Abstract
One of the most important factors affecting the quality of PCR is the choice of primers. In general, the longer the PCR product the more difficult it is to select efficient primers and set appropriate designing primers, and in general, the more DNA sequence information is available, the better the ch0ance of finding an optimal primer pair. Efficient primers can be designed by avoiding the following flaws: primer-dimer formation, self-complementarity, too lowT m of the primers, and/or their incorrect internal stability profile. Tips on subcloning PCR products, calculating duplex stability (predicting dimer formation strength), and designing degenerate primers are given.
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References
Breslauer, K. J., Frank, R., Blocker, H., and Markey, L. A. (1986) Predicting DNA duplex stability from the base sequence.Proc. Natl. Acad. Sci. USA 83, 3746–3750.
Freier, S. M., Kierzek, R., Jaeger, J. A., Sugimoto, N., Caruthers, M. H., Neilson, T., and Turner, D. H. (1986) Improved free-energy parameters for predictions of RNA duplex stability.Proc. Natl. Acad. Sci. USA 83, 9373–9377.
Groebe, D. R. and Uhlenbeck, O. C. (1988) Characterization of RNA hairpin loop stability.Nucleic Acids Res. 16, 11,725–11,735.
Cheng, S., Fockler, C., Barnes, W. M., and Russell, H. (1994) Effective amplification of long targets from clones inserts and human genomic DNA.Proc. Natl. Acad. Sci. USA 91, 5696–5699.
Wu, D. Y., Ugozzoli, L., Pal, B. K., Qian, J., and Wallace, R. B. (1991) The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction.DNA Cell Biol.,10, 233–238.
Rychlik, W., Spencer, W. J., and Rhoads, R. E. (1990) Optimization of the annealing temperature for DNA amplification in vitro.Nucleic Acids Res. 18, 6409–6412.
Rychlik, W. and Rhoads, R. E. (1989) A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA.Nucleic Acids Res. 17, 8543–8551.
Lee, C. C. and Caskey, C. T. (1990) cDNA cloning using degenerate primers, inPCR Protocols ( Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J. eds.), Academic, New York, pp. 46–53.
Kwok, S., Kellogg, D. E., McKinney, N., Spasic, D., Goda, L., Levenson, C., and Sninsky, J. J. (1990) Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies.Nucleic Acids Res. 18, 999–1005.
Eckert, K. A. and Kunkel, T. A. (1990) High fidelity DNA synthesis by theThermus aquaticus DNA polymerase.Nucleic Acids Res. 18, 3739–3744.
Petruska, J., Goodman, M. F., Boosalis, M. S., Sowers, L. C., Cheong, C., and Tinoco, I., Jr. (1988) Comparison between DNA melting thermodynamics and DNA polymerase fidelity.Proc. Natl. Acad. Sci. USA 85, 6252–6256.
Kawasaki, E. (1990) Amplification of RNA, inPCR Protocols (Innis, M. A., Gelfand, D. H., Sninsky, J.J., and White, T. J. eds.), Academic, New York, pp. 21–27.
White, B. A., ed. (1993)Methods in Molecular Biology, vol. 15: PCR Protocols, Humana, Totowa, NJ.
New England BioLabs, 1993–1994 Catalog, “Cleavage close to the end of DNA fragments,” p. 180.
Jung, V., Pestka, S. B., and Pestka, S. (1990) Efficient cloning of PCR generated DNA containing terminal restriction endonuclease recognition sites.Nucleic Acids Res. 18, 6156.
Eckert, K. A. and Kunkel, T. A. (1991) The fidelity of DNA polymerase used in PCR, inPolymerase Chain Reaction: A Practical Approach ( McPherson, M. J., Quirke, P., and Taylor, G.R. eds.), IRL, Oxford, UK, pp. 227–246.
Marchuk, D., Drumm, M., Saulino, A., and Collins, F. S. (1991) Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR productsNucleic Acids Res. 19, 1154.
Holton, T. A. and Graham, M. W. (1991) A simple and efficient method for direct cloning of PCR products using ddT-tailed vectorsNucleic Acids Res. 19, 1156.
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Rychlik, W. Selection of primers for polymerase chain reaction. Mol Biotechnol 3, 129–134 (1995). https://doi.org/10.1007/BF02789108
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DOI: https://doi.org/10.1007/BF02789108