96 7 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.

AuNPs (20 and 40 nm) were obtained from BBI Solutions (Cardiff, UK). All oligonucleotides (Probe-A, Probe-B, ssDNA-15, ssDNA-20, agDNA-15, agDNA-20, RNA-15, NA, NB, and MB) shown in Figure S1 were purchased from FASMAC (Kanagawa, Japan). T7 RNA polymerase was purchased from Takara Bio Inc. (Shiga, Japan). 96-7 DNA polymerase and RNase inhibitor were purchased from Nippon Gene (Tokyo, Japan). Deoxynucleotide solution mix (dNTPs) and ribonucleotide solution mix (rNTPs) were purchased from New England Biolabs Inc. (Ipswich, ME, USA). Human α-thrombin was purchased from Funakoshi (Tokyo, Japan). Transferrin, human immunoglobulin G (IgG), human serum albumin (HSA), and bovine serum albumin (BSA) were purchased from Merck (Munich, Germany). NAP-5 columns were provided with the oligonucleotides. Absorption spectra and fluorescence intensities were measured using microplate reader (TECAN, Zürich, Switzerland). All photographs were taken using a digital camera (SONY, Tokyo, Japan).