3.3 Primers

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:

Other considerations which must be taken into account are:

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Figure 7: Hairpin loops and primer dimers

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:

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.

© SCBC 2007