Vent exo dna polymerase

CMG assembly and activation on small DNA circles were performed as in ‘unwinding assays’ with the following modifications. The buffer for DNA relaxation and MCM loading was 25 mM Tris-Cl pH 7.2, 100 mM K-glutamate, 10 mM magnesium acetate, 0.02% NP-40-S, 5 mM ATP. After phosphorylation with DDK, buffer was added to give a final concentration 250 mM K-glutamate, 25 mM Tris-Cl, 10 mM Mg-acetate, 0.02 % NP-40-S, 400 μg/ml BSA, 5 mM ATP and 25 nM Topo I. After 10 min CMG assembly at 30°C, KMnO₄ was added to 3 mM for 4 min before the reaction was quenched with 1 M beta-mercaptoethanol (Sigma) and stop mix, and DNA processed as described in ‘unwinding assays’. The DNA pellet was resuspended in 48 μl 1x cutsmart buffer (NEB), digested with 40 U EcoRI-HF (NEB) for 20 min at 37°C, extracted once with phenol:chloroform:isoamylalcohol (25:24:1), ethanol precipitated, and analysed by primer extension. Primer extension reactions contained ³²P end-labelled primer oMD167 and 70 U/ml Vent (exo-) DNA polymerase (NEB), and were carried out for 26 cycles. Reactions were quenched with stop mix, ethanol precipitated and separated on a denaturing 5 % urea-bis-polyacrylamide gel.

Briefly, a RPER (20 μL) was set up in 1 X ThermoPol Buffer (20 mM Tris-HCl, 10 mM (NH₄)₂SO₄, 10 mM KCl, 2 mM MgSO₄, 0.1% Triton X-100, pH 8.8 at 25 °C; NEB, MA, USA) with 3 μL of each ExoSAP-treated RT-PCR product, 5′-biotinylated external (common) Reverse AEGIS primer (0.2μM), and Vent (exo-) DNA polymerase (1 unit per reaction, NEB, MA, USA). Without conversion (an “extension” reaction), dNTPs (final 0.2 mM each) were added. For the dZ incorporation into the final amplicon (“conversion”), nucleoside triphosphates (dATP, dTTP, dGTP, and dZTP, final concentration 0.2 mM of each) were added. The “extension” and “conversion” reaction mixtures were incubated in DNA Engine^® Multi-Bay Thermal Cyclers (BioRad, Hercules, CA, USA) at 95 °C for 1 min, followed by 20 cycles (94 °C for 20 seconds, 55 °C for 30 seconds, 72 °C for 30 seconds). A final incubation was run at 72 °C for 1 minute. Reaction mixtures were then quenched with 4 mM EDTA.
For standard RT-PCR products, a set of 22 reverse target-specific primers (0.2 μM each) was added to the “extension” or “conversion” reactions. The other reaction components were the same as above.

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).