Douglas Young, working in Alexander Deiters’s group at NC State, describes a new method for introducing targeted mutations into plasmids. Unlike previous site-directed mutagenesis methods, this one requires no restriction enzymes or ligases, and uses only two oligonucleotide primers. The key trick is to put photocleavable tags on some of the primer nucleotides. These tags block polymerase readthrough and prevent unwanted primer hybridizations. After PCR, all UV irradiation is all you need to cleave the tags, letting those single-stranded overhangs hybridize. The resulting nicked plasmid can be transformed directly into E. coli. The authors show that in one step, their method can delete huge sections of plasmid, or, even better, insert completely new sequences up to 17 bp in length, at any arbitrary position in the plasmid.
I have two questions. One is whether the authors have tried doing more than one round mutagenesis/insertion completely in-vitro. After annealing to create the nicked plasmid, a quick enzyme treatment can repair the nicks. Does this give rise to a substrate ready for the next round of mutagenic PCR, with no need for transformation and outgrowth of E. coli?
The other is, how long will it take till this technology is commercialized? I hope that the authors hear from custom oligonucleotide synthesis providers (maybe NEB, Invitrogen, IDT, or others) soon.