Csa dna polymerase

RCA reactions were performed to amplify the repetitive sequences using DNA polymerases with strand-displacement activity. The four thermostable DNA polymerases were as follows: Bst DNA polymerase Large Fragment (Bst-LF) (New England Biolabs, NEB), Bst DNA polymerase, Csa DNA polymerase, and 96–7 DNA polymerase (Nippon Gene). All RCA reactions were performed in a final volume of 50 μL at the optimum temperature of each enzyme in a 0.2-mL polypropylene tube (QSP) using a thermal-cycler (TaKaRa). Reactions were initiated after raising the temperature from 13°C to each initial temperature (60°C or 55°C) for 2 min. The control RCA reaction contained a dNTP mixture (each 2.5 mM), 10 pmol each of two primers, buffer specific for each DNA polymerase, eights units of each DNA polymerase, and 60 ng of each circular template–primer complex. Bst-LF was incubated in ThermoPol Buffer (20 mM Tris–HCl, 10 mM (NH₄)₂SO₄, 10 mM KCl, 2 mM MgSO₄, and 0.1% Triton X-100; NEB) at 60°C for 20–60 min. The Bst DNA polymerase (Nippon Gene) was incubated in the Bst reaction buffer (8 mM Mg²⁺) at 60°C for 20–60 min. The Csa DNA polymerase (Nippon Gene) was incubated in the Csa reaction buffer (8 mM Mg²⁺) at 60°C for 10–60 min. The 96–7 DNA polymerase (Nippon Gene) was incubated in the 96–7 reaction buffer (9.5 mM Mg₂⁺) at 55°C for 10–180 min. A thermal-cycler was used to inactivate each DNA polymerase.