PCR-amplification of GC-rich regions: 'slowdown PCR'.
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.
Frey
Ulrich H
UH
Insititut für Pharmakogenetik, Universität Duisburg Essen, Universitätsklinikum, Essen D-45122, Germany. ulrich.frey@uk-essen.de
Bachmann
Hagen S
HS
Peters
Jürgen
J
Siffert
Winfried
W
D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D020862:GC Rich Sequence; D006801:Humans; D007202:Indicators and Reagents; D016133:Polymerase Chain Reaction / Q000379:methods*; D048493:Proto-Oncogene Proteins B-raf / Q000737:chemistry* / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D013696:Temperature
D017931:DNA Primers; D007202:Indicators and Reagents; C045550:7-deaza-2'-deoxyguanosine; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine