The Polymerase Chain Reaction, or PCR, is a powerful molecular biology technique used to create millions or even billions of copies of a specific segment of DNA. This process allows researchers to amplify a particular DNA sequence from a small initial sample, making it detectable and usable for various downstream applications. PCR essentially mimics the natural DNA replication process that occurs within living cells, but it does so in a controlled laboratory setting. The ability to generate large quantities of specific DNA has made PCR an indispensable tool in fields ranging from medical diagnostics to forensic analysis.
Essential Components for PCR
Setting up a PCR requires several specific ingredients, each playing a distinct role in the amplification process. The DNA template is the original DNA molecule containing the target sequence to be copied; it can originate from various sources, such as genomic DNA, complementary DNA (cDNA), or plasmid DNA. DNA polymerase, particularly a heat-stable version like Taq polymerase, isolated from the bacterium Thermus aquaticus, is responsible for synthesizing new DNA strands and can withstand the high temperatures required during the PCR cycling.
Primers are short, single-stranded DNA fragments, typically 18 to 22 nucleotides long, that bind to specific regions on either side of the DNA segment to be amplified. Two primers, a forward and a reverse primer, are used to define the exact starting points for DNA synthesis and ensure only the desired region is copied. Deoxynucleotide triphosphates (dNTPs), which include dATP, dCTP, dGTP, and dTTP, are the individual building blocks (nucleotides) that the DNA polymerase uses to construct new DNA strands. These are typically added in equal molar concentrations to ensure balanced DNA synthesis.
A reaction buffer provides the optimal chemical environment, including the necessary pH and salt concentrations, for the DNA polymerase to function effectively, often containing magnesium ions (Mg²⁺) which are cofactors for the enzyme. Essential equipment for setting up PCR includes sterile PCR tubes, precise pipettes for accurate liquid handling, and a thermocycler, which is the machine that controls the temperature changes throughout the reaction.
Preparing the PCR Reaction
Accurate preparation of the PCR reaction mixture is important for successful amplification. The components are typically added to small, thin-walled PCR tubes, which are designed for efficient heat transfer within the thermocycler.
A common approach for preparing multiple reactions is to create a “master mix” containing all common reagents except the DNA template and primers. This strategy helps minimize errors and contamination.
The typical order of adding reagents to the tube or master mix involves starting with water, followed by the reaction buffer, dNTPs, and then the primers. The DNA template is usually added next, and the DNA polymerase is added last to ensure its activity is preserved. After all components are added, the mixture should be gently mixed and then briefly centrifuged to collect all liquids at the bottom of the tube.
Running the Thermocycler Program
Once the PCR reaction mixture is prepared, the tubes are placed into a thermocycler, a specialized instrument that precisely controls temperature changes. The PCR process involves repeated cycles of three main temperature-dependent steps: denaturation, annealing, and extension. Denaturation involves heating the reaction to a high temperature, typically 94–98°C, for about 15-30 seconds, which breaks the hydrogen bonds holding the double-stranded DNA template together, separating it into two single strands.
Following denaturation, the temperature is lowered to an annealing temperature, usually between 50–65°C, for 20-40 seconds. During this phase, the forward and reverse primers bind, or “anneal,” to their complementary sequences on the single-stranded DNA templates.
The final step is extension, where the temperature is raised to an optimal level for the DNA polymerase, typically around 72°C. At this temperature, the Taq polymerase synthesizes new DNA strands by adding dNTPs to the primers, extending them in the 5′ to 3′ direction along the template strand. These three steps are repeated for 25 to 40 cycles, leading to an exponential amplification of the target DNA segment.
Verifying PCR Results
After the thermocycler program is complete, the amplified DNA products, often called amplicons, need to be visualized to confirm the success of the PCR. The primary method for checking PCR results is gel electrophoresis.
This technique separates DNA fragments based on their size and electrical charge. DNA molecules are negatively charged, so when an electric current is applied across a gel matrix, they migrate towards the positive electrode. Smaller DNA fragments navigate through the gel’s pores more quickly and travel further than larger fragments, resulting in a separation by size.
To interpret the results, the gel is typically stained with a DNA-binding dye, which makes the DNA bands visible under ultraviolet light. A “DNA ladder” or marker, consisting of DNA fragments of known sizes, is run alongside the samples to estimate the size of the amplified PCR products. The presence of a distinct band at the expected size indicates successful amplification of the target DNA. Conversely, the absence of a band might suggest the PCR failed, while multiple bands or smears could indicate non-specific amplification or the presence of primer dimers, which are small fragments formed by primers binding to each other.