Primer (molecular biology)

A primer is a strand of nucleic acid that serves as a starting point for DNA replication. 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). This RNA is produced by primase, and is later removed and replaced with DNA by a repair polymerase.

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 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.

Uses of synthetic primers
DNA sequencing is used to determine the nucleotides in a DNA strand; the chain termination method (dideoxy sequencing or Sanger method) uses a primer as a start marker for the chain reaction.

In PCR, primers are used to determine the DNA fragment to be amplified by the PCR process. The length of primers is usually not more than 30 nucleotides, and they match exactly the beginning and the end of the DNA fragment to be amplified. They direct replication towards each other - the extension of one primer by polymerase then becomes the template for the other, leading to an exponential increase in the target segment.

It is worth noting that primers are not essentially always necessary for DNA synthesis and can in fact be used by viral polymerases, e.g. influenza, for RNA synthesis.

PCR primer design
The melting temperature of a primer is defined as the temperature at which 50% of that same DNA molecule species form a stable double helix and the other 50% have been separated to single strand molecules. The melting temperature required increases with the length of the primer. Primers that are too short would anneal at several positions on a long DNA template, which would result in non-specific copies. On the other hand, the length of a primer is limited by the temperature required to melt it. Melting temperatures that are too high, i.e., above 80°C, can also cause problems since the DNA polymerases used for PCR are less active at such temperatures. The optimum length of a primer is generally from 20 to 30 nucleotides with a melting temperature between about 55°C and 65°C.

Pairs of primers should have the similar melting temperatures as annealing in a PCR reaction occurs for both simultaneously. 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 Tm significantly lower than the annealing temperature may fail to anneal and extend at all.

Primer sequences need to be chosen to uniquely select for a region of DNA, avoiding the possibility of mishybridization to a similar sequence nearby. Mononucleotide repeats should be avoided, as loop formation can occur and contribute to mishybridization. Primers should not easily anneal with other primers in the mixture (either other copies of same or the reverse direction primer); this phenomenon can lead to the production of 'primer dimer' products contaminating the mixture. Primers should also not anneal strongly to themselves, as internal hairpins and loops could hinder the annealing with the template DNA.

Degenerate primers
Sometimes degenerate primers are used. These are actually mixtures of similar, but not identical, primers. They may be convenient if the same gene is to be amplified from different organisms, as the genes themselves are probably similar but not identical. The other use for degenerate primers is when primer design is based on protein sequence. As several different codons can code for one amino acid, it is often difficult to deduce which codon is used in a particular case. Therefore primer sequence corresponding to the amino acid isoleucine might be "ATH", where A stands for adenine, T for thymine, and H for adenine, thymine, or cytosine, according to the genetic code for each codon, using the IUPAC symbols for degenerate bases. Use of degenerate primers can greatly reduce the specificity of the PCR amplification. The problem can be partly solved by using touchdown PCR.

Degenerate primers are widely used and extremely useful in the field of microbial ecology. They allow for the amplification of genes from thus far uncultivated microorganisms or allow the recovery of genes from organisms where genomic information is not available. Usually, degenerate primers are designed by aligning gene sequencing found in GenBank. Differences among sequences are accounted for by using IUPAC degeneracies for individual bases. PCR primers are then synthesized as a mixture of primers corresponding to all permutations.