PCR-amplification of GC-rich regions: 'slowdown PCR'
PCR-amplification of GC-rich regions: 'slowdown PCR'
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DOI：
10.1038/nprot.2008.112
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Siffert, Winfried
中科院分区：
文献类型：
--
作者：
Frey, Ulrich H.;Bachmann, Hagen S.;Siffert, Winfried
聚合酶链式反应(PCR)技术已成为分子研究中不可缺少的手段。然而，富含GC的模板的PCR扩增经常受到发夹等二级结构的形成和较高的熔融温度的阻碍。我们提出了一种新的方法，称为‘减速聚合酶链式反应’，它允许成功的聚合酶链式反应扩增极富GC(483%)的DNA靶标。该方案依赖于添加7-去氮-2‘-脱氧鸟苷、聚合酶链式反应混合物中的dGTP类似物和一种新的随温度变化的标准化循环方案。后者包括通常较低的斜坡率2.5℃的S(-1)和达到退火温度的1.5℃S(-1)的低冷却速度，并运行48个循环。我们建立了这个方法作为一种通用的方法，不仅可以扩增非常丰富的GC区域，而且还可以用于不同退火温度模板的常规DNA诊断和药物遗传学。该方案需要5小时才能完成。
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 (483%) 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 degrees C s(-1) and a low cooling rate of 1.5 degrees 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.