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A Tool for Multiple Targeted Genome Deletions that Is Precise, Scar-Free, and Suitable for Automation

Fig 1

The seamless gene deletion method.

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.

Fig 1