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 Laboratory Manual, Dieffenbach, C.W, and Dveksler, G.S., Ed., Cold Spring Harbor Laboratory Press, New York, 1995, 133-155.
2.Innis, M.A., and Gelfand, D.H., Optimization of PCRs, in PCR protocols, A Guide to Methods and Applications, Innis, M.A., Gelfand, D.H., Sninsky, J.J., and White, T.J., Ed., CRC Press, London, 1994, 5-11.
3.Sharrocks, A.D., The design of primers for PCR, in PCR Technology, Current Innovations, Griffin, H.G., and Griffin, A.M, Ed., CRC Press, London, 1994, 5-11.
4.Suggs, S.V., Hirose, T., Miyake, E.H., Kawashima, M.J., Johnson, K.I., and Wallace, R.B., Using Purified Genes, in ICN-UCLA Symp. Developmental Biology, Vol. 23, Brown, D.D. Ed., Academic Press, New York, 1981, 683.
5.Kwok, S., Kellog, D.E. McKinney, N., Spasic, D., Goda, L., Levenson, C., and Sninsky, J.J., Effects of primer-template mismatches on the polymerase chain reaction: Human Immunodeficiency Virus 1 model studies. Nucleic Acids Res. 18:999-1005, 1990.
* PCR Disclaimer: PCR is licensed under U.S. patent numbers 4,683,202, 4,683,195, 4,965,188 and 5,075,216 or their foreign counterparts, owned by Hoffmann-La Roche Inc. and F. Hoffmann-La Roche Ltd.
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