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Figure 1 | BMC Biotechnology

Figure 1

From: Site-specific genomic (SSG) and random domain-localized (RDL) mutagenesis in yeast

Figure 1

Introduction of mutations into the genome by site-specific genomic (SSG) and random domain-localized (RDL) mutagenesis. Both methods involve two sequential transformations. The first transformation (Transform. 1 in center of diagram) disrupts the targeted sequence with a PCR fragment containing URA3. The left and right ends of the fragment share 40 bp of homology to either side of the insertion site. In SSG mutagenesis (left side of figure), the fragment is inserted into the target region, and in RDL mutagenesis (right side of figure) the fragment replaces the target sequence. Transformants are selected on medium lacking uracil. The second transformation (Transform. 2) replaces URA3 with a PCR fragment containing one or more mutations (represented by an asterisk). In SSG mutagenesis, the mutations are at one end of the fragment and were introduced on the primer. In RDL mutagenesis, the mutations are introduced by error-prone PCR at random in the fragment. Transformants are selected on medium containing 5-fluoroorotic acid (5-FOA) to select for loss of URA3.

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