Q5 hot start polymerase

Genomic DNA (gDNA) was isolated from the leaves of T₀–T₃ plants using a modified CTAB method [85 (link)]. Putative transgenic plants were verified by the PCR amplification of T-DNA fragments using primers hpt-F-205 and hpt-R-205 (Table S1) specific for the hptII gene.
To detect mutations in the target gene, transgenic plants were analyzed using T7 Endonuclease I (T7EI). For this purpose, the target region was amplified by PCR using primers gsk1.1_Ontarg_L and gsk1.1_Ontarg_R (Table S1) and Q5 hot-start polymerase (New England Biolabs, Ipswich, MA, USA). After amplification, 10 μL samples of the PCR mixtures of the tested and WT plants were mixed in a 1:1 ratio to form the heteroduplexes of amplicons, and the heteroduplex mixture was subjected to enzyme digestion with T7EI (New England Biolabs, Ipswich, MA, USA) according to protocol described by [86 ]. The digested amplicons were separated on 3% agarose gels (Micropor Delta, Prona, Madrid, Spain) and visualized on a Kodak Gel Logic 200 Imaging System. The same analysis was performed for predicted off-target sites.

MDS was performed as previously described (Jee et al., 2016 (link)). In brief, samples of 10 ml from the last cultures of the four B. subtilis strains were spun down and resuspended in 1 ml Tris-EDTA buffer (pH7.5) and incubated −80°C overnight. Genomic DNA extraction was performed using Qiagen genomic tip (100G) and quantified using Nanodrop. To generate each library, 2 μg of genomic DNA were independently treated with NlaIII (leading strand) or Hpy166III (lagging strand) restriction enzyme, which cleaves and delimits the Region Of Interest (ROI). The primers design was performed considering the cut sequence of NlaIII or Hpy166III for leading or lagging strand, respectively. A single PCR cycle was performed with 500 ng of restriction enzyme treated genomic DNA, 500 μM barcoded forward adapter primers annealing to the 3′ end of the ROI and Q5 Hot Start polymerase (New England Biolabs; Ipswich, MA, United States). Unused barcodes were removed with ExoI. The forward library was amplified using a forward adapter (described below in “Primer schema”) and performing 14 cycles of PCR with Q5 Hot Start high-fidelity DNA polymerase. Reverse adapter primers were used to define the ROI. Finally reverse adapters were used to amplify the libraries in 15 cycles of PCR. All libraries were resolved in an 8% acrylamide gel and purified with Ampure XP beads (see Supplementary Figures S2, S9).