Deep vent exo

DNA was transcribed in 100 μl transcription buffer as aforementioned except that DTT was excluded and 1 mM SBED-GMP^{8 (link)} was added to the transcription buffer. After transcription at 37 °C for 1 hour, the samples were irradiated and subjected to primer extension as the previous studies^{1 (link),4 (link)}. DNA was purified by Wizard SV Gel and PCR Clean-Up System (Promega) followed by primer extension with Deep Vent (exo-) (NEB, M0259) and FAM-5’-TGACAGCGATGCGTAGAATCGCTAG. The G ladder was obtained by extension of non-transcribed DNA in the presence of Acy-CTP (NEB, N0460).

DNA polymerases were purchased from New England Biolabs (Vent, Vent (exo-), DeepVent (exo-), Bst 2.0, and Therminator) and Thermo Scientific (Taq, Pfu, and Phusion). The reaction mixture (25 μL) contained either 4 μM primer and template or 4 μM hairpin oligonucleotides. The dNTP concentrations were 40 or 200 μM (each) in corresponding buffer. ThermoPol^® buffer (New England Biolabs) was used for the Vent, Vent (exo-), DeepVent (exo-), Bst 2.0, and Therminator polymerases. A buffer supplied by the manufacturers was used in the reactions with Taq, Pfu, and Phusion polymerases. One and a half microliters of 25 mM MgCl₂were added to the reaction mixtures with Taq polymerase. Two units of enzyme were used for all polymerases, except Bst2.0 (8 units). To initiate pyrophosphorolysis, PPi was added to the reaction mixture at 0.4 mM if not otherwise stated. The reaction mixtures were heated to 95 °C for 1 min and subsequently cooled to 55 °C for 1 min. PE or hairpin modification reactions were performed at 64 or 72 °C for varying amounts of time.