Polymerase chain reaction (PCR) is a laboratory technique for producing millions to billions of copies of a selected DNA segment. It uses short synthetic primers to identify the target and repeated rounds of enzyme-mediated copying to amplify it. PCR is a foundational method in molecular biology, enabling analysis of genetic material that would otherwise be present in quantities too small for many laboratory procedures. Its applications include research, identification, and clinical testing. (genome.gov)
Development
PCR was developed by Kary Mullis and was first publicly presented in 1985. In 1993, Mullis received half of the Nobel Prize in Chemistry for inventing the method; the other half went to Michael Smith for separate work on site-directed mutagenesis. The award recognized methods that substantially expanded experimental manipulation and analysis of DNA. (nobelprize.org)
An important practical development was the use of Taq polymerase, a heat-resistant DNA-copying enzyme originally isolated from the bacterium Thermus aquaticus. Its thermal stability allows it to function through repeated heating cycles. Automated instruments called thermal cyclers control these temperature changes, making amplification reproducible and reducing manual handling. Subsequent improvements have included polymerases with greater accuracy and reagents adapted to difficult templates. (thermofisher.com)
Molecular principle and components
PCR depends on complementary base pairing and the activity of DNA polymerase, an enzyme that synthesizes a new DNA strand using an existing strand as a template. Two primers bind to opposite template strands at sites flanking the selected region. Their orientation allows synthesis toward and through the target, establishing the boundaries of the amplified fragment, or amplicon. Polymerase extends each primer from its 3′ end. (thermofisher.com)
A typical reaction contains template DNA, both primers, a thermostable polymerase, the four deoxynucleoside triphosphates, and a buffer containing magnesium ions. The nucleotide triphosphates supply the building blocks for new strands. Magnesium supports polymerase activity, while buffer composition and pH influence reaction performance. Primer sequence, template quality, and reagent balance affect specificity and yield. (thermofisher.com)
Unlike amplification inside a living cell, PCR operates in a laboratory reaction mixture. Newly synthesized DNA becomes template material in subsequent cycles, creating the chain-like repetition that gives the method its name. (genome.gov)
Thermal cycling and product detection
Conventional PCR repeats three principal stages:
- Denaturation: heating, commonly around 94–98°C, separates double-stranded DNA into single strands.
- Annealing: cooling allows primers to bind complementary sequences. The temperature depends on primer composition and reaction conditions.
- Extension: polymerase adds nucleotides to the bound primers. For many commonly used enzymes, this occurs around 68–72°C. (videos.thermofisher.com)
A run commonly includes roughly 25–35 cycles, although the number varies with the assay. Under ideal conditions, amplification approaches a doubling per cycle during the exponential phase. Actual efficiency is lower or variable, and amplification eventually slows as reaction conditions become limiting. The final plateau therefore prevents conventional endpoint yield from serving as a straightforward measure of the starting DNA quantity. (thermofisher.com)
Endpoint products are often examined using gel electrophoresis, which separates DNA fragments by size. A band near the expected size supports successful amplification, but size alone does not establish sequence identity. Products can subsequently be used in DNA sequencing, cloning, or other analyses. (videos.thermofisher.com)
Major variants
Reverse-transcription PCR (RT-PCR) begins with RNA. Reverse transcriptase converts RNA into complementary DNA, which is then amplified. This makes RNA targets accessible to PCR-based analysis, including messenger RNA and viral RNA. “RT” denotes reverse transcription rather than real-time measurement; the two processes can nevertheless be combined in RT-qPCR. (bio-rad.com)
Quantitative real-time PCR (qPCR) monitors amplification through fluorescent signals during cycling. The cycle at which a signal crosses a defined threshold provides information about the initial target quantity. Interpretation depends on amplification efficiency, calibration or normalization, and appropriate controls. This differs from measuring accumulated product only after amplification has finished. (bio-rad.com)
Digital PCR partitions a sample into many separate reactions. After amplification, partitions are classified as positive or negative. Poisson statistics account for partitions containing more than one target molecule, allowing estimation of target concentration without a conventional standard curve. Applications include rare-sequence detection and copy-number analysis. (bio-rad.com)
Applications
PCR supplies material for investigating genes, identifying mutations, and preparing fragments for sequencing or recombinant DNA work. In genetics, it supports analysis of inherited variation. It also contributed extensively to genome-mapping procedures in the Human Genome Project. (videos.thermofisher.com)
Forensic applications include DNA fingerprinting from small samples. Clinical assays use PCR-based methods in the diagnosis of genetic disorders and detection of bacteria or viruses. Such tests detect particular nucleic-acid sequences rather than directly observing an entire organism. For influenza, detection of viral RNA does not necessarily demonstrate viable virus or ongoing viral replication. (genome.gov)
Limitations and quality control
PCR can amplify unintended DNA introduced through contamination. Poor primer specificity can produce unrelated fragments, while damaged templates, inhibitory substances, or unsuitable reaction conditions can prevent amplification. Primers may also interact with one another, forming primer dimers instead of the intended product. (neb.com)
Controls distinguish these problems from genuine results. A no-template control helps detect contamination; a positive control checks that amplification can occur. In RNA-based experiments, a control lacking reverse transcriptase can reveal genomic DNA contamination. Internal controls can help identify inhibition or sample-quality differences. (bio-rad.com)
Polymerases also differ in copying accuracy. Taq lacks the proofreading activity found in some higher-fidelity enzymes. Proofreading polymerases can remove incorrectly incorporated bases through 3′-to-5′ exonuclease activity, reducing sequence errors in applications where accurate copies are important. (neb.com)