Phi29 dnap

Multiple displacement amplification assay was performed using the plasmid pUC19 (NEB, USA) as substrate. In brief, the pUC19 plasmid (NEB, USA) was mixed with random hexamer primers (NEB, USA) and incubated at 95 °C for 3 min and cooled to room temperature. The formed primed pUC19 DNA was added to the reaction mixture (25 μL, pH 7.5) contained 100 nM IME199 DNAP or 10 U phi29 DNAP (NEB, USA), 50 mM Tris -HCl, 10 mM (NH₄)₂SO₄, 10 mM MgCl₂, 500 μM dNTPs, 4 mM DTT, and incubated at 30 °C for 2 h. Electrophoresis and analysis were performed as described above.
The fidelity of IME199 DNAP was measured as previously described [15 (link)] with some modifications. In brief, IME199 DNAP or phi29 DNAP was incubated with 1 ng pUC19 (NEB, USA) at 30 °C for 8 h according to the above system. Amplification products were digested with restriction endonuclease SalI-HF (NEB, USA), heat inactivated, ligated, transformed into XL-10 competent cells (Vazyme, China), and plated onto Luria–Bertani solid plates containing ampicillin (50 μg/mL), X-Gal (0.8 mg) and IPTG (0.8 mg). Plates were incubated at 37 °C for 16 h and then the blue and white colonies were counted. In addition, the plasmids extracted from the white clones were sent to Ruibiotech (Beijing, China) for sequencing to identify the mutation site.

Strand displacement assay using single-stranded circular M13mp18 (NEB, USA) as the template. The assay was carried out in the presence of 100 nM IME199 DNAP or 10 U phi29 DNAP (NEB, USA) or 10U T4 DNAP (NEB, USA), 10 mM MgCl₂, 50 mM Tris–HCl, 10 mM (NH₄)₂SO₄, 250 μM dNTPs, 4 mM DTT, 25 ng M13mp18 (NEB, USA) and 1 μM primer Site1-P4 (5′-TCGTAATCATGGTCATAGCTGTTTCCTG -3′) in a 25 μL reaction. After incubation for the indicated times at 30 °C, the reaction was stopped by adding 25 mM EDTA and 0.5% SDS. DNA replication products were analyzed by electrophoresis in 1.0% agarose contained Genecolour II nucleic acid dyes under electrophoretic conditions at 120 V for 50 min, and the gel was analyzed using fluorescence imaging with the Tanon Imaging System (Tanon 5200 Multi, Biotanon, China).
In addition, the characteristics of IME 199DNAP were investigated in RCA using different primers with M13mp18 (NEB, USA) as the substrate according to the above method. The concentration of DNA was measured using the DNA HS Assay Kit (Cat# FS-T1002, Beijing Foreverstar Biotech Co., Ltd, China) according to the manufacturer’s instructions. The yield of DNA was calculated using the equation Yield = v × c, where v is the reaction volume and c is the concentration of detected DNA.

First, the forked DNA template (20 pM in Replication Buffer) was loaded into the channel at a rate of 70 μl/min in the presence of 150 nM SYTOX orange, allowing for direct visualisation. After 1 minute or after a density of 0.3–0.7 molecules/μm² on the surface was reached, 80 μl of fluorescently labelled RPA (AF647-RPA, 20 nM in Replication Buffer supplemented with 150 nM SYTOX orange) was loaded at a rate of 70 μl/min. Before the reaction was initiated, initial fluorescence intensities were recorded to determine the base line of RPA intensity at the fork. Next, 5 units of Phi29 DNAp (NEB) was loaded in the presence of 20 nM RPA and the specified concentration of dNTPs.