Reverse transcription polymerase chain reaction (RT-PCR)

SIGNIFICANCE: This technique uses ribonucleic acid (RNA) and a heat-resistant enzyme to produce many copies of deoxyribonucleic acid (DNA). The development of RT-PCR has greatly advanced genetics research and is used in the detection of many inherited disorders and infections.

The Basics of RT-PCR

DNA, the genetic material of most organisms, is used to make a form of nucleic acid called ribonucleic acid (RNA) that provides the blueprint to actually manufacture molecules such as proteins. This process is called transcription. Reverse transcription (RT) involves the use of RNA as the template to make DNA. This involves an enzyme called reverse transcriptase.

In RT-PCR, a strand of RNA is reverse transcribed to produce what is called complementary DNA (cDNA). It is the cDNA that is subsequently amplified in number in the PCR reaction.

RT-PCR used to be done by hand. Now, the process is carried out in an automated machine that can produce tens of thousands of copies of the selected sequence of DNA within hours. Organizations such as The Institute for Genomic Research (TIGR) that have dozens of PCR machines are capable of producing millions of copies of DNA in a day.

The Components of PCR

PCR relies on the presence of a template, deoxyribonucleotides, a pair of primers, and DNA polymerase. The template is the RNA strand.

Deoxyribonucleotides are the building blocks of DNA. There are four deoxyribonucleotides: adenine, thymine, cytosine, and guanine. Their three-dimensional structures are such that adenine will bond with thymine, and cytosine will bond with guanine. In the double strand of DNA that forms the helical structure found in the cell, the two strands will be complementary to one another; thus, a sequence of adenine-thymine-guanine on one strand will be mirrored by thymine-adenine-cytosine on the other strand.

Primers are sequences of deoxyribonucleotides that have been deliberately constructed to be complementary with the sequences of a portion of each DNA strand. This causes the primers to associate with one or the other of the DNA strands. Binding of primer to DNA is important in the PCR process.

DNA polymerase is an enzyme that catalyzes the construction of DNA from the building blocks that are supplied. PCR is carried out at an elevated temperature that helps keep the DNA strands separated from each other, so the polymerase must be capable of functioning at the higher temperatures (many proteins, including enzymes, change their structure at higher temperatures, causing loss of function). PCR became possible with the discovery of Taq polymerase, an enzyme made by the thermophilic (“heat-loving”) bacterium Thermus aquaticus, which naturally lives in hot springs.

The Three Steps of PCR

Part of the sequence of the targeted DNA has to be known in order to design the primers. In the first step, the targeted double-stranded DNA is heated to more than 90 degrees Celsius (194 degrees Fahrenheit). During this process, the two strands of the targeted DNA separate from each other. Each strand is capable of being a template.

The second step involves a gradual decrease to a temperature at which the two DNA strands would normally reassociate, but primers are also present in the solution at a greater concentration than DNA. This causes reassociation to occur between the primers and the specific complementary regions on each DNA strand. Complete reassociation of the DNA strands cannot occur. The association between the primers and the DNA will produce a short region of two strands, with a longer region having a single strand of the DNA.

The third step, amplification, focuses on these single strands of DNA. In the presence of the necessary deoxyribonucleotides and other compounds, DNA polymerase catalyzes the manufacture of a complementary strand of DNA. At the end of this process, the newly constructed strands can be separated at high temperature to begin the cycle again.

As the cycles are repeated again and again, the number of each target sequence doubles with each cycle (a logarithmic increase).

PCR reactions are carried out in an apparatus called a thermocycler, which is designed to change temperatures automatically. The various conditions of temperature and reaction times can be programmed, and the procedure occurs without operator assistance.

Impact

American molecular biologist Kary Mullis developed PCR in the 1970s. The ability to quickly amplify DNA revolutionized molecular biology. In 1993, Mullis received the Nobel Prize in Physiology or Medicine. RT-PCR has also greatly advanced genetics research. For example, the technique was invaluable in the sequencing of the human genome. RT-PCR is also widely used in the detection of many inherited disorders. Many people first became aware of the technique during the COVID-19 pandemic, when PCR testing for the SARS-CoV-2 virus was widely used.

Key terms

• deoxyribonucleotide: the building block of DNA consisting of a nitrogen-containing molecule, deoxyribose-containing sugar, and phosphate(s)
• enzyme: a protein that allows a reaction to proceed more quickly or with less supplied energy, but which is not altered by the reaction
• protein: a molecule composed of amino acids
• primer: a fragment of nucleic acid that is used to initiate the replication of DNA
• reverse transcriptase: enzyme that catalyzes the formation of DNA from RNA
• RNA: ribonucleic acid; a form of genetic material composed of nucleotides

Bibliography

"COVID-19 and PCR Testing." Cleveland Clinic, 21 Jan. 2025, my.clevelandclinic.org/health/diagnostics/21462-covid-19-and-pcr-testing. Accessed 11 Dec. 2025.

Hodge, Russ. The Future of Genetics: Beyond the Human Genome Project. New York: Facts On File, 2009.

Read, Andrew. New Clinical Genetics. Oxfordshire, England: Scion, 2007.

Strachan, Tom. Human Molecular Genetics. London: Garland Science, 2003.

Tagarro, Alfredo, et al. "Dynamics of Reverse Transcription-Polymerase Chain Reaction and Serologic Test Results in Children with SARS-CoV-2 Infection." The Journal of Pediatrics, vol. 241, 2022, pp. 126-132.e3, doi.org/10.1016/j.jpeds.2021.09.029. Accessed 11 Dec. 2025.