Polymerase chain reaction

The polymerase chain reaction, or PCR, is a lab technique for quickly making many copies of a specific DNA sequence, which allows for detailed analysis. It was created in 1983 by American biochemist Kary Mullis while he worked at Cetus Corporation. In 1993, Mullis shared the Nobel Prize in Chemistry with Michael Smith, who developed other key methods for working with DNA. PCR is essential for many genetic tests, research projects, the study of ancient DNA, and identifying disease-causing agents. The method works by taking a tiny amount of DNA and, through repeated temperature changes, making millions of copies in an exponential chain reaction. It is now a routine and often necessary tool in medical labs, biomedical research, and forensic science.

Most PCR methods rely on thermal cycling, which repeatedly heats and cools the reaction mixture. These temperature changes allow for two key steps: separating the DNA strands (melting) and building new strands using an enzyme. The main ingredients are primers—short, single-stranded DNA fragments called oligonucleotides that match the target DNA sequence—and a heat-stable DNA polymerase. First, the double helix is split apart at high temperature (denaturation). Next, the temperature is lowered so the primers can attach to their matching sequences on the DNA. Then, the DNA polymerase uses free nucleotides to build a new strand along each template. As the process repeats, the newly made DNA also becomes a template, creating a chain reaction that rapidly multiplies the original target.

Nearly all PCR uses a heat-stable DNA polymerase, most commonly Taq polymerase, which comes from the thermophilic bacterium *Thermus aquaticus*. If the polymerase were sensitive to heat, it would be destroyed during the denaturation step. Before Taq was available, fresh polymerase had to be added manually each cycle, which was slow and expensive. PCR is used for many purposes: cloning DNA for sequencing, manipulating genes, creating mutations, building evolutionary trees, analyzing gene function, diagnosing and tracking genetic disorders, amplifying ancient DNA, creating genetic fingerprints for forensics and parentage testing, and detecting pathogens in infectious disease tests.

**Principles**

PCR targets a specific region of a DNA strand. Most methods amplify fragments between 0.1 and 10 kilo-base pairs (kbp), though some can handle up to 40 kbp. The amount of product is limited by the available ingredients, which run out as the reaction proceeds. A basic PCR setup includes: a DNA template containing the target region; a DNA polymerase (often heat-resistant Taq, or Pfu polymerase, which is more accurate because it proofreads but works slower—mixing a little Pfu with Taq combines speed and accuracy); two primers that match the 3' ends of both strands of the target (DNA polymerase needs a double-stranded starting point, so primers provide that); deoxynucleoside triphosphates (dNTPs), the building blocks for new DNA; a buffer to keep the right chemical conditions; and bivalent cations like magnesium (Mg²⁺) or manganese (Mn²⁺)—Mg²⁺ is most common, while Mn²⁺ can increase errors for mutagenesis—plus monovalent cations like potassium (K⁺). The reaction is usually done in 10–200 µL volumes inside small tubes (0.2–0.5 mL) placed in a thermal cycler. Modern cyclers use a Peltier device to heat and cool the block by reversing the electric current. Thin-walled tubes help heat transfer quickly, and most cyclers have heated lids to stop condensation; older models without heated lids need a layer of oil or a wax ball on top.

**Procedure**

A typical PCR runs 20–40 cycles of temperature changes, each with two or three distinct steps. The process often starts with a single high-temperature step (above 90 °C) and ends with a final hold for product extension or short-term storage. The exact temperatures and times depend on the specific reaction.