C12015: Efficient, Expansive, User-defined DNA MutagenesisNovelty:
This invention provides a leap forward in saturation mutagenesis called PFunkel. PFunkel is a next-generation Kunkel mutagenesis strategy that can, for the first time, efficiently and cheaply produce libraries containing user-defined mutations, including sequentially permuted genes. This technique is rapidly being adopted by academic labs and can be adopted to reinforce commercial, directed evolution service offerings.
Value Proposition:
There is currently no method for creating extensive libraries with a researcher-defined mutational composition spanning across an entire gene. Further, methods to make multiple mutations and methods for site-directed mutagenesis suffer from low efficiency. The PFunkel mutagenesis method overcomes the pitfalls of current research methods. This novel method provides a simple, single tube procedure for the creation of single mutations approaching 100% efficiency, the creation of multiple mutations at high efficiency, and the creation of expansive, user-defined libraries of mutations consisting of all possible mutations of a gene or genes, or a user-defined subset of all mutations of a gene or genes. Additional advantages of this method include:
• Higher efficiency than currently practiced techniques.
• Method is performed in a single tube and can be executed in a single day.
• Possibility to produce a novel library mutagenesis kit.
Technical Details:
Johns Hopkins researchers have developed a novel method for mutagenesis. In this method the mutations are introduced via oligonucleotides that anneal and prime synthesis on a single- or double-stranded uracil-containing DNA template. The reaction makes use of five enzymes and undergoes multiple temperature cycles that ultimately result in double-stranded closed circular plasmid DNA ready for transformation, each molecule encoding a user-defined mutation. The protocol can be optimized for introducing one or more mutations per gene and the frequency of mutated variants approaches 100%. Importantly, the method is rapid, taking a single day and performed in a single tube.
Looking for Partners:
To develop and commercialize the PFunkel mutagenesis method as a novel library mutagenesis kit.
Stage of Development:
Discovery
Data Availability:
Under CDA/NDA
Publications/Associated Cases:
Firnberg, E. and M. Ostermeier. "PFunkel: efficient, expansive, user-defined mutagenesis." PLoS One
