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Short answer: In a PCR reaction, primers are short DNA molecules that bind to the flanking regions of the target DNA and give the DNA polymerase a 3′ end to extend. Without a primer that has annealed to the template, the polymerase has nothing to start from. The pair of primers also selects which segment of the genome is amplified.
Definition and scope
What is polymerase chain reaction (PCR)?
The National Human Genome Research Institute (NHGRI) glossary defines PCR as “a laboratory technique for rapidly producing (amplifying) millions to billions of copies of a specific segment of DNA, which can then be studied in greater detail.” It relies on multiple rounds of DNA synthesis to amplify that segment.
What is a PCR reaction?
A PCR reaction is one tube or well in which that amplification runs: template DNA, primers, a DNA polymerase, nucleotides and buffer are cycled through repeated temperature steps in a thermal cycler. For a setup walkthrough, see PCR protocol basics, and for the template side of the reaction, PCR template DNA.
What is a primer in PCR?
A primer is a short, synthetic DNA fragment. NHGRI describes PCR as using “short synthetic DNA fragments called primers to select a segment of the genome to be amplified.” Thermo Fisher’s PCR basics page describes the same molecules as “short DNA molecules called primers” that “bind to flanking regions of the target DNA.”
What is the function of primers in a PCR reaction?
Primers do two jobs.
- They mark where copying starts. Thermo Fisher explains that polymerases work through “the incorporation of nucleotides to extend primers at their 3′ ends in the 5′ to 3′ direction.” The polymerase extends a primer; it does not begin from bare template. Thermo Fisher’s end-point PCR primer page describes the extension step as the point where the polymerase binds and synthesizes a new strand of DNA once the primers have annealed to their complementary base-pair regions.
- They choose which segment is amplified. NHGRI says primers “select a segment of the genome to be amplified.” Because they bind to the flanking regions of the target, the DNA copied in each cycle is the stretch those binding sites enclose, and each round of synthesis adds to the number of copies of that segment.
What primers do at each step of a PCR cycle
A cycle has three stages. The table below sets out what happens to the DNA and to the primers at each stage.
| Stage | What happens to the DNA | What the primers do |
|---|---|---|
| Denaturation | “double-stranded DNA templates are heated to separate the strands.” (Thermo Fisher, PCR basics) | Nothing yet: the template strands are being made single-stranded so the primers can reach them. |
| Annealing | The temperature is lowered (Thermo Fisher gives 50-65 °C) so primers can anneal to their complementary base-pair regions (Thermo Fisher, end-point PCR primers). | Bind to the flanking regions of the target DNA. |
| Extension | The temperature rises to 72 °C, allowing the polymerase to bind and synthesize a new strand (Thermo Fisher, end-point PCR primers). | Their 3′ ends are extended by the DNA polymerase along the template strands. |
Repeating the cycle is what produces “multiple rounds of DNA synthesis” (NHGRI).
Forward and reverse primers, and related concepts
Thermo Fisher describes primers binding to flanking regions, meaning regions on either side of the target. That is why a standard reaction is set up with a pair of primers rather than one. For the practical rules on choosing them, see how to choose primers for PCR and, for checking specificity against a genome, NCBI BLAST for primers. For primers that pair with each other instead of the template, see qPCR melt curve analysis and primer dimers.
Where it matters for labs: a primer record checklist
Because the primers set both the start point and the target, the primer details belong in the lab’s reaction record. Thermo Fisher’s end-point PCR primer guidance gives the parameters worth writing down, with its own figures:
- Length: “In general, a length of 18-30 nucleotides for primers is good.”
- Melting temperature (Tm): Thermo Fisher advises making the Tm of the primers “between 65 °C and 75 °C, and within 5 °C of each other.”
- Annealing temperature: “Annealing temperatures are generally about 5 °C below the Tm of the primers.”
- Sequence, concentration and lot or order reference: record these alongside the above so a run can be repeated; these are record-keeping fields, not values from the sources.
The Tm range is the vendor’s own guidance for its products and chemistry. Other published guidance uses different ranges (Thermo Fisher itself gives 50-65 °C for the annealing step), and the CASRAI primer-choice guide shows them, so record which source and which polymerase system your Tm and annealing values came from rather than treating one range as universal.
Common misunderstandings
- “Primers are used only once.” Repeated cycles of primer binding and extension are what amplify the segment (NHGRI: “multiple rounds of DNA synthesis”).
- “Primers start working at the denaturation step.” Denaturation separates the strands; primers bind at annealing.
- “The polymerase finds the target on its own.” The polymerase extends the 3′ end of a primer; the primers are what direct it to the target region.
- “One Tm range applies to every primer.” The 65-75 °C range above is one vendor’s guidance, not a universal rule.
FAQ
What is the function of primers in a PCR reaction?
They anneal to the flanking regions of the target DNA and supply the 3′ end the DNA polymerase extends, which also selects the segment that is amplified.
What is a primer in PCR?
A short synthetic DNA fragment that binds to a complementary region of the template and is extended by the polymerase.
What is PCR?
A laboratory technique for rapidly producing millions to billions of copies of a specific segment of DNA (NHGRI).
What is a PCR reaction?
The mixture of template, primers, polymerase, nucleotides and buffer that is cycled through denaturation, annealing and extension.
Sources
- NHGRI, Polymerase Chain Reaction, genetics glossary, checked 29 October 2026.
- Thermo Fisher Scientific, PCR Basics, checked 29 October 2026.
- Thermo Fisher Scientific, End-point PCR primers support: getting started, checked 29 October 2026.
