A novel metric to improve mismatched primer selection and quantification accuracy in amplifying DNA repeats for quantitative polymerase chain reactions.
A novel metric to improve mismatched primer selection and quantification accuracy in amplifying DNA repeats for quantitative polymerase chain reactions.
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DOI：
10.1371/journal.pone.0292559
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发表时间：
2023
期刊：
影响因子：
3.7
通讯作者：
中科院分区：
文献类型：
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作者：
在定量聚合酶链反应（qPCR）实验中，含有相对于模板的错配的引物广泛用于测量重复DNA元件。引物-模板错配可能由于低效的退火和扩增而导致低估输入样品量。但是引物-模板错配如何影响定量准确性尚未得到严格的研究。在这项研究中，我们进行了一系列的qPCR实验，其中我们测试了三对错配端粒引物（tel 1/tel 2，tel 1b/tel 2b和telg/泰尔克）和两对完全匹配的参考基因引物（36 B4-F/-R和IFNB 1-F/-R）在三个不同的引物浓度在四个循环条件下。使用的模板是来自两个人细胞系的基因组DNA和含有端粒序列、参考基因序列或两者的寡聚双链体。我们证明，来自含有错配引物的反应的输入样品量的低估不是由于较低的扩增效率（E），而是由于输入样品的无效使用。我们定义了一个新的概念，扩增效率（f）量化的有效性输入样品扩增引物。我们已经修改了传统的qPCR动力学公式，以包括f，其校正了引物错配的影响。我们证明，含有错配端粒引物对的反应具有相似的效率（E），但与具有完全匹配基因引物对的反应相比，效率（f）不同程度地降低。使用定量参数f，可以调整端粒引物对初始靶标的低估以提供更准确的测量。此外，我们发现浓度为500 nM和900 nM的tel 1b/tel 2b引物组表现出最佳的扩增效率f。该研究提供了一种将扩增功效的评估纳入qPCR分析的新方法。反过来，它提高了使用qPCR方法扩增DNA重复序列时的错配引物选择和定量准确性。
In quantitative polymerase chain reaction (qPCR) experiments, primers containing mismatches with respect to the template are widely used in measuring repetitive DNA elements. Primer-template mismatches may lead to underestimation of the input sample quantity due to inefficient annealing and amplification. But how primer-template mismatches affect quantification accuracy has not been rigorously investigated. In this study, we performed a series of qPCR experiments in which we tested three pairs of mismatched telomere primers (tel1/tel2, tel1b/tel2b and telg/telc) and two pairs of perfect-match reference gene primers (36B4-F/-R and IFNB1-F/-R) at three different primer concentrations under four cycling conditions. Templates used were genomic DNA from two human cell lines and oligo duplexes which contained telomere sequences, reference gene sequences, or both. We demonstrated that the underestimation of input sample quantity from reactions containing mismatched primers was not due to lower amplification efficiency (E), but due to ineffective usage of the input sample. We defined a novel concept of amplification efficacy (f) which quantifies the effectiveness of input sample amplification by primers. We have modified the conventional qPCR kinetic formula to include f, which corrects the effects of primer mismatches. We demonstrated that reactions containing mismatched telomere primer pairs had similar efficiency (E), but varying degrees of reduced efficacy (f) in comparison to those with the perfect-match gene primer pairs. Using the quantitative parameter f, underestimation of initial target by telomere primers can be adjusted to provide a more accurate measurement. Additionally, we found that the tel1b/tel2b primer set at concentration of 500 nM and 900 nM exhibited the best amplification efficacy f. This study provides a novel way to incorporate an evaluation of amplification efficacy into qPCR analysis. In turn, it improves mismatched primer selection and quantification accuracy in amplifying DNA repeats using qPCR methods.
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