Polymerase Chain Reaction, or PCR, is a laboratory method used to make millions to billions of copies of a specific DNA segment. This technique allows scientists to amplify a very small amount of DNA to a quantity large enough for study. The process is analogous to a molecular photocopier that isolates and reproduces a particular DNA sequence. PCR is a fundamental procedure in molecular biology and biomedical research.
The Key Ingredients for PCR
A successful PCR experiment relies on several components, with the first being the DNA template. This is the original DNA sample containing the target sequence of interest, which can be sourced from materials like blood, saliva, or ancient biological samples. The quality and quantity of the template DNA can influence the outcome of the reaction.
To specifically target a region for amplification, two types of primers are required. Primers are short, single-stranded DNA fragments, known as oligonucleotides, custom-designed to be complementary to the sequences at the beginning and end of the target region. By binding to the template DNA, these primers define the exact segment that will be copied, which determines the specificity of the PCR reaction.
The enzyme responsible for synthesizing new DNA is a DNA polymerase. In PCR, a special heat-stable polymerase, most famously Taq polymerase, is used. This enzyme was originally isolated from Thermus aquaticus, a bacterium that lives in hot springs, and its ability to withstand high temperatures allows for the repeated heating and cooling cycles of PCR without adding new enzyme each cycle.
The final components are the deoxynucleoside triphosphates (dNTPs), which are the building blocks of DNA. These are the individual units of adenine (A), guanine (G), cytosine (C), and thymine (T). The DNA polymerase uses these dNTPs to construct the new DNA strands in an order that is complementary to the DNA template.
The Three Stages of a PCR Cycle
The amplification of DNA through PCR is achieved by subjecting the reaction mixture to a series of repeated temperature changes. These cycles are managed by a machine called a thermal cycler, which precisely controls the temperature and duration of each step. A typical PCR process consists of 25 to 35 of these cycles, with each cycle comprising the three distinct stages.
The first stage is denaturation, which occurs at a high temperature, typically between 94 and 98 degrees Celsius. This intense heat breaks the hydrogen bonds holding the two strands of the template DNA double helix together, causing them to separate into single strands. This provides the single-stranded templates for the synthesis of new DNA.
Following denaturation, the temperature is lowered for the annealing stage, usually to a range between 50 and 65 degrees Celsius. This reduction in temperature allows the primers to bind, or anneal, to their complementary sequences on the single-stranded DNA templates. The specific annealing temperature depends on the primer composition and is optimized for specific binding.
The final stage of the cycle is extension, or elongation, carried out at a temperature of around 72 degrees Celsius. At this temperature, the Taq polymerase enzyme is optimally active. It attaches to the primer-template complex and begins to add dNTPs, synthesizing a new DNA strand complementary to the template. This process effectively doubles the number of copies of the target DNA sequence.
Achieving Exponential Amplification
PCR generates an exponential number of DNA copies because the DNA strands synthesized during one cycle become the templates for the next. After the first cycle, what was one double-stranded DNA molecule becomes two. Those two molecules are then denatured, and in the second cycle, they serve as templates to create four molecules.
This doubling process continues with each subsequent cycle. Four copies become eight, eight become sixteen, and so on. This geometric progression allows for the rapid accumulation of the target DNA. Within a few hours, a process of 25 to 35 cycles can amplify a single DNA molecule into millions or billions of copies.
The exponential nature of this amplification makes PCR a highly sensitive technique. It can detect and amplify a target sequence from even minuscule starting amounts of DNA. However, the reaction does not continue indefinitely; eventually, components like dNTPs or primers become depleted, or the polymerase loses activity, causing the amplification to plateau.
Visualizing the Amplified DNA
After the PCR process is complete, a common method used to determine if the target DNA was successfully amplified is gel electrophoresis. This technique allows for the separation of DNA fragments based on their size, providing a visual confirmation of the PCR product.
In gel electrophoresis, the PCR products are loaded into a porous gel matrix, typically made of agarose. An electric current is applied across the gel, causing the negatively charged DNA molecules to move toward the positive electrode. Shorter DNA fragments navigate the pores of the gel more easily and therefore travel farther than longer fragments.
To see the separated DNA fragments, the gel is stained with a fluorescent dye that binds to DNA. When the gel is exposed to ultraviolet (UV) light, the DNA fragments appear as bands. If the PCR was successful, a distinct band corresponding to the expected size of the amplified target sequence will be visible, confirming the reaction produced the correct DNA product.