Triplex DNA: A new platform for polymerase chain reaction-based biosensor

Abstract

Non-specific PCR amplification and DNA contamination usually accompany with PCR process, to overcome these problems, here we establish a sensor for thrombin by sequence-specific recognition of the PCR product with molecular beacon through triplex formation. Probe A and probe B were designed for the sensor, upon addition of thrombin, two probes hybridized to each other and the probe B was extended in the presence of Klenow Fragment polymerase and dNTPs. The PCR amplification occurred with further addition of Taq DNA Polymerase and two primers, the PCR product was recognized by molecular beacon through triplex formation. The fluorescence intensity increased with the logarithm of the concentration of thrombin over the range from 1.0 × 10(-12) M to 1.0 × 10(-7) M, with a detection limit of 261 fM. Moreover, the effect of DNA contamination and non - specific amplification could be ignored completely in the proposed strategy.

Figure 1. Scheme of the biosensor for thrombin.

Figure 2

(A) Fluorescence spectroscopy of molecular beacon after hybridization with different cycle of PCR product. The PCR amplification was carried out under the condition of 30 s denaturation at 94 °C, 30 s of annealing temperature at 60 °C, and 10 s of extending temperature at 72 °C. MB: 200 nM, spermine: 0.4 mM, pH: 5.0. (B) Circular dichroism (CD) spectroscopy of Oligonucleotides under different conditions. a. MB: 1.0 × 10⁻⁵ M; b. R1·R2: 1.0 × 10⁻⁵ M; c. Mixture of 1.0 × 10⁻⁵ M MB and 1.0 × 10⁻⁵ M R1·R2. Spermine: 0.4 mM, pH: 5.0.

Figure 3. Calibration curve for the biosensor.

45 cycles of PCR amplification was carried out by using 30 s of denaturation at 94 °C, 30 s of annealing temperature at 60 °C, and 10 s of extending temperature at 72 °C. MB: 200 nM, spermine: 0.4 mM, pH:5.0. Each point was the mean of three measurements. The error bars are the standard deviation.

Figure 4. Effect of lambda - DNA on the fluorescence of the PCR product of 1.0 × 10⁻⁸ M thrombin.

(A) Detecting PCR product by using SYRB - Green; SYRB - Green: 1 × , spermine: 0.4 mM, pH:5.0. (B) Detecting PCR product with the proposed method (detecting PCR product through triplex formation); MB: 200 nM, spermine:0.4 mM, pH:5.0.

Figure 5. Selectivity of the sensor and effect of interference molecules.

(A) a: blank; b: 1.0 × 10⁻⁷ M of lysozyme; c: 1.0 × 10⁻⁷ M of hemoglobin; d: 1.0 × 10⁻⁷ M of apo-transferrin human; e: 1.0 × 10⁻⁸ M of thrombin; f: mixture of 1.0 × 10⁻⁸ M of thrombin and 1.0 × 10⁻⁷ M of lysozyme; g: mixture of 1.0 × 10⁻⁸ M of thrombin and 1.0 × 10⁻⁷ M of hemoglobin; h: mixture of 1.0 × 10⁻⁸ M of thrombin and 1.0 × 10⁻⁷ M of apo-transferrin human. Every point was the mean of three measurements, error bar was the standard deviation. (B) Electrophoresis photograph of PCR product. 1. 500 bp DNA ladder; 2. Blank; 3. 1.0 × 10⁻⁸ M of thrombin; 4. 1.0 × 10⁻⁷ M of lysozyme; 5. 1.0 × 10⁻⁷ M of hemoglobin; 6. 1.0 × 10⁻⁷ M of apo-transferrin human; 7. mixture of 1.0 × 10⁻⁸ M of thrombin and1.0 × 10⁻⁷ M of lysozyme, hemoglobin, apo-transferrin human; EB: 0.5 μg/mL; Agarose: 3%.