P3a site-specific and cassette mutagenesis for seamless protein, RNA and plasmid engineering (IMAGE)
Caption
P3a cassette mutagenesis directed by Ultramer primer pairs.
(A) Cartoon showing a pair of Ultramer oligo primers for introducing insertion up to 0.36 kb. The current up limit of an Ultramer is 0.2 kb. (B) Insertion of the 300-bp coding sequence for SHRT (an artificial protein that neutralize a snake toxin) into a mammalian expression vector. (C) Sequence chromatograms of four representative plasmids sequenced for engineering the coding sequence for SHRT. Eleven colonies were analyzed by restriction digestion and 4 were found to contain an insertion, resulting in an efficiency of 4/11 (36.4%). (D) Efficiency of P3a cassette mutagenesis to insert gene fragments via Ultramer primers. (E) Summary of different parameters for direct comparison of P3 and P3a mutagenesis methods, with the latter being much faster, more convenient and efficient.
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Copyright: © 2025 Yang. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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