Influence of design probe and sequence mismatches on the efficiency of fluorescent RPA
Influence of design probe and sequence mismatches on the efficiency of fluorescent RPA
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DOI：
10.1007/s11274-019-2620-2
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发表时间：
2019-06-01
影响因子：
4.1
通讯作者：
Lan, Quanxue
中科院分区：
文献类型：
--
作者：
Liu, Xiaoqing;Yan, Qiongying;Lan, Quanxue
重组酶聚合酶扩增 (RPA) 是一种等温扩增技术。由于其检测周期短、特异性高，已在各个领域得到应用。然而，探针设计对 RPA 效率的影响尚不清楚，寡核苷酸序列错配对 RPA 性能的影响也很少讨论。在本研究中，我们发现具有相同探针的不同引物对荧光RPA的效率影响较小，而具有相同扩增区域的不同探针对荧光RPA的效率影响较大。通过对实验数据的分析，我们总结了适合荧光RPA的探针的设计规则。规则是探针中荧光基团之间的最佳距离为1-2个碱基，并且G含量应尽可能减少。此外，我们通过设计一系列探针验证了这一规则。此外，我们发现探针的碱基错配对 RPA 有显着影响，这可能导致假阳性并改变扩增效率。然而，覆盖引物序列中心的1-3个错配仅影响RPA的扩增效率，而不影响其特异性。并且随着引物错配数量的增加，RPA的效率也会相应降低。本研究表明荧光RPA的效率与探针密切相关。我们建议在设计荧光探针时，必须考虑密切相关的非靶标和特定碱基的存在。
Recombinase polymerase amplification (RPA) is an isothermal amplification technique. Because of its short detection cycle and high specificity, it has been applied in various fields. However, the design of probe on the efficiency of RPA is not well understood and the effect of sequence mismatches of oligonucleotides on the performance of RPA is rarely discussed. In this study, we found that different primers with the same probe have a slight effect on the efficiency of fluorescent RPA, and different probes with the same amplified region have a great influence on the efficiency of fluorescent RPA. We summarized the design rules of probes suitable for fluorescent RPA by analyzing the experimental data. The rule is that the best distance between fluorescent groups in the probe is 1-2 bases, and the G content should be reduced as far as possible. In addition, we verified this rule by designing a series of probes. Furthermore, we found the base mismatches of the probe had a significant effect on RPA, which can lead to false positives and can change the amplification efficiency. However, 1-3 mismatches covering the center of the primer sequence only affect the amplification efficiency of RPA, not its specificity. And with an increase in the number of primer mismatches, the efficiency of RPA will decrease accordingly. This study suggests that the efficiency of fluorescent RPA is closely related to the probe. We recommend that when designing a fluorescent probe, one must consider the presence of closely related non-targets and specific bases.