Figure 1.

MO-MAGE method for targeted whole genome mutagenesis. 130 base oligonucleotides were designed and synthesized on a DNA microarray, which can be ordered from several commercial vendors. The oligos can be designed with different barcodes, which allow selective PCR amplification of a desired subpool. One of the primers are 5′ phosphorylated, which allow the degradation of only one of the strands by λ-exonuclease, resulting in single stranded oligos. The barcodes are removed by enzymatic treatment with endonuclease VIII (cutting the barcode by removing a uracil from the modified primer), DpnII and a guide primer (to make a double stranded cut site for DpnII). The final 90 bp single stranded oligos are directly applicable for MAGE.