Successful amplification of extremely GC-rich promoterregions using a novel ‘slowdown PCR’techniqueHagen S. Bachmann, Winfried Siffert, Ulrich H. FreyObjectives PCR has become a routine technique in DNAgenotyping for diagnostic or pharmacogenetics purposes.Promoter regions of genes are in the main focus fordetecting novel regulatory single nucleotidepolymorphisms (rSNPs). However, due to very high GCcontent PCR setup procedures can be very timeconsuming and not infrequently amplification of theregions of interest fail.Methods We developed a novel method termed‘Slowdown’ PCR which allows the successful amplificationof extremely GC-rich (> 83%) targets. The method relieson combination of a novel standardized cycling protocolwith varying temperature ramping rates plus the additionof 7-deaza-29-deoxyguanosine, a dGTP analogue.Moreover, we provide essential practical hints for optimalprimer and template length design for such GC-richtargets.Results Using this setup we successfully amplified andsequenced GC-rich DNA regions such as the 59-upstreamregions of the genes GNAS1 and GNAQ as well as exon1of the BRAF gene which could not be amplified by others.Conclusion Here we show that ‘Slowdown’ PCR is aversatile method not only for amplification of extremelyGC-rich regions but also for routine DNA diagnostics andpharmacogenetics for templates with different annealingtemperatures. Pharmacogenetics 13:759– 766 & 2003LippincottWilliams& WilkinsPharmacogenetics2003, 13:759–766Keywords: PCR, promoter, touchdown, rSNPInstitut fu¨r Pharmakologie, Universita¨tsklinikum, D-45122 Essen, Germany.Corresponding author: Dr med. Ulrich H. Frey, Institut fu¨r Pharmakologie,Universita¨tsklinikum Essen, D-45122 Essen, Germany.Tel: +49 -201 -723 3459; fax: +49- 201...
