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PCR Protocols pp 89–99Cite as

Optimization of Polymerase Chain Reactions

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 226))

Abstract

The polymerase chain reaction (PCR) is a powerful method for fast in vitro enzymatic amplifications of specific DNA sequences. PCR amplifications can be grouped into three different categories: standard PCR, long PCR, and multiplex PCR. Standard PCR involves amplification of a single DNA sequence that is less than 5 kb in length and is useful for a variety of applications, such as cycle sequencing, cloning, mutation detection, etc. Long PCR is used for the amplification of a single sequence that is longer than 5 kb and up to 40 kb in length. Its applications include long-range sequencing; amplification of complete genes; PCR-based detection and diagnosis of medically important large-gene insertions or deletions; molecular cloning; and assembly and production of larger recombinant constructions for PCR-based mutagenesis (1,2). The third category, multiplex PCR, is used for the amplification of multiple sequences that are less than 5 kb in length. Its applications include forensic studies; pathogen identification; linkage analysis; template quantitation; genetic disease diagnosis; and population genetics (3–5). Unfortunately, there is no single set of conditions that is optimal for all PCR. Therefore, each PCR is likely to require specific optimization for the template/primer pairs chosen. Lack of optimization often results in problems, such as no detectable PCR product or low efficiency amplification of the chosen template; the presence of nonspecific bands or smeary background; the formation of “primer-dimers” that compete with the chosen template/primer set for amplification; or mutations caused by errors in nucleotide incorporation.

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References

  1. Higuchi, R. (1989) Using PCR to engineer DNA, in PCR Technology: Principles and Applications for DNA Amplifications (Erlich, H. A., ed.), Stockton Press, Inc., New York, pp. 61–70.

    Google Scholar 

  2. Foord, O. S. and Rose, E. A. (1995) Long-distance PCR, in PCR Primer (Dieffenback, C. W. and Dveksler, G. S., ed.), Cold Spring Harbor Laboratory Press, Cold Spring, NY, pp. 63–77.

    Google Scholar 

  3. Edwards, A., Civitello, A., Hammond, H. A., and Caskey, C. T. (1991) DNA typing and genetic mapping with trimeric and tetrameric tandem repeats. Am. J. Hum. Genet. 49, 746–756.

    PubMed  CAS  Google Scholar 

  4. Edwards, A., Hammond, H. A., Jin, L., Caskey, C. T., and Chakroborty, R. (1992) Genetic-variation at five trimeric and tetrameric tandem repeat loci in four human population groups. Genomics 12, 241–253.

    Article  PubMed  CAS  Google Scholar 

  5. Klimpton, C. P., Gill, P., Walton, A., Urquhart, A., Millican, E. S., and Adams, M. (1993) Automated DNA profiling employing multiplex amplification of short tandem repeat loci. PCR Methods Appl. 3, 13–21.

    Google Scholar 

  6. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

    Google Scholar 

  7. Lindahl, T. and Nyberg, B. (1972) Rate of depurination of native deoxyribonucleic acid. Biochemistry 11, 3610–3618.

    Article  PubMed  CAS  Google Scholar 

  8. 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.

    Article  PubMed  CAS  Google Scholar 

  9. Lowe, T. M. J., Sharefkin, J., Yang, S. Q., and Dieffenback, C. W. (1990) A computer program for selection of oligonucleotide primers for polymerase chain reaction. Nucleic Acids Res. 18, 1757–1761.

    Article  PubMed  CAS  Google Scholar 

  10. O’Hara, P. J. and Venezia, D. (1991) PRIMGEN, a tool for designing primers from multiple alignments. CABIOS 7, 533–534.

    Google Scholar 

  11. Montpetit, M. L., Cassol, S., Salas, T., and O’shaughnessy, M. V. (1992) OLIGOSCAN: A computer program to assist in the design of PCR primers homologous to multiple DNA sequences. J. Virol. Methods 36, 119–128.

    Article  PubMed  CAS  Google Scholar 

  12. Suggs, S. V., Hirose, T., Myake, D. H., Kawashima, M. J., Johnson, K. I., and Wallace, R. B. (1981) Using purifed genes, ICN-UCLA Symp. Mol. Cell. Biol. 23, 683–693.

    CAS  Google Scholar 

  13. Breslauer, K. J., Ronald, F., Blocker, H., and Marky, L. A. (1986) Predicting DNA duplex stability from the base sequence. Proc. Natl. Acad. Sci. USA 83, 3746–3750.

    Article  PubMed  CAS  Google Scholar 

  14. Freier, S. M., Kierzek, R., Jaeger, J. A., Sugimoto, N., Caruthers, M. H., Neilson, T., et al. (1986) Improved free-energy parameters for predictions of RNA duplex stability. Proc. Natl. Acad. Sci. USA 83, 9373–9377.

    Article  PubMed  CAS  Google Scholar 

  15. Innis, M. A. and Gelfand, D. H. (1990) Optimization of PCRs, in PCR Protocols: A Guide to Methods and Applications (Gelfand, D. H., Sninsky, J. J., Innis, M. A., and White, H., eds.), Academic Press, San Diego, CA, pp. 3–12.

    Google Scholar 

  16. Innis, M. A., Myambo, K. B., Gelfand, D. H., and Brow, M. A. D. (1988) DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplifed DNA. Proc. Natl. Acad. Sci. USA 85, 9436–9440.

    Article  PubMed  CAS  Google Scholar 

  17. Ohler, L. and Rose, E. A. (1992) Optimization of long distance PCR using a transposonbased model system. PCR Methods Appl. 2, 51–59.

    PubMed  CAS  Google Scholar 

  18. Gelfand, D. H. (1989) Taq DNA polymerase, in PCR Technology: Principles and Applications for DNA Amplification (Erlich, H. A., ed.), Stockton Press, New York, pp. 17–22.

    Google Scholar 

  19. Cheng, S. (1995) Longer PCR amplifications, in PCR Strategies (Innis, M. A., Gelfand, D. H., and Sninsky, J. J., eds.) Academic Press, San Diego, CA, pp. 313–324.

    Chapter  Google Scholar 

  20. Brock, T. D. and Freeze, H. (1969) Thermus aquaticus gene, a non-sporulating extreme thermophile. J. Bacteriol. 98, 289–297.

    Article  PubMed  CAS  Google Scholar 

  21. Giebel, L. B. and Spritz, R. A. (1990) Site-directed mutagenesis using the double-stranded DNA fragment as a PCR primer. Nucleic Acids Res. 18, 4947.

    Article  PubMed  CAS  Google Scholar 

  22. Saike, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R. Horu, G. T., Mullis, K. B., and Erlich, H. A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–491.

    Article  Google Scholar 

  23. Flaman, J.-M., Frebourg, T., Moreau, V., Charbonnier, R., Martin, C., Ishioka, C., et al. (1994). A rapid PCR fidelity assay. Nucleic Acids Res. 22, 3259–3260.

    Article  PubMed  CAS  Google Scholar 

  24. Cline, J., Braman, J. C., and Hogrefe, H. H. (1996) PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res. 24, 3546–3551.

    Article  PubMed  CAS  Google Scholar 

  25. Sardelli, A. D. (1993) Plateau effect-understanding PCR limitations, in Amplifications: A Forum for PCR Users. Perkin-Elmer Corp., Norwald, CT, pp. 1.

    Google Scholar 

  26. Saiki, R. K. (1989) The design and optimization of the PCR, in PCR Technology (Erlich, H. A., ed.), Stockton Press, New York, pp. 7–16.

    Google Scholar 

  27. Kim, H. S. and Smithies, O. (1988) Recombinant fragment assay for gene targeting based on the polymerase chain reaction. Nucleic Acids Res. 16, 8887–8903.

    Article  PubMed  CAS  Google Scholar 

  28. McConlogue, L., Brow, M. D., and Innis, M. A. (1988) Structure-independent DNA amplification by PCR using 7-deaza-2′-deoxyguanosine. Nucleic Acids Res. 16, 9869.

    Article  PubMed  CAS  Google Scholar 

  29. Mytelka, D. S. and Chamberlin, M. J. (1996) Analysis and suppression of DNA polymerase pauses associated with a trinucleotide consensus. Nucleic Acids Res. 24, 2774–2781.

    Article  PubMed  CAS  Google Scholar 

  30. Pomp, D. and Medrano, J. F. (1991) Organic solvents as facilitators of polymerase chain reaction. BioTechniques 10, 58–59.

    PubMed  CAS  Google Scholar 

  31. Newton, C. R. and Graham, A. (1994) PCR, Bios Scientific, Publishers Ltd., Oxford.

    Google Scholar 

  32. Levinson, G., Fields, R. A., Harton, G. L., Palmer, F. T., Maddleena, A., Fugger, E. F., et al. (1992) Reliable gender screening for human preimplantation embryos, using multiple DNA target-sequences. Hum. Reprod. 7, 1304–1313.

    PubMed  CAS  Google Scholar 

  33. Chamberlain, J. S., Gibbs, R. A., Ranier, J. E., Nguyen, P. N., and Caskey, C. T. (1988) Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res. 16, 11,141–11,156.

    Article  PubMed  CAS  Google Scholar 

  34. Uggozoli, L. and Wallace, B. (1992) Application of an allele-specific polymerase chain reaction to the direct determination of ABO blood group genotypes. Genomics 12, 670–674.

    Article  Google Scholar 

  35. Henke, W., Herdel, K., Jung, K., Schnorr, D., and Loening, S. A. (1997) Betaine improves the PCR amplification of GC-rich DNA sequences. Nucleic Acids Res. 25, 3957–3958.

    Article  PubMed  CAS  Google Scholar 

  36. Hengen, P. N. (1997) Optimizing multiplex and LA-PCR with betaine. Trends Biochem. Sci. 22, 225–226.

    Article  PubMed  CAS  Google Scholar 

  37. Bassam, B. J. and Caetano-Anolles, G. (1993) Automated “hot start” PCR using mineral oil and paraffin wax. BioTechniques 14, 30–34.

    PubMed  CAS  Google Scholar 

  38. Wainwright, L. A. and Seifert, H. S. (1993) Paraffin beads can replace mineral oil as an evaporation barrier in PCR. BioTechniques 14, 34–36.

    PubMed  CAS  Google Scholar 

  39. Rous, K. H. (1995) Optimization and troubleshooting in PCR, in PCR Primer (Diefferbach, C. W. and Dveksler, G. S. eds.), CSH Press, New York, pp. 53–62.

    Google Scholar 

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Author information

Authors and Affiliations

  1. Epicentre Technologies Corp., Madison, WI

    Haiying Grunenwald

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  1. Haiying Grunenwald
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Editors and Affiliations

  1. Department of Surgery, Glasgow Royal Infirmary, Glasgow, UK

    John M. S. Bartlett

  2. Department of Hematology, Royal Infirmary of Edinburgh, Edinburgh, UK

    David Stirling

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© 2003 Humana Press Inc.

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Grunenwald, H. (2003). Optimization of Polymerase Chain Reactions. In: Bartlett, J.M.S., Stirling, D. (eds) PCR Protocols. Methods in Molecular Biology™, vol 226. Humana Press. https://doi.org/10.1385/1-59259-384-4:89

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  • DOI: https://doi.org/10.1385/1-59259-384-4:89

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-642-0

  • Online ISBN: 978-1-59259-384-2

  • eBook Packages: Springer Protocols

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