Primer (molecular biology)

A primer is a short, single-stranded nucleic acid used by all living organisms in the initiation of DNA synthesis.

Later, the RNA strands must be removed accurately and replace them with DNA nucleotides forming a gap region known as a nick that is filled in using an enzyme called ligase.

A class of enzymes called primases add a complementary RNA primer to the reading template de novo on both the leading and lagging strands.

Unlike in the leading strand, this method results in the repeated starting and stopping of DNA synthesis, requiring multiple RNA primers.

The DNA polymerase component of reverse transcriptase requires an existing 3' end to begin synthesis.

[1] In prokaryotes, DNA polymerase I synthesizes the Okazaki fragment until it reaches the previous RNA primer.

Then the enzyme simultaneously acts as a 5′→3′ exonuclease, removing primer ribonucleotides in front and adding deoxyribonucleotides behind.

[3] Nick translation refers to the synchronized activity of polymerase I in removing the RNA primer and adding deoxyribonucleotides.

At the end, when all the RNA primers have been removed, nicks form between the Okazaki fragments that are filled-in with deoxyribonucleotides using an enzyme known as ligase1, through a process called ligation.

In solution, the primer spontaneously hybridizes with the template through Watson-Crick base pairing before being extended by DNA polymerase.

The ability to create and customize synthetic primers has proven an invaluable tool necessary to a variety of molecular biological approaches involving the analysis of DNA.

[1] The polymerase chain reaction (PCR) uses a pair of custom primers to direct DNA elongation toward each other at opposite ends of the sequence being amplified.

These primers are typically between 18 and 24 bases in length and must code for only the specific upstream and downstream sites of the sequence being amplified.

Regions high in mononucleotide and dinucleotide repeats should be avoided, as loop formation can occur and contribute to mishybridization.

One application for this practice is for use in TA cloning, a special subcloning technique similar to PCR, where efficiency can be increased by adding AG tails to the 5′ and the 3′ ends.

Degenerate primers may not perfectly hybridize with a target sequence, which can greatly reduce the specificity of the PCR amplification.