The DNA fragment to be amplified is determined by selecting primers. Primers are short, artificial DNA strands -usually 18-25 base pairs long and not often more than 50 bp - that are complementary to opposite strands of the DNA at the beginning and the end of the fragment to be amplified. The primers anneal to opposite strands of the DNA template and point towards each other so when the DNA polymerase synthesises the new DNA the strands are extended towards each other.
When designing a primer several factors have to be taken into consideration. It is not always possible to make the ideal primer. Major factors playing a role in primer design are:
- Length of the primer: as this decreases so does the annealing temperature and the chances of a short primer binding non-specifically are increased. The optimum length of a primer is generally from 15 to 40 nucleotides with a melting temperature between 55°C and 65°C;
- The melting temperature (Tm): this is defined as the temperature at which half of the primer binding sites are occupied. As the length of the primer increases so does its melting temperature. Too high a temperature, i.e. above 80°C, causes problems as the DNA polymerase becomes less active.
Other considerations which must be taken into account are:
- The GC-content which should be between 40-60% with the bases evenly distributed to reduce the chances of non-specific binding. It also makes a distinction between the annealing and elongation temperatures;
- The calculated Tm for both primers used in a reaction should not differ by more than 5°C and the Tm of the amplification product should not differ from that of the primers by >10°C;
- The annealing temperature is usually 5°C below the calculated lower Tm, but is refined by observed results for individual primer sets;
- Possible intra- and intermolecular base pairing should be avoided so no hairpin loops or primer dimers are formed;
- The primer 3' terminus is critical to PCR success since the primer extends from the 3' end. The 3' end should not be complementary over greater than 3-4 bases to any region of the other primer (or even the same primer) used in the reaction and must provide correct base matching to the template.
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Sometimes degenerate primers are used. These are mixtures of primers which vary at certain defined positions. They are designed to isolate DNA from a species:
- Where the sequence is unknown but data has been determined for the homologous gene in other species. In such a case the genes are probably similar but not identical;
- Where only the protein sequence is known. Several different codons can code for one amino acid, for example the 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.
Use of degenerate primers reduces the specificity of the PCR amplification but this can be partly resolved by using touchdown PCR where the initial annealing temperature is higher than required and is lowered over subsequent cycles of the reaction (see Section on Developments of the PCR technique). This increases the chance of a primer to bind specifically especially in cases where the DNA has a number of sequences similar to that of the primer.
To help with primer design there are many computer programmes available. These usually require the sequence of the gene to be amplified to be entered or the gene database accession number to be known. For examples of these programmes see the following websites:
Primer design at www.invitrogen.com;
Primer3Plus at www.bioinformatics.nl;
The PCR Jump Station at www.horizonpress.com.