Successful amplification of extremely GC-rich promoter regions using a novel ???slowdown PCR??? technique
2003; Lippincott Williams & Wilkins; Volume: 13; Issue: 12 Linguagem: Inglês
10.1097/00008571-200312000-00006
ISSN1473-561X
AutoresHagen S. Bachmann, Winfried Siffert, Ulrich H. Frey,
Tópico(s)RNA Research and Splicing
ResumoObjectives 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. Conclusion 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.
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