A Tool for Multiple Targeted Genome Deletions that Is Precise, Scar-Free, and Suitable for Automation
Three PCR amplifications use two sets of primers that have complex dependencies. The PCR1 primers (1 and 2) are chosen so that the amplicon generated is a 500–1000 bp segment immediately adjacent to and upstream from the target gene. PCR2 generates target gene-specific variants of the marker gene(s) for fusion to the product of PCR1 and a 40 bp repeat sequence (R) copied from a region immediately downstream of the target sequence. The PCR3 is a SOE-ing reaction that fuses the products of PCR1 and PCR2 to generate a complete deletion cassette flanked by repeat sequences native to the target. Marker excision is induced by growing transformants on a minimal medium containing 5-fluoroorotic acid—a pyrimidine analogue. The repeat sequences recombine to cleanly excise the marker gene without the addition of extraneous sequences to the genome.