Figure 1.

Features of one-step genome modification using MIRAGE. (a) 45 bp flanking ends of ‘neck’ and ‘tail’ target the mutagenesis cassette into the yeast chromosome (gDNA), simultaneously replacing the undesired allele (white diamond) with the desired mutation (white triangle). The ura3-IR confers transformants with uracil prototrophy. Spontaneous and precise excision of the IR occurs within the 25 bp DR, leaving behind only the desired mutation. Since the mechanism of MIRAGE excision is not known, the IR is drawn with hyphenated lines. Omitting the mutation in the ‘neck’ primer will lead to deletion, rather than replacement. (b) Creation of the mutagenesis cassette requires preparation of each ura3 fragment separately. After PCR amplification and digestion with EcoRI and MfeI, the two halves are ligated together. Ligation of compatible EcoRI and MfeI ends abolishes both restriction sites. Redigestion with EcoRI and MfeI, followed by gel purification removes palindromic ligation products.