Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

PCR-amplification of GC-rich regions: 'slowdown PCR'

Nature Protocols volume 3, pages 1312–1317 (2008)Cite this article

This article has been updated

Abstract

The polymerase chain reaction (PCR) technique has become an indispensable method in molecular research. However, PCR-amplification of GC-rich templates is often hampered by the formation of secondary structures like hairpins and higher melting temperatures. We present a novel method termed 'Slowdown PCR', which allows the successful PCR-amplification of extremely GC-rich (>83%) DNA targets. The protocol relies on the addition of 7-deaza-2′-deoxyguanosine, a dGTP analog to the PCR mixture and a novel standardized cycling protocol with varying temperatures. The latter consists of a generally lowered ramp rate of 2.5 °C s−1 and a low cooling rate of 1.5 °C s−1 for reaching an annealing temperature and is run for 48 cycles. We established this protocol as a versatile method not only for amplification of extremely GC-rich regions, but also for routine DNA diagnostics and pharmacogenetics for templates with different annealing temperatures. The protocol takes 5 h to complete.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Comparison of different cycling methods.
Figure 2: PCR from the promoter region of GNAS1 with extremely high GC content (83.8%).
Figure 3: (Slowdown PCR) with dc7GTP.

Similar content being viewed by others

Change history

  • 13 August 2009

    In the version of this article initially published, the center and right-hand panels of Figure 1 were incorrectly drawn; the orientation of the cycles and the placement of the connecting arrows were incorrect. Also, in the bottom cycle of the center panel, the annealing temperature 58 °C should have been presented as "y °C". Finally, in the last sentence of the legend for Figure 1, "from 70 to 53 °C" should have read ""from 70 to 54 °C". These errors have been corrected in the HTML and PDF versions of the article.

References

  1. Mullis, K.B. & Faloona, F.A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 155, 335–350 (1987).

    Article  CAS  PubMed  Google Scholar 

  2. Saiki, R.K. et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–491 (1988).

    Article  CAS  PubMed  Google Scholar 

  3. Roux, K.H. Optimization and troubleshooting in PCR. PCR Methods Appl. 4, S185–S194 (1995).

    Article  CAS  PubMed  Google Scholar 

  4. McDowell, D.G., Burns, N.A. & Parkes, H.C. Localised sequence regions possessing high melting temperatures prevent the amplification of a DNA mimic in competitive PCR. Nucleic Acids Res. 26, 3340–3347 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Varadaraj, K. & Skinner, D.M. Denaturants or cosolvents improve the specificity of PCR amplification of a G + C-rich DNA using genetically engineered DNA polymerases. Gene 140, 1–5 (1994).

    Article  CAS  PubMed  Google Scholar 

  6. Hapgood, J.P., Riedemann, J. & Scherer, S.D. Regulation of gene expression by GC-rich DNA cis-elements. Cell Biol. Int. 25, 17–31 (2001).

    Article  CAS  PubMed  Google Scholar 

  7. Bachmann, H.S., Siffert, W. & Frey, U.H. Successful amplification of extremely GC-rich promoter regions using a novel 'slowdown PCR' technique. Pharmacogenetics 13, 759–766 (2003).

    Article  CAS  PubMed  Google Scholar 

  8. Frey, U.H. et al. A novel promoter polymorphism in the human gene GNAS affects binding of transcription factor upstream stimulatory factor 1, Galphas protein expression and body weight regulation. Pharmacogenet. Genomics 18, 141–151 (2008).

    Article  CAS  PubMed  Google Scholar 

  9. Frey, U.H. et al. Characterization of the GNAQ promoter and association of increased Gq expression with cardiac hypertrophy in humans. Eur. Heart J. 29, 888–897 (2008).

    Article  CAS  PubMed  Google Scholar 

  10. Bachmann, H.S. et al. The AA genotype of the regulatory BCL2 promoter polymorphism (938C>A) is associated with a favorable outcome in lymph node negative invasive breast cancer patients. Clin. Cancer Res. 13, 5790–5797 (2007).

    Article  CAS  PubMed  Google Scholar 

  11. Nuckel, H. et al. Association of a novel regulatory polymorphism (-938C>A) in the BCL2 gene promoter with disease progression and survival in chronic lymphocytic leukemia. Blood 109, 290–297 (2007).

    Article  PubMed  Google Scholar 

  12. Riemann, K. et al. Comparison of manual and automated nucleic acid extraction from whole-blood samples. J. Clin. Lab Anal. 21, 244–248 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Saccone, S., De, S.A., Della, V.G. & Bernardi, G. The highest gene concentrations in the human genome are in telomeric bands of metaphase chromosomes. Proc. Natl. Acad. Sci. USA 89, 4913–4917 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Rychlik, W., Spencer, W.J. & Rhoads, R.E. Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res. 18, 6409–6412 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Hube, F., Reverdiau, P., Iochmann, S. & Gruel, Y. Improved PCR method for amplification of GC-rich DNA sequences. Mol. Biotechnol. 31, 81–84 (2005).

    Article  CAS  PubMed  Google Scholar 

  16. Musso, M., Bocciardi, R., Parodi, S., Ravazzolo, R. & Ceccherini, I. Betaine, dimethyl sulfoxide, and 7-deaza-dGTP, a powerful mixture for amplification of GC-rich DNA sequences. J. Mol. Diagn. 8, 544–550 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Sahdev, S., Saini, S., Tiwari, P., Saxena, S. & Singh, S.K. Amplification of GC-rich genes by following a combination strategy of primer design, enhancers and modified PCR cycle conditions. Mol. Cell Probes 21, 303–307 (2007).

    Article  CAS  PubMed  Google Scholar 

  18. Sarkar, G., Kapelner, S. & Sommer, S.S. Formamide can dramatically improve the specificity of PCR. Nucleic Acids Res. 18, 7465 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Winship, P.R. An improved method for directly sequencing PCR amplified material using dimethyl sulphoxide. Nucleic Acids Res. 17, 1266 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Pomp, D. & Medrano, J.F. Organic solvents as facilitators of polymerase chain reaction. Biotechniques 10, 58–59 (1991).

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Chester, N. & Marshak, D.R. Dimethyl sulfoxide-mediated primer Tm reduction: a method for analyzing the role of renaturation temperature in the polymerase chain reaction. Anal. Biochem. 209, 284–290 (1993).

    Article  CAS  PubMed  Google Scholar 

  24. Lee, C.H., Mizusawa, H. & Kakefuda, T. Unwinding of double-stranded DNA helix by dehydration. Proc. Natl. Acad. Sci. USA 78, 2838–2842 (1981).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Fernandez-Rachubinski, F., Eng, B., Murray, W.W., Blajchman, M.A. & Rachubinski, R.A. Incorporation of 7-deaza dGTP during the amplification step in the polymerase chain reaction procedure improves subsequent DNA sequencing. DNA Seq. 1, 137–140 (1990).

    Article  CAS  PubMed  Google Scholar 

  26. Don, R.H., Cox, P.T., Wainwright, B.J., Baker, K. & Mattick, J.S. 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 19, 4008 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hecker, K.H. & Roux, K.H. High and low annealing temperatures increase both specificity and yield in touchdown and stepdown PCR. Biotechniques 20, 478–485 (1996).

    Article  CAS  PubMed  Google Scholar 

  28. Davies, H. et al. Mutations of the BRAF gene in human cancer. Nature 417, 949–954 (2002).

    Article  CAS  PubMed  Google Scholar 

  29. Jung, A., Ruckert, S., Frank, P., Brabletz, T. & Kirchner, T. 7-Deaza-2′-deoxyguanosine allows PCR and sequencing reactions from CpG islands. Mol. Pathol. 55, 55–57 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Freier, S.M. et al. Improved free-energy parameters for predictions of RNA duplex stability. Proc. Natl. Acad. Sci. USA 83, 9373–9377 (1986).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Rychlik, W., Spencer, W.J. & Rhoads, R.E. Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res. 18, 6409–6412 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Caulfield, M. et al. Linkage of the angiotensinogen gene to essential hypertension. N. Engl. J. Med. 330, 1629–1633 (1994).

    Article  CAS  PubMed  Google Scholar 

  33. Cambien, F. et al. Deletion polymorphism in the gene for angiotensin-converting enzyme is a potent risk factor for myocardial infarction. Nature 359, 641–644 (1992).

    Article  CAS  PubMed  Google Scholar 

  34. Siffert, W. et al. Association of a human G-protein beta3 subunit variant with hypertension. Nat. Genet. 18, 45–48 (1998).

    Article  CAS  PubMed  Google Scholar 

  35. Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Insititut für Pharmakogenetik, Universität Duisburg Essen, Universitätsklinikum, Essen, D-45122, Germany

    Ulrich H Frey, Hagen S Bachmann & Winfried Siffert

  2. Klinik für Anästhesiologie und Intensivmedizin, Universität Duisburg Essen, Universitätsklinikum, Essen, D-45122, Germany

    Ulrich H Frey & Jürgen Peters

Authors
  1. Ulrich H Frey
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hagen S Bachmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jürgen Peters
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Winfried Siffert
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ulrich H Frey.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Frey, U., Bachmann, H., Peters, J. et al. PCR-amplification of GC-rich regions: 'slowdown PCR'. Nat Protoc 3, 1312–1317 (2008). https://doi.org/10.1038/nprot.2008.112

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2008.112

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing