Primers in the polymerase chain reaction. Primers in the polymerase chain reaction. Primers are short, artificial DNA strands of about 18 to 25 nucleotides that match the beginning and end of the DNA fragment to be amplified. The primers anneal to the single-stranded DNA template at these points. Once the primers bind, 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 (Tm) depends on a number of considerations. The melting temperature of a primer is defined as the temperature at which half of the primer binding sites are occupied. The melting temperature increases with the length of the primer. Primers that are too short would anneal non-specifically at several positions on a long DNA template, resulting in non-specific copies. The length of a primer is limited by the temperature required to melt it. Melting temperatures that are too high, for example above 80°C, are not advisable as DNA-polymerase is less active at these temperatures. The optimum length of a primer is generally between twenty and forty nucleotides with a melting temperature between 60°C and 75°C.
Degenerate primers Sometimes degenerate primers are used. These are 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 will be similar but not identical. Degenerate primers are also used when the DNA sequence is unknown and primer design is based on the protein sequence. As several different codons can code for one amino acid it is not possible to design spefific primers. The use of degenerate primers can greatly reduce the specificity of the PCR amplification. Touchdown PCR may be used in this case.
Considerations in Primer Design GC-content should be between 40-60. Calculated Tm for both primers used in the reaction should not differ >5°C and Tm of the amplification product should not differ from primers by >10°C. Annealing temperature should be 5°C lower than the calculated lower Tm. However it should be chosen empirically for individual conditions. Inner self-complementary hairpins of >4 and of dimers >8 should be avoided. 3' terminus is extremely case sensitive - it must not be complementary to any region of the other primer used in the reaction and must provide correct base matching to template.