Successful amplification of extremely GC-rich promoter regions using a novel 'slowdown PCR' technique.
OBJECTIVE
PCR has become a routine technique in DNA genotyping for diagnostic or pharmacogenetics purposes. Promoter regions of genes are in the main focus for detecting novel regulatory single nucleotide polymorphisms (rSNPs). However, due to very high GC content PCR setup procedures can be very time consuming and not infrequently amplification of the regions of interest fail.


METHODS
We developed a novel method termed 'Slowdown' PCR which allows the successful amplification of extremely GC-rich (> 83%) targets. The method relies on combination of a novel standardized cycling protocol with varying temperature ramping rates plus the addition of 7-deaza-2'-deoxyguanosine, a dGTP analogue. Moreover, we provide essential practical hints for optimal primer and template length design for such GC-rich targets.


RESULTS
Using this setup we successfully amplified and sequenced GC-rich DNA regions such as the 5'-upstream regions of the genes GNAS1 and GNAQ as well as exon1 of the BRAF gene which could not be amplified by others.


CONCLUSIONS
Here we show that 'Slowdown' PCR is 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.
Bachmann
Hagen S
HS
Snstitut für Pharmakologie, universitätsklinikum, Essen, Germany.
Siffert
Winfried
W
Frey
Ulrich H
UH
D001483:Base Sequence; D017931:DNA Primers; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic
D017931:DNA Primers