Abstract

The hairpin probe is characterized by a higher fluorescence quenching efficiency as compared with the linear one under the conditions of real-time PCR, which leads to a lower background level of fluorescence and, consequently, a higher signal to noise ratio during real-time PCR. An experimental comparison of the fluorescence-quenching efficiency of two oligonucleotide probes in different conformations (hairpin in a molecular beacon format and linear in a TaqMan format) was made. There is a difference in the interaction between the quencher and fluorophore for the probes of different conformations. For a linear probe, quenching occurs through the mechanism of inductive-resonance energy transfer (Förster resonance energy transfer, FRET), while that for a hairpin probe quenching occurs through contact quenching through a closer arrangement of the fluorophore and quencher, but a resonant energy transfer according to the Förster mechanism is also possible. It was demonstrated that the absorption spectrum for the linear probe almost coincides with the absorption spectrum of an oligonucleotide representing a probe without a quencher, which indicates a dynamic (Förster) mechanism of energy transfer. On the contrary, the absorption spectra for the hairpin probe and oligonucleotide representing the probe without a quencher differ significantly, which indicates a contact mechanism of energy transfer between the fluorophore and fluorescence quencher. The fluorescence spectra of the probes and their complexes with the oligonucleotide complementary to the linear probe (and the loop of the hairpin probe) and the amplicon (200 bp in length containing a DNA target for the probes) allowed for comparison of these two probes by comparing the energy migration radii, the efficiency of donor fluorescence quenching. The energy migration radius R calculated by the experimental data was 32.4 Å for the hairpin probe and 47.3 Å for the linear probe.

中文翻译：

---

用于实时 PCR 的线性和发夹探针中荧光团-猝灭剂系统的分子内相互作用

摘要

与线性探针相比，发夹探针的特点是在实时 PCR 条件下具有更高的荧光猝灭效率，从而导致荧光背景水平更低，因此在实时 PCR 期间具有更高的信噪比. 对两种不同构象（分子信标形式的发夹和 TaqMan 形式的线性）的寡核苷酸探针的荧光猝灭效率进行了实验比较。对于不同构象的探针，淬灭剂与荧光团之间的相互作用存在差异。对于线性探头，猝灭是通过感应共振能量转移（Förster 共振能量转移，FRET）机制发生的，虽然发夹探针淬灭是通过荧光团和淬灭剂的更紧密排列通过接触淬灭发生的，但根据 Förster 机制的共振能量转移也是可能的。结果表明，线性探针的吸收光谱几乎与代表没有猝灭剂的探针的寡核苷酸的吸收光谱一致，这表明能量转移的动态 (Förster) 机制。相反，发夹探针和代表没有猝灭剂的寡核苷酸的吸收光谱明显不同，这表明荧光团和荧光猝灭剂之间存在能量转移的接触机制。探针及其与线性探针（和发夹探针的环）互补的寡核苷酸和扩增子（长度为 200 bp，包含探针的 DNA 目标）的复合物的荧光光谱允许通过以下方式比较这两个探针比较能量迁移半径、供体荧光猝灭效率。实验数据计算得到的能量迁移半径R为发夹探针32.4 Å，线探针47.3 Å。