扩增高GC含量模板的优化程序.pdfVIP

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Original article 759 Successful amplification of extremely GC-rich promoter regions using a novel ‘slowdown PCR’ technique Hagen S. Bachmann, Winfried Siffert, Ulrich H. Frey Objectives PCR has become a routine technique in DNA sequenced GC-rich DNA regions such as the 59-upstream genotyping for diagnostic or pharmacogenetics purposes. regions of the genes GNAS1 and GNAQ as well as exon1 Promoter regions of genes are in the main focus for of the BRAF gene which could not be amplified by others. detecting novel regulatory single nucleotide polymorphisms (rSNPs). However, due to very high GC Conclusion Here we show that ‘Slowdown’ PCR is a content PCR setup procedures can be very time versatile method not only for amplification of extremely consuming and not infrequently amplification of the GC-rich regions but also for routine DNA diagnostics and regions of interest fail. pharmacogenetics for templates with different annealing temperatures. Pharmacogenetics 13:759–766 2003 Methods We developed a novel method termed Lippincott Williams Wilkins ‘Slowdown’ PCR which allows the successful amplification of extremely GC-rich ( 83%) targets. The method relies Pharmacogenetics 2003, 13:759–766 on combination of a novel standardized cycling protocol Keywords: PCR, promoter, touchdown, rSNP with varying temperature ramping rates plus the addition ¨

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