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

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

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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.

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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.

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.

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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

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  1. Ulrich H Frey
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  4. Winfried Siffert
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Correspondence to Ulrich H Frey.

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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

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