Polymerase Chain Reaction
A Typical Pcr Reaction
A typical PCR reaction consists of the following components, mixed together in a solution with a total volume of between 25 and 100 microliters. The solution must include the template DNA, the primers, nucleotides to serve as building blocks for the newly forming DNA, DNA polymerase to catalyze the synthesis, and buffers and salts, usually including magnesium, that are required for optimal activity of the DNA polymerase. The template can be an unpurified mixture of DNA, such as DNA extracted from a swab of cheek cells from a patient or crime suspect.
To perform the PCR reaction, the tube containing the solution is placed into a machine called a DNA thermal cycler. Thermal cyclers are basically programmable heating blocks. They usually contain a thick aluminum block with holes in which PCR reaction tubes can fit snugly. The block can be rapidly cooled or heated to specific temperatures, for specific lengths of time, under programmable computer control. Each cycle in a PCR reaction is controlled by changing the temperature of the block and, therefore, of the reaction mixture.
The first step in PCR is to heat the mixture to a high temperature, usually 94 to 95 °C, for about five minutes. The hydrogen bonds that hold together the two strands of a double helix are broken at these temperatures, and the DNA separates into single strands. This process is termed denaturation.
In the second step, the PCR mixture is cooled to a lower temperature, typically between about 50 °C and 65 °C. This allows the primers to anneal to their specific complementary sequences in the template DNA. The temperature for this step is chosen carefully to be just low enough to allow the primers to bind, but no cooler. A lower annealing temperature might allow the primers to bind to regions in the template DNA that are not perfect complements, which could lead to the amplification of non-specific sequences.
The optimal annealing temperature for a set of primers can be determined by a formula that is based on the nucleotide composition of the primers, but it is often a matter of trial and error to find the best annealing temperature. The annealing step usually takes about fifteen to thirty seconds, an amazingly short time considering that the primers must "scan" through the template DNA to find their proper binding sites.
In the third step, the reaction is heated again, usually to about 72 °C, the temperature at which the DNA polymerase is most active. Most enzymes are destroyed at 72 °C. In the early days of PCR, scientists used a DNA polymerase that was derived from the bacterium Escherichia coli, which itself is most active at human body temperature, 37 °C. But the E. coli polymerase was destroyed at the high temperatures required for the denaturation and annealing steps, and the polymerase therefore had to be added anew to the reaction, during each PCR cycle.
To solve this problem, scientists purified DNA polymerases from microorganisms that live in hot springs or in deep-sea thermal vents. These organisms' enzymes are most active at high temperatures. The most commonly used enzyme for PCR is called Taq DNA polymerase, which was originally purified from the hot-spring bacterium Thermus aquaticus. (Most commercially available preparations today are recombinant versions, produced in engineered E. coli strains.)
At 72 °C, Taq DNA polymerase adds nucleotides to the 3′ ends of annealed primers at the rate of about two thousand nucleotides per minute. Therefore, to amplify a sequence that is one thousand nucleotides long, the primer extension step must last about thirty seconds at 72 °C. By the end of this step, each template strand has a new complementary strand. This completes the first cycle of the PCR reaction.
The cycle can be repeated, at that point, by restarting the denaturation step. In the next cycle, the original two DNA strands will serve again as templates, as will the two newly synthesized strands. In this way, the number of templates has doubled, and it will double again with each successive cycle.
At the end of the reaction, the tube contains DNA fragments that are almost solely copies of the target DNA. The original template DNA mixture is still present, but for the purpose of most applications (with the exception of subsequent PCR experiments), it is present in negligible amounts compared to the PCR product. The amplified DNA can be analyzed by gel electrophoresis, ligated into a cloning vector, labeled for use as a hybridization probe, or used in numerous other experimental procedures.
- Polymerase Chain Reaction - Contamination In Pcr Reactions
- Polymerase Chain Reaction - Designing Primers
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