Primer Design

An overview of the science behind primers as well as how to design primers on the Benchling platform.

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Primers are key ingredients in DNA synthesis, a process that occurs in sequencing, cloning, PCR, and other molecular biology methods in the lab.

With Benchling, teams can easily access shared primer libraries, upload new primer sequences, or design brand new primers. Link primer information directly in the Benchling Notebook and Benchling Registry providing full traceability for every experiment where a primer was used. Be able to easily attribute results from experiments with the exact set of primers used, or see which sequences a primer is associated with. Once primers are designed, run in silico PCR, or use them to plan critical tasks such as restriction cloning, Golden Gate assembly, and Gibson cloning.

What are primers?

Primers are simple but key ingredients for DNA synthesis both within our bodies and within scientific experiments. Primers can also be called oligonucleotides and are literally small pieces of single-stranded nucleotides, generally about 5 – 22 base pairs in length. The main property of primers is they must be complementary to the DNA template strand, serving to “prime” the strand for DNA polymerase to bind to and initiate DNA synthesis.

What types of primers are there? RNA vs DNA primers

Living organisms solely use RNA primers, while primers used in the lab are usually DNA primers. Scientists use DNA primers instead of RNA primers for a variety or reasons. DNA primers are far more stable and easier to store, and they require less hard-to-come-by enzymes to initiate synthesis (see Chapter 2, Figure 1).

DNA Primers


In vitro: PCR amplification, DNA sequencing, cloning, and more


Amplification is temperature-dependent, requiring fewer proteins


18 – 24 base pairs


Chemically synthesized by scientists


Longer-lived, more stable

RNA Primers


In vivo: DNA replication


Replication is enzyme-dependent catalytic reaction, requiring several proteins


10 – 20 base pairs


Primase (a type of RNA polymerase)


Shorter-lived, more reactive

The binding of DNA or RNA primers to the template strand initiates the enzyme responsible for DNA synthesis, DNA polymerase, to begin adding nucleotides to the reactive 3’-hydroxyl end (called the “3 prime end”) of a existing nucleic acid on the primer, elongating and replicating the parent strand. 

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