( t can promote non-specific annealing. P...)General Notes on Primer Design in PCR*

t can promote non-specific annealing. Poly A and Poly T stretches are also to be avoided as these will breath and open up stretches of the primer-template complex. This can lower the efficiency of amplification. Polypyrimidine (T, C) and polypurine (A, G) stretches should also be avoided. Ideally the primer will have a near random mix of nucleotides, a 50% GC content and be ~20 bases long. This will put the Tm in the range of 56C 62C(1).

3-end Sequence

It is well established that the 3' terminal position in PCR primers is essential for the control of mis-priming(5). We have already explored the problem of primer homologies occurring at these regions. Another variable to look at is the inclusion of a G or C residue at the 3' end of primers. This GC Clamp helps to ensure correct binding at the 3' end due to the stronger hydrogen bonding of G/C residues. It also helps to improve the efficiency of the reaction by minimizing any breathing that might occur.

Conclusion

It is essential that care is taken in the design of primers for PCR. Several parameters including the length of the primer, %GC content and the 3' sequence need to be optimized for successful PCR. Certain of these parameters can be easily manually optimized while others are best done with commercial computer programs. In any event, careful observance of the general rules of primer design will help ensure successful experiments.

References

1. Dieffenbach, C.W., Lowe, T.M.J., Dveksler, G.S., General Concepts for PCR Primer Design, in PCR Primer, A Labo
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