Kunkel Mutagenesis

overview

Kunkel mutagenesis, a form of site-directed mutagenesis, efficiently inserts, deletes or mutates one or more nucleotides. This method uses a phage-derived uracil-containing ssDNA template and mutagenic oligonucleotides to confer targeted mutations to a plasmid of interest. Colonies with desired mutation(s) are identified by rolling circle amplification (RCA) and Sanger sequencing. 

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This protocol takes a phage-derived uracil-containing ssDNA template of your choice along with mutagenic oligonucleotides based on your desired mutation(s), and delivers sequencing data and plasmid DNA ready for your analysis and downstream assays - 32 at a time. 

methodology

Mutagenic oligonucleotides specified by the customer and synthesized by IDT are diluted to 100 µM and kinased with T4 Polynucleotide Kinase. Next, kinased mutagenic oligos are annealed to the dU-ssDNA template provided by the customer in a reaction containing T4 ligase buffer (Figure 1). 

Figure 1  dU-ssDNA template with 1 annealed mutagenic oligo. 

Figure 1  dU-ssDNA template with 1 annealed mutagenic oligo. 

A polymerization reaction catalyzed by T7 polymerase and T4 ligase generates a heterocomplex containing a dU-containing template and a dT- mutant strand (Figure 2). 

Figure 2  Heterodimer complex of dU-ssDNA parent strand and strand containing mutation. 

Figure 2  Heterodimer complex of dU-ssDNA parent strand and strand containing mutation. 

Transformation of the heterocomplex into an ung+ dut+ bacterial strain degrades the dU-containing template strand, leaving only the dT-mutant strand to be replicated (Figure 3). Colonies with desired mutation(s) are identified by RCA and Sanger sequencing. 

Figure 3  Double stranded plasmid containing mutation on both strands that will get transformed into ung+ dut+ bacteria to generate the final plasmid. 

Figure 3  Double stranded plasmid containing mutation on both strands that will get transformed into ung+ dut+ bacteria to generate the final plasmid. 

validation

To validate this Transcriptic protocol, 32 constructs were assembled as described, and 10 final assemblies were chosen at random for testing. DH10B cells (Zymo Research Corporation) were transformed with each of these assembled constructs and grown for 16 hours on selective media. Three colonies for each of the 10 assemblies were picked and validated using RCA followed by Sanger sequencing.

For every assembly tested, at least 1 out of 3 picked colonies contained the expected mutagenic sequence (Figure 4). Overall, 10 out of 10 reactions contained the desired mutation site - yielding a 100% success rate. 

Figure 4 Pie chart summarizing observed cloning efficiencies. 

Figure 4 Pie chart summarizing observed cloning efficiencies. 

Conclusion

Transcriptic’s automated platform provides a robust, high-throughput method for executing Kunkel mutagenesis on demand. Mutants are assembled, transformed and sequencing data is provided to the customer for analysis and identification of correct mutants. Correct clones are ready-to-go for plasmid prep and other downstream applications. 


VALIDATION DATE:  October 2015
PROTOCOLS EXECUTED:  Kunkel Mutagenesis; Transform, Spread, Pick; Sequencing; Plasmid Prep