Sensitive fluorescence detection of nucleic acids based on isothermal circular strand-displacement polymerization reaction

Abstract

Here we have developed a sensitive DNA amplified detection method based on isothermal strand-displacement polymerization reaction. This method takes advantage of both the hybridization property of DNA and the strand-displacement property of polymerase. Importantly, we demonstrate that our method produces a circular polymerization reaction activated by the target, which essentially allows it to self-detect. Functionally, this DNA system consists of a hairpin fluorescence probe, a short primer and polymerase. Upon recognition and hybridization with the target ssDNA, the stem of the hairpin probe is opened, after which the opened probe anneals with the primer and triggers the polymerization reaction. During this process of the polymerization reaction, a complementary DNA is synthesized and the hybridized target is displaced. Finally, the displaced target recognizes and hybridizes with another probe, triggering the next round of polymerization reaction, reaching a target detection limit of 6.4 x 10(-15) M.

Figure 1.

The mechanism of isothermal amplified detection of DNA based on strand-displacement polymerization reaction. In the presence of target DNA, the hairpin fluorescence probe recognizes and hybridizes with it and undergoes a conformational change, leading to stem separation (Step 1). Following this, the primer anneals with the open stem of the hairpin fluorescence probe and triggers a polymerization reaction in the presence of dNTP/polymerase (Step 2). Next, in the process of primer extension, the target is displaced by the polymerase with strand-displacement activity, after which a complementary DNA is synthesized, forming a probe–cDNA complex (Step 3). Finally, to renew the cycle, the displaced target hybridizes with another hairpin fluorescence probe, which triggers yet another polymerization reaction (Step 4).

Figure 2.

Hairpin fluorescence probe hybridization with target. The mixture containing 5.0 × 10⁻⁸ M probe with 0 M (curve a), 5.0 × 10⁻⁸ M (curve b) and 1.0 × 10^–7 M (curve c) target, respectively. All samples were incubated at 37°C.

Figure 3.

Thermal profiles of hairpin fluorescence probe and the hybrid with its target. The mixture containing 5.0 × 10^–8 M probe with 0 M (curve a) and 5.0 × 10^–8 M (curve b) target, respectively. The temperature was increased from 40°C to 90°C in increment of 2°C, with each increment lasting 2 min.

Figure 4.

Verification of isothermal strand-displacement polymerization reaction. The mixture containing 5.0 × 10⁻⁸ M probe and 5.0 × 10^–8 M primer with 6.25 × 10^–9 M target (curve a), 6.25 × 10^–9 M random DNA (curve c) added at t₀ in the presence of dNTPs/polymerase respectively; curve b: no target added.

Figure 5.

Non-denaturing PAGE analysis of the products by the isothermal strand-displacement polymerization reaction. a–c curves: products generated by the isothermal strand-displacement polymerization reaction containing 2.0 × 10^–7 M probe, 2.0 × 10^–7 M primer and 2.0 × 10⁻⁸ M target at different time intervals in the presence of dNTPs/polymerase, (a) t = 60, (b) t = 20, (c) t = 0 min. All samples were incubated at 37°C.

Figure 6.

Detection of different concentrations of target based on isothermal strand-displacement polymerization reaction. Experiments were performed in the presence of 15U polymerase Klenow fragment exo⁻ and 100 μM dNTPs with 5 × 10^–8 M probe, 5 × 10^–8 M primer, and different concentrations of target. (A) Monitoring the fluorescence intensity of this amplified DNA detection method over a range of target DNA concentrations. The curves from a to i contain the target with 1.0 × 10^–10, 2.0 × 10^–11, 4.0 × 10^–12, 8.0 × 10^–13, 1.6 × 10^–13, 3.2 × 10^–14, 6.4 × 10^–15, 1.28 × 10^–15 and 0 M, respectively. All samples were incubated at 37°C. (B) The relationship of the rate of fluorescence enhancement with target DNA concentration.

Figure 7.

The hairpin fluorescence probe response to the target. Experiments were performed in the absence of polymerase and dNTPs with 5 × 10^–8 M probe and different concentrations of target. The curves from a to e contain the target with 1.25 × 10^–8, 2.5 × 10^–9, 5 × 10^–10, 1 × 10^–10 and 0 M, respectively. All samples were incubated at 37°C.