Figure 2

Comparison of affinity star minimizing and maximizing reaction conditions on SIN/DMSO-induced TaqII REase specificity change. (A) Effect of SIN and DMSO under star minimizing conditions. The influence of SIN and DMSO on TaqII activity was evaluated in reaction conditions minimizing star activity, as we reported previously [29]. 0.3 μg (1.2-pmol GACCGA recognition sites) PCR(SINGLE) substrate was digested with 6 pmol TaqII in the reaction buffer: 40 mM Tris–HCl, pH 8.0, at 65°C, 10 mM (NH₄)₂SO₄, 10 mM MgCl₂, 1 mM DTT, BSA 100 μg/ml, 100 μM SIN in the DMSO concentration range from 0 to 50% for 16 h at 65°C. Lane M, Sigma PCR 20-bp Low Ladder (selected bands marked); lane K, undigested PCR fragment; lanes 1–9, digested PCR fragment in the presence of increasing DMSO concentrations: lane 1, 0% DMSO; lane 2, 2.5%; lane 3, 5%; lane 4, 10%; lane 5, 15%; lane 6, 20%; lane 7, 30%; lane 8, 40%; lane 9, 50%. (B) Effect of SIN and DMSO under star stimulating conditions. The influence of SIN and DMSO on TaqII activity was evaluated in reaction conditions stimulating star activity, as we reported previously [29]. The reaction was conducted as described in A in the reaction buffer: 40 mM Tris–HCl, pH 6.0, at 65°C, 10 mM MgCl₂, 1 mM DTT, BSA 100 μg/ml, 100 μM SIN. Lanes M, K and 1–7 are as described above in A.