Localised sequence regions possessing high melting temperatures prevent the amplification of a DNA mimic in competitive PCR
Localised sequence regions possessing high melting temperatures prevent the amplification of a DNA mimic in competitive PCR
复制标题
DOI：
10.1093/nar/26.14.3340
复制
发表时间：
1998-07-15
影响因子：
14.9
通讯作者：
Parkes, HC
中科院分区：
文献类型：
--
作者：
McDowell, DG;Burns, NA;Parkes, HC
聚合酶链式反应是一种非常强大的识别和检测技术。竞争性 PCR 越来越多地被用作定量的基础。然而，序列长度、解链温度和一级序列均已被证明会影响 PCR 系统中的扩增效率，因此可能会影响竞争性 PCR 中靶标和模拟物所需的等效共扩增。这里讨论的工作不仅说明了设计竞争性 PCR 测定时平衡长度和解链温度的必要性，而且还强调了仔细检查富含 GC 结构域和其他序列的重要性，这些序列产生稳定的二级结构，这些结构可能通过充当暂停或终止位点来降低扩增效率。我们提供的数据证实，在特定情况下，这种局部序列、高熔解温度区域可以充当永久终止位点，并为这种效应的严重性提供了解释，这种效应导致竞争性 PCR 中 DNA 模拟物的扩增被阻止。还证明，当在甜菜碱或校对酶存在下使用Tao DNA聚合酶时，效果可能会降低或消除。
The polymerase chain reaction is an immensely powerful technique for identification and detection purposes. Increasingly, competitive PCR is being used as the basis for quantification. However, sequence length, melting temperature and primary sequence have all been shown to influence the efficiency of amplification in PCR systems and may therefore compromise the required equivalent co-amplification of target and mimic in competitive PCR. The work discussed here not only illustrates the need to balance length and melting temperature when designing a competitive PCR assay, but also emphasises the importance of careful examination of sequences for GC-rich domains and other sequences giving rise to stable secondary structures which could reduce the efficiency of amplification by serving as pause or termination sites. We present data confirming that under particular circumstances such localised sequence, high melting temperature regions can act as permanent termination sites, and offer an explanation for the severity of this effect which results in prevention of amplification of a DNA mimic in competitive PCR. It is also demonstrated that when Tao DNA polymerase is used in the presence of betaine or a proof reading enzyme, the effect may be reduced or eliminated.