Polymerase Chain Reaction (PCR) is a laboratory technique that functions like a molecular photocopier to amplify a specific segment of DNA. It generates millions of copies from a minimal starting sample, producing enough DNA for applications like forensic investigations, genetic testing, and diagnosing infectious diseases.
The Thermal Cycler
The thermal cycler is the instrument at the center of the PCR process, designed to precisely control and rapidly change temperatures. Reaction tubes with the DNA sample and reagents are placed into a thermal block inside the machine. This block heats and cools with speed and uniformity, cycling through a series of temperature stages dozens of times to achieve amplification.
Each PCR cycle has three steps defined by temperature. The first is denaturation, where the thermal cycler heats the block to approximately 95°C. This high temperature breaks the hydrogen bonds holding the DNA double helix together, causing the two strands to separate and provide templates for creating new DNA.
The second step is annealing, where the machine cools to between 50°C and 65°C. At this lower temperature, short DNA sequences called primers bind to their complementary sequences on the separated strands. These primers define the specific segment of DNA that will be copied.
The final step is extension. The temperature is raised to around 72°C, the optimal temperature for the DNA polymerase enzyme to function. The polymerase attaches to the primers and adds nucleotides, the building blocks of DNA, to create a new strand. This process doubles the amount of target DNA and is repeated for 30 to 40 cycles, resulting in massive amplification.
Essential Tools for Sample Preparation
Before amplification, sample preparation requires tools to handle minute liquid volumes with high precision. The primary instruments are micropipettes, which accurately measure and transfer microliter volumes of PCR reagents like the DNA template, primers, and polymerase. Accurate pipetting ensures the reaction components are in the correct concentrations for success.
After combining reagents in PCR tubes, a microcentrifuge is used. This device spins the tubes at high speeds, using centrifugal force to pull all liquid droplets to the bottom. This ensures all components are collected in the reaction mixture and none are left on the sides of the tube.
A vortex mixer may be used to ensure the components are thoroughly mixed. This tool rapidly agitates the tube, creating a vortex that homogenizes the solution for an efficient and uniform reaction. All preparation steps are performed in a sterile environment, like a laminar flow hood, to prevent contamination from foreign DNA.
Visualizing PCR Results
After PCR is complete, the DNA products are analyzed to confirm the reaction was successful by separating the fragments based on size. This is accomplished using gel electrophoresis, which requires its own equipment. This step is performed in a post-PCR area to avoid contaminating pre-PCR reagents.
The main apparatus is the gel electrophoresis box, which is connected to a power supply. A gel is placed into the tank and filled with a conductive buffer solution. The DNA samples are mixed with a loading dye and pipetted into wells at one end of the gel. The power supply creates an electrical field, causing the negatively charged DNA to migrate through the gel.
Shorter DNA fragments move more quickly through the porous gel than longer ones, resulting in separation by size. To make the DNA visible, the gel is stained with a fluorescent dye that binds to it. A UV transilluminator then emits ultraviolet light, causing the dye to fluoresce and reveal the DNA as distinct bands, which are captured by an imaging system. A band at the expected size indicates successful amplification.
Real-Time PCR Systems
Real-time PCR systems, or qPCR machines, are an advanced method for analysis. These instruments integrate DNA amplification and detection into a single process. A qPCR machine contains an optical module that measures fluorescence inside each reaction tube during every cycle, eliminating the need for post-PCR analysis like gel electrophoresis.
Real-time PCR uses fluorescent reporters, such as DNA-binding dyes or probes, in the reaction mix. As the amount of amplified DNA increases with each cycle, the fluorescence signal grows proportionally. The machine’s optical system excites the reporters with a light source and a detector measures the emitted fluorescence, which is plotted by a computer in real time.
This integration of amplification and detection provides the main advantage of qPCR: quantification. The system determines not just the presence of a DNA sequence, but also its initial quantity in the sample. The cycle at which fluorescence crosses a certain threshold is used to calculate the starting amount of DNA. This quantitative capability makes real-time PCR a useful tool for applications like measuring gene expression or determining viral load.