Figure 2.4.

Detailed view of a primer (3f or 5r) used for assembly of the knockout cassette and one end of the hph cassette used for gene deletions, showing the location of the bar code. The circle represents the restriction enzyme Mme1 binding to its recognition sequence, and the attached curved arrow points to the position where it will cut genomic DNA containing a disrupted gene. After cutting with Mme1, the “gene-specific 20-mer” comprises a molecular bar code that may be detected through ligation of a linker (shown as a gray bar on the right) followed by PCR using primers (horizontal thick black arrows) specific for the linker and the hph cassette.