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

Nat Protoc. 2008;3(8):1312-7. doi: 10.1038/nprot.2008.112.

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 degrees C s(-1) and a low cooling rate of 1.5 degrees 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.

Publication types

  • Validation Study

MeSH terms

  • DNA Primers
  • DNA-Directed DNA Polymerase / chemistry
  • Deoxyguanosine / analogs & derivatives
  • Deoxyguanosine / chemistry
  • Electrophoresis, Agar Gel
  • GC Rich Sequence*
  • Humans
  • Indicators and Reagents
  • Polymerase Chain Reaction / methods*
  • Proto-Oncogene Proteins B-raf / chemistry*
  • Proto-Oncogene Proteins B-raf / genetics
  • Sequence Analysis, DNA / methods*
  • Temperature

Substances

  • DNA Primers
  • Indicators and Reagents
  • 7-deaza-2'-deoxyguanosine
  • BRAF protein, human
  • Proto-Oncogene Proteins B-raf
  • DNA-Directed DNA Polymerase
  • Deoxyguanosine