Protocol abstract
Nature Protocols 3, - 1312 - 1317 (2008)
Published online: 24 July 2008 | Corrected online: 13 August 2009 | doi:10.1038/nprot.2008.112
Subject Category: Nucleic acid based molecular biology
PCR-amplification of GC-rich regions: 'slowdown PCR'
Ulrich H Frey1,2, Hagen S Bachmann1, Jürgen Peters2 & Winfried Siffert1
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.
- Insititut für Pharmakogenetik, Universität Duisburg Essen, Universitätsklinikum, Essen D-45122, Germany.
- Klinik für Anästhesiologie und Intensivmedizin, Universität Duisburg Essen, Universitätsklinikum, Essen D-45122, Germany.
Correspondence to: Ulrich H Frey1,2 e-mail: ulrich.frey@uk-essen.de
nature-products
A-Z product listing
- 10
Taq buffer with KCl and 15 mM MgCl2
(Fermentas) - 6 DNA loading dye(Fermentas)
- 7-Deaza-2'-deoxy-guanosine-5'-triphosphate(Roche Applied Sciences)
- Agarose(Fermentas)
- dATP(Fermentas)
- dCTP(Fermentas)
