The Effect of Primer-Template Mismatches on the Detection and Quantification of Nucleic Acids Using the 5′ Nuclease Assay
The Effect of Primer-Template Mismatches on the Detection and Quantification of Nucleic Acids Using the 5′ Nuclease Assay
复制标题
DOI：
10.2353/jmoldx.2010.090035
复制
发表时间：
2010-01-01
影响因子：
4.1
通讯作者：
Schutten, Martin
中科院分区：
文献类型：
--
作者：
Stadhouders, Ralph;Pas, Suzan D.;Schutten, Martin
实时聚合酶链反应（PCR）是当前用于检测和定量核酸的选择方法，特别是用于分子诊断。引物和模板之间的互补性对于PCR应用通常是至关重要的，因为错配会严重降低引发效率。然而，这些不匹配的影响的定量数据很少。我们使用5 '-核酸酶测定定量研究了3'端引物区域内引物-模板错配对实时PCR的影响。我们的结果表明，单个错配引起广泛的影响，范围从对PCR扩增的较小（7.0循环阈值，例如A-A、C-A、A-G、C-C）。发现了特异性错配类型、位置和影响之间的明确关系，这对于DNA与RNA扩增以及Taq/Moloney鼠白血病病毒与基于rTth的扩增保持一致。使用的各种主混合物之间的影响的总体大小差异很大（高达七倍），对于某些主混合物，观察到反向或正向引物特异性影响，强调了使用的实验条件的重要性。总之，这些数据表明，错配影响遵循一致的模式，并使我们能够制定几个指导方针，用于预测引物-模板错配行为时，使用特定的5-核酸酶测定主混合物。我们的研究提供了新的见解错配行为，并应允许更优化的实时PCR检测涉及引物模板错配的发展。（J Mol Diagn 2010，12：109-117; DOI. 10.2353/jmoldx.2010.090035）
Real-time polymerase chain reaction (PCR) is the current method of choice for detection and quantification of nucleic acids, especially for molecular diagnostics. Complementarity between primers and template is often crucial for PCR applications, as mismatches can severely reduce priming efficiency. However, little quantitative data on the effect of these mismatches is available. We quantitatively investigated the effects of primer-template mismatches within the 3'-end primer region on real-time PCR using the 5'-nuclease assay. our results show that single mismatches instigate a broad variety of effects, ranging from minor (7.0 cycle threshold, eg, A-A, C-A, A-G, C-C) on PCR amplification. A clear relationship between specific mismatch types, position, and impact was found, which remained consistent for DNA versus RNA amplifications and Taq/Moloney murine leukemia virus versus rTth based amplifications. The overall size of the impact among the various master mixes used differed substantially (up to sevenfold), and for certain master mixes a reverse or forward primer-specific impact was observed, emphasizing the importance of the experimental conditions used. Taken together these data suggest that mismatch impact follows a consistent pattern and enabled us to formulate several guidelines for predicting primer-template mismatch behavior when using specific 5-nuclease assay master mixes. Our study provides novel insight into mismatch behavior and should allow for more optimized development of real-time PCR assays involving primer-template mismatches. (J Mol Diagn 2010,12:109-117; DOI. 10.2353/jmoldx.2010.090035)