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Primer


This article is about molecular biology for alternate uses, see primer (disambiguation).

A primer is a nucleic acid strand (or related molecule) that serves as a starting point for DNA replication. A primer is required because most DNA polymerases (enzymes that catalyze the replication of DNA) cannot begin synthesizing a new DNA strand from scratch, but can only add to an existing strand of nucleotides.

In most natural DNA replication, the ultimate primer for DNA synthesis is a short strand of RNA. This RNA is produced by an RNA polymerase, and is later removed and replaced with DNA by a DNA polymerase.

Many laboratory techniques of molecular biology that involve DNA polymerases, such as DNA sequencing and polymerase chain reaction (PCR), require primers. The primers used for these techniques are usually short (about 20 bases), artificially synthesized DNA molecules. The actual construction of such primers starts with 3'-hydroxyl nucleosides attached to a so-called controlled-pore glass (CPG). The 5'-hydroxyl of the nucleosides is covered dimethoxytrityl (DMT), which prevents the building of a nucleotide chain. To add a nucleotide, DMT is chemically removed, and the nucleotide is added. The 5'-hydroxyl of the new nucleotide is blocked by DMT, preventing the addition of more than one nucleotide to each chain. After that, the cycle is repeated for each nucleotide in the primer. This is a simplified description, the actual process is quite complicated. For that reason, most laboratories do not make primers themselves, but order them by specialized companies.

DNA sequencing is used to determine the nucleotides in a DNA strand. A sequencing method called dideoxy sequencing (also known as chain termination method or Sanger method) uses a primer as a start marker for the chain reaction.

In polymerase chain reaction (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 50 nucleotides (Since DNA is usually double-stranded, its length is measured in base pairs. The length of single-stranded DNA is measured in bases or nucleotides), and they match exactly the beginning and the end of the DNA fragment to be amplified. They anneal (adhere) to the DNA template at these starting and ending points, where the DNA-Polymerase binds and begins the synthesis of the new DNA strand.

The choice of the length of the primers and their melting temperature depends on a number of considerations. The melting (or annealing) temperature of a primer is defined as the temperature below which the primer will anneal to the DNA template and above which the primer will dissociate (break apart) from the DNA template. 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 80C, can also cause problems since the DNA-Polymerase is less active at such temperatures. The optimum length of a primer is generally from 30 to 40 nucleotides with a melting temperature between about 60C and 75C. There are several ways to calculate the melting temperature (TM) of primers. (A, G, C and T are the number of nucleotides in the primer, respectively. [Na+] is the concentration of Na+ in the PCR vial.)

  • "GC"-method : Fast and simple, for primers with more than 13 nucleotides.
  • "Salt-adjusted"-method : More accurate than GC, for primers with more than 13 nucleotides.
  • Base-stacking calculation : Most accurate, but complicated
where
ΔH is the enthalpy of base stacking interactions adjusted for helix initiation factors
ΔS is the entropy of base stacking adjusted for helix initiation factors and for the contributions of salts to the entropy
R is the universal gas constant

Also, a primer should not easily anneal with itself or others of its kind, building loops or hairpins in the process. This could hinder the annealing with the template DNA. However, small hairpins are usually unavoidable.

Sometimes degenerate primers are used. These are actually mixtures of similar, but not identical, primers. They may be convenient if 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. (See genetic code for further details about codons) Use of degenerate primers can greatly reduce the spesificity of the PCR amplification. The problem can be partly solved by using touchdown PCR.

References

An earlier version of the above article was posted on Nupedia.

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