PCR primer

A primer is a short strand of RNA or DNA (generally about 18-22 bases) that serves as a starting point for DNA synthesis. It is required for DNA replication because the enzymes that catalyze this process, 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 vivo DNA replication utilizes short strands of RNA called RNA primers to initiate DNA synthesis on both the leading and lagging strands — DNA primers are not seen in vivo in humans. These RNA primers can be made de novo.

On the other hand, many of the in vitro laboratory techniques that involve DNA polymerase in biochemistry and molecular biology (such as DNA sequencing and the polymerase chain reaction), use DNA primers because they are more temperature stable. In experiments, it is often important to use a primer with a similar Tm (melting temperature) to the template strand it will be hybridizing to. A primer with a Tm significantly higher than the reaction's annealing temperature may mishybridize and extend at an incorrect location along the DNA sequence, while one with a Tm significantly lower than the annealing temperature may fail to anneal and extend at all. 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.

The lagging strand of DNA 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.

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