a College of Food Science and Engineering , Ocean University of China , Qingdao , China.
b Laboratory for Marine Drugs and Bioproducts , Qingdao National Laboratory for Marine Science and Technology , Qingdao , China.
J Biomol Struct Dyn. 2019 Jul;37(11):2867-2874. doi: 10.1080/07391102.2018.1498804. Epub 2019 Jan 18.
In the current design of quantitative polymerase chain reaction (qPCR) systems, the sequences of primers are the primary concerns. The secondary structures of DNA templates have not been much considered, although they should be also critically important. In this paper, various hairpins with different stem lengths and loop sizes are placed near primer-binding sites, and their effects on the amplification efficiency of qPCR are systematically investigated. When a hairpin is formed either in the inside of the amplicon or in its outside, the amplification is notably suppressed. The magnitudes of suppression increase with the increase in stem length and the decrease in loop size, and are especially significant for the hairpins formed inside the amplicon. With very long stems (e.g., 20-bp), the effect is still more drastic, and no targeted amplification products are formed. On the basis of melting temperature () measurements, these suppression effects of hairpins have been mostly ascribed to competitive inhibition of primer binding to the template. It has been concluded that, in order to design precise and reliable qPCR systems, at least 60-bp sequences around primer-binding sites, both inside and outside the amplicons, must be analyzed to confirm that stable secondary structures are not formed in the vicinity of primer-binding sites. Communicated by Ramaswamy H. Sarma.
在当前的定量聚合酶链反应（qPCR）系统设计中，引物序列是主要关注点。尽管 DNA 模板的二级结构也应该非常重要，但它们并没有得到太多考虑。在本文中，我们在引物结合位点附近放置了各种具有不同茎长和环大小的发夹，并系统地研究了它们对 qPCR 扩增效率的影响。当发夹在扩增子内部或外部形成时，扩增会明显受到抑制。抑制的程度随着茎长的增加和环大小的减小而增加，对于在扩增子内部形成的发夹尤为显著。当茎非常长（例如 20 个碱基）时，效果更为明显，并且没有形成靶向扩增产物。基于熔解温度（Tm）测量，这些发夹的抑制效应主要归因于引物与模板结合的竞争抑制。因此得出结论，为了设计精确可靠的 qPCR 系统，必须分析引物结合位点周围至少 60 个碱基的序列，包括扩增子内部和外部，以确认在引物结合位点附近不会形成稳定的二级结构。由 Ramaswamy H. Sarma 传达。
