Out with the old, in with the new : Allele Replacement

Source: Gray, M., Piccirillo, S., and Honigberg, S.M. Two-step method for constructing unmarked insertions, deletions and allele substitutions in the yeast genome. 2005. FEMS Microbiology Letters, 248: 31-6

In the spirit of the recent election, I decided to focus on a paper about change.

The authors developed a two-step process for removing, inserting or replacing regions in the yeast genome. They first remove the gene in question, replacing it with URA3. This is possible by flanking the URA3 with sequences homologous to the sequences flanking the gene in question. Due to recombination, URA3 is inserted in place of the gene. Selection for this occurs by growing cells on media lacking uracil ("Yes We Can...complete pyrimidine synthesis!"). The next step involves taking the replacement gene and flanking it with the same homologous sequences. Again, recombination replaces URA3 with this new insertion through recombination. Selection for this occurs by growing cells on media containing uracil and 5-FOA, a chemical that mimics uracil but is toxic. If a cell still has URA3 it will attempt to metabolize 5-FOA and kill itself. This method can be used to insert a new sequence (flankers originally touch), delete a sequence (URA3 replacement is just touching flankers), or replace a sequence. To insure replacement worked, primers from the replacing strand may contain a point mutation so that PCR would not amplify the inserted sequence and a gel-run would fail to show any DNA. Sequencing is always necessary to confirm due to the likelihood that any URA gene may mutate between functionality and pointless at any stage.