Primer (molecular biology)

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Primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. They are required because the enzymes that catalyze replication, DNA polymerases, can only add new nucleotides to an existing strand of DNA. The polymerase starts replication at the 3'-end of the primer, and copies the opposite strand.

In most cases of natural DNA replication, the primer for DNA synthesis and replication is a short strand of RNA (which can be made de novo).

Many of the laboratory techniques of biochemistry and molecular biology that involve DNA polymerase, such as DNA sequencing and the polymerase chain reaction (PCR), require DNA primers. These primers are usually short, chemically synthesized oligonucleotides, with a length of about twenty bases. They are hybridized to a target DNA, which is then copied by the polymerase.

Contents

Mechanism in vivo

The lagging strand is that strand of the DNA double helix that is orientated in a 5' to 3' manner. Therefore, its complement must be synthesized in a 3'→5' manner. Because DNA polymerase III cannot synthesize in the 3'→5' direction, the lagging strand is synthesized in short segments known as Okazaki fragments. Along the lagging strand's template, primase builds RNA primers in short bursts. DNA polymerases are then able to use the free 3'-OH groups on the RNA primers to synthesize DNA in the 5'→3' direction.

The RNA fragments are then removed by DNA polymerase I for prokaryotes or DNA polymerase δ for eukaryotes (different mechanisms are used in eukaryotes and prokaryotes) and new deoxyribonucleotides are added to fill the gaps where the RNA was present. DNA ligase then joins the deoxyribonucleotides together, completing the synthesis of the lagging strand.

Primer removal

In eukaryotic primer removal, DNA polymerase δ extends the Okazaki fragment in 5' to 3' direction, and when it encounters the RNA primer from the previous Okazaki fragment, displacing the 5′ end of the primer into a single-stranded RNA flap, which is removed by nuclease cleavage. Cleavage of the RNA flaps involves either endonuclease 1 (FEN1) cleavage of short flaps, or coating of long flaps by the single-stranded DNA binding protein replication protein A (RPA) and sequential cleavage by Dna2 nuclease and FEN1.[1]

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