Exosap it enzymes mixture

To destroy excess primers and deactivate dNTPs prior to reverse primer extension reaction, ExoSAP-IT enzymes mixture (2 μl; Affymetrix, Cleveland, OH, USA) was added to aliquots (5 μl) of standard-AEGIS or SAMRS–AEGIS nested PCR. The mixtures were incubated at 37 °C (30 min). The enzymes were inactivated by heating at 80 °C (20 min). The treated PCR products (3 μl) were then added directly to the RPER. Briefly, an RPER (20 μl) was done in 1 × ThermoPol Buffer (20 mM Tris–HCl, 10 mM (NH₄)₂SO₄, 10 mM KCl, 2 mM MgSO₄, and 0.1% Triton X-100, pH 8.8) at 25 °C (NEB) with 5′-biotinylated external (common) reverse AEGIS primer (0.2 μM), and Vent (exo-) DNA polymerase (1 U per reaction; NEB). To perform extension without transliteration, dNTPs (final 0.2 mM each) were added. To incorporate dZ into the final amplicon with transliteration (where Z replaces C due to primer extension with mismatching of dZTP opposite template G), nucleoside triphosphates (dATP, dTTP, dGTP, and dZTP, final concentration 0.2 mM each) were added without dCTP. Both were incubated in DNA Engine Multi-Bay Thermal Cyclers (Bio-Rad, Hercules, CA, USA) at 95 °C (1 min), followed by 20 cycles (94 °C for 20 s, 55 °C for 30 s, and 72 °C for 30 s) with a final incubation cycle at 72 °C (1 min). Reaction mixtures were then quenched with 4 mM ethylenediaminetetraacetic acid (EDTA).