A Study of PCR Inhibition Mechanisms Using Real Time PCR
A Study of PCR Inhibition Mechanisms Using Real Time PCR
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DOI：
10.1111/j.1556-4029.2009.01245.x
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发表时间：
2010-01-01
影响因子：
1.6
通讯作者：
McCord, Bruce R.
中科院分区：
文献类型：
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作者：
Opel, Kerry L.;Chung, Denise;McCord, Bruce R.
本项目采用实时聚合酶链反应（real - time polymerase chain reaction， PCR）技术，通过检测扩增子长度、熔融温度和序列的影响来研究PCR抑制的机制。设计了三种不同扩增子长度和三种不同熔点温度的引物，用于定位HUMTH01基因座上的一个纯合等位基因。通过在反应混合物中加入不同浓度的各种PCR抑制剂来确定对每个引物对扩增效率的影响。结果表明，根据共提取抑制剂的类型，PCR过程中可能发生多种抑制机制。这些包括Taq抑制、DNA模板结合和对反应效率的影响。此外，一些抑制剂似乎以不止一种方式影响反应。总的来说，我们发现扩增子大小和熔化温度在某些抑制机制中很重要，而在其他机制中则不重要，了解PCR抑制的关键问题是确定干扰物质的身份。
In this project, real time polymerase chain reaction (PCR) was utilized to study the mechanism of PCR inhibition through examination of the effect of amplicon length, melting temperature, and sequence. Specifically designed primers with three different amplicon lengths and three different melting temperatures were used to target a single homozygous allele in the HUMTH01 locus. The effect on amplification efficiency for each primer pair was determined by adding different concentrations of various PCR inhibitors to the reaction mixture. The results show that a variety of inhibition mechanisms can occur during the PCR process depending on the type of co-extracted inhibitor. These include Taq inhibition, DNA template binding, and effects on reaction efficiency. In addition, some inhibitors appear to affect the reaction in more than one manner. Overall we find that amplicon size and melting temperature are important in some inhibition mechanisms and not in others and the key issue in understanding PCR inhibition is determining the identity of the interfering substance.