method

PCR

PCR is a method for amplifying specific DNA sequences through repeated cycles of heating and cooling.
32 related articles

Description

Polymerase chain reaction (PCR) is an enzymatic method of amplification (multiple copying) of DNA in a test tube (in vitro). It allows selectively synthesizing a huge number of copies of a given DNA fragment, using a thermostable DNA polymerase (an enzyme that withstands high temperatures) and temperature cycling. The method revolutionized molecular biology, medicine, and forensics.

History

The concept of DNA amplification via repeated replication cycles was proposed by Kary Mullis in 1983 while working at the biotechnology company Cetus Corporation. The idea came to him during an evening drive along a California highway. The first experiments using the Klenow fragment (unstable at high temperatures) were labor-intensive, as the enzyme had to be added after each denaturation cycle. The key breakthrough was the use of thermostable Taq DNA polymerase, isolated from thermophilic bacteria of Yellowstone, which allowed automation of the process. In 1993, Mullis received the Nobel Prize in Chemistry. Since then, the method has been continuously improved: real-time PCR (qPCR), digital PCR, and other variants have emerged.

How it works

The process consists of three main stages: (1) Denaturation at ~95°C — breaking hydrogen bonds between complementary DNA strands to form single-stranded templates. (2) Primer annealing at ~50–65°C — specific binding of short oligonucleotide primers to complementary sites on the templates. (3) Elongation at ~72°C — thermostable DNA polymerase synthesizes a new DNA strand starting from the 3' end of the primer, using deoxynucleotide triphosphates (dNTPs) as building blocks. The cycles are repeated 25–40 times in a thermal cycler. A key parameter is specificity, ensured by primer design and optimization of the annealing temperature. The quantitative version (qPCR) uses fluorescent probes to monitor product accumulation in real time, allowing determination of the initial template quantity.

In practice

Foundation of COVID-19 tests, paternity establishment, and criminal detection from a drop of blood.

Fun fact

The sensitivity of PCR is such that theoretically a single molecule of DNA can be detected in a sample. That is why coronavirus tests could detect infection at the earliest stages.

Concept graph →

Related articles · 36

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