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Nucleic acid based molecular biology

Author(s): Ulrich H Frey
Affiliation(s): Insititut für Pharmakogenetik, Universität Duisburg Essen, Universitätsklinikum
DOI: 10.1038/nprot.2008.112

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 °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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