The Polymerase Chain Reaction (PCR) is a laboratory technique used to create millions to billions of copies of a specific DNA segment. This process, often referred to as “molecular photocopying,” rapidly amplifies DNA for detailed study. Developed in 1983 by Kary Mullis, PCR has fundamentally transformed molecular biology.
Understanding PCR
PCR’s primary goal is to produce a large quantity of a specific DNA sequence from a very small initial sample. This amplification is essential for molecular and genetic analyses, as original samples often lack sufficient DNA. For instance, studying a specific gene or detecting a pathogen would be nearly impossible without it.
PCR allows researchers to isolate DNA fragments for sequencing, analyze genetic markers, or identify the presence of infectious agents. The technique’s sensitivity allows for the isolation and amplification of DNA from as little as a single cell.
The Essential Ingredients
PCR requires several specific components for DNA amplification. These include the DNA template, the original DNA segment for copying. Two short DNA sequences, called primers, typically 20 to 30 bases long, bind to either side of the target DNA, marking the start and end points.
Deoxynucleotide triphosphates (dNTPs) — A, G, C, and T — serve as building blocks for new DNA strands. A heat-stable enzyme, Taq polymerase, adds these dNTPs to synthesize new DNA. A buffer solution provides the optimal chemical environment for the reaction.
The PCR Cycle: Three Core Steps
PCR relies on thermal cycling, using repeated temperature changes to drive DNA amplification. A thermocycler precisely controls these shifts through multiple cycles. Each cycle consists of three distinct steps: denaturation, annealing, and extension.
Denaturation, the first step, involves heating the mixture to 94-98°C, separating the double-stranded DNA template into two single strands. Next, the temperature lowers to 50-65°C for annealing, where primers bind to their complementary sequences on the single-stranded DNA.
Finally, at 72°C, the extension step occurs. Taq DNA polymerase synthesizes new DNA strands by adding dNTPs to the primers, building a complementary sequence. This cycle repeats 20-40 times, theoretically doubling target DNA with each cycle, generating millions to billions of copies.
Visualizing the Results
After PCR, amplified DNA must be detected and visualized to confirm successful amplification and determine fragment size. Gel electrophoresis is a common method. Samples are loaded into a gel, which acts as a molecular sieve.
An electric current causes negatively charged DNA fragments to migrate towards the positive electrode. Smaller fragments move faster and further, while larger fragments move slower. After separation, DNA fragments are typically stained with a fluorescent dye, allowing viewing under ultraviolet light as distinct bands, indicating presence and size.
Where PCR Makes a Difference
In forensics, PCR is widely used for DNA fingerprinting, allowing investigators to identify individuals from tiny samples found at crime scenes. Medical diagnostics heavily rely on PCR for detecting pathogens, such as viruses and bacteria, and for diagnosing genetic disorders by identifying specific gene mutations. The technology was, for instance, a benchmark method for detecting the SARS-CoV-2 virus during the COVID-19 pandemic.
In research, PCR is indispensable for gene cloning, studying gene expression, and analyzing genetic variations. It also plays a significant role in paternity testing, confirming biological relationships by comparing DNA profiles.