Mutations are created using a variation of ligation during-amplification(1) in which the plasmid is PCR-amplified with phosphorylated primers and the product ends are ligated together to convert the annealed DNA strands into closed circular DNA molecules (Fig 1). These closed circular DNA molecules then become template molecules in subsequent rounds of the PCR amplification. The desired mutations (insertions, deletions, or substitutions) are created by incorporating them into the primer sequence. USB’s FideliTaq Polymerase is used to ensure faithful duplication of the plasmid sequence and successful longdistance PCR. Thermostable DNA ligase is utilized for efficient creation of circular plasmid DNA during each round of PCR amplification. Together these enzymes generate exponentially amplified, circular, mutated plasmid DNA. To enhance selection of the mutated plasmid from the parental plasmid, the PCR amplification reaction is digested with Dpn I prior to transformation into competent cells.
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| Fig. 1. Change-IT™ Mutagenesis Method. Phosphorylated oligonucleotides are annealed to template plasmid. One oligonucleotide bears the desired sequence changes while the other anneals to a common sequence, such as the β-lactamase ORF. FideliTaq™ DNA Polymerase extends the oligonucleotides and DNA ligase seals the nascent DNA strands creating a replicated plasmid bearing the desired mutation. This process is repeated in a PCR amplification for 15 to 30 cycles, generating exponentially amplified, mutated, double-stranded plasmid as product for subsequent transformation. |
Convenience
Minimal hands-on time is required – set up an amplification reaction, a restriction enzyme digest, and a transformation. Kit includes optimized reaction buffer, phosphorylated common primers to β-lactamase, and enzymes.
High Fidelity and Large Plasmids
Faithful amplification of plasmids is ensured by FideliTaq Polymerase and an optimized Change-IT Buffer. Mutate plasmids as large as 15 kb.
High Efficiency
Exponential amplification of the mutated plasmid provides for high mutagenesis efficiency, even with multiple mutations.
The efficiency for the creation of one, two, or three mutations in a single template were determined for pUC19 (Table 1). The single mutation blocked expression of β-galactosidase (Fig. 2). The double mutation blocked expression of β-galactosidase and created a Xho I site. The triple mutation created an Nco I site, a Xho I site, and blocked expression of β-galactosidase. The single and double mutations were created with the greatest efficiencies. The triple mutation was present in 80% of the plasmid product, a lower rate than the double mutation as expected for this technique.
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| Creation of Multiple Mutations. Single, double, or triple mutation reactions were performed. The single mutation inactivated LacZα, the double mutation inactivated LacZα and added a Xho I site, and the triple mutation inactivated LacZα and added both an Nco I site and a Xho I site. Images of plates demonstrate that the LacZα inactivating mutation was present in all conditions (i.e. white mutated colonies predominate). Colony PCR amplifications were then analyzed by restriction digests. The product from the single mutation condition lacked both Nco I and Xho I sites, as expected (single band visible on agarose gel). Restriction analysis of the double mutation condition revealed two bands in all colonies screened, indicating all carried the Xho I site insertion. Restriction analysis of the triple mutation condition revealed that 9 of 10 colonies incorporated all three site-directed mutations. |
References
1. Chen, Z. and Ruffner, D.E. (1998) Nucl. Acids Res. 26(4), 1126-1127. | 
