Figure 6.

Physical characterization of YAC and BAC clones containing the human NBS1 gene isolated by CRISPR/Cas9-mediated TAR cloning from total genomic DNA. (a) The NBS1 gene contains 16 exons and maps at 8q21. E13F/E13R are diagnostic primers for exon 13 used to screen yeast transformants to find gene-positive clones. Positions of Cas9 sites relative to 5’ and 3’ hooks are indicated. (b) The presence of different NBS1 coding regions in three independent His⁺ TAR/YAC/BAC clones was examined by PCR. M corresponds to GenRuler DNA Ladder Low Range (Sigma, cat. no. D0428). (c) Alu-profiles of two YACs containing the NBS1 gene in yeast. (d) Characterization of three BAC clones containing the NBS1 gene. BAC DNAs were isolated, linearized, separated by CHEF, and visualized by staining with EtBr. Predicted size of the NBS1-containing fragments are observed. M1 correspond to CHEF DNA Size Standard Ladder (BIO-RAD, cat no. 1703635) (e) Restriction digestion profiles of BAC DNA isolated from three NBS1-positive E. coli colonies. BAC DNA was digested by HindIII (lanes 1–3) and EcoRI (lanes 4–6). Identical profiles were observed for each restriction endonuclease. M2 corresponds to GenRuler 1 kb DNA Ladder (Thermo Fisher Scientific, cat. no. SM0311).

This Figure was adapted from (Lee et al., 2015).