase is extracted, is one ofthose that live in hot springs. The enzyme is called Taq polymerase. We saw that the biotechnology industry eagerly searches for organisms that live under extreme conditions, and here we have an example of why it does so.
At the start of the process, the two DNA strands are separated by heating, after which short oligonucleotide primers are added in large excess and, via cooling, are allowed to anneal to the DNA strands. These primers are complementary to the ends of the DNA chosen for amplification and serve the same purpose as the RNA primers in normal replication. Once the primers have annealed to the DNA, the temperature is raised again to optimize the activity of Taq polymerase, which begins synthesizing the new DNA from the 3' end of the primer. The two complementary strands grow in the 5' to 3' direction (Figure 13.23), and the Taq polymerase is allowed to work until the desired length of DNA has been synthesized.
This first round doubles the amount of the desired DNA. The process of unwinding the two strands, annealing the primers, and copying the complementary strand is repeated, bringing about a second doubling of the selected double-stranded DNA. It is not necessary to add more primer because it is present in large excess. The whole process is automated. Control of the temperature to which the strands are heated to separate them is crucial, as is the temperature chosen for annealing the primers.
The amount of DNA continues to double in subsequent rounds of ampli-fication. After about an hour, and 25 to 40 cycles of replication, one obtains millions to hundreds of millions of copies of the desired DNA segment, usually a few hundred to a few thousand base pairs long (Figure 13.23). Other DNA sequences are not amplified and do not interfere with the reaction or subse-quent use of the amplified DNA.
The most important part of the science behind PCR is the design of the primers. They have to be sufficiently long to be specific for the target sequence but not so long that they are too expensive. Usually, the primers are 18 to 30 bases long. They must also have optimal binding properties, such as an amount of G and C that is sufficient to allow them to anneal before the entire DNA renatures. In addition, the two primers should contain similar amounts of G and C so that they have the same melting temperature. The sequence of the primer must not lead to secondary structures within a primer or between the two different primers; otherwise, the primers bind to themselves instead of to the DNA being amplified. For example, if a primer had the sequence AAAAATTTTT, it would form a hairpin loop with itself and would not be avail-able to bind the DNA.
Amplification of the amounts of DNA in extremely small samples has made it possible to obtain accurate analyses that were not possible earlier. Forensic applications of the technique have resulted in positive identifications of crime victims and suspects. Even minuscule amounts of ancient DNA, such as those available from Egyptian mummies, can now be researched after amplification. The following Biochemical Connections box describes some forensic uses of DNA technology.
Polymerase chain reaction (PCR) is a sophisticated, automated technique for amplifying DNA from very small amounts of sample.
The DNA to be amplified is mixed with specific primers, dATPs, dCTP, dGTP, dTTP, and a heat-stable form of DNA polymerase.
The mixture undergoes 20 to 40 rounds of DNA polymerization via cycling the temperatures so that the DNA strands separate, the primers anneal, and the polymerase fills in the DNA. Each cycle doubles the target DNA.
The technique has revolutionized forensic science, as DNA can be ampli-fied from just a few cells and then the DNA analyzed and identified.
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