Generate Multiple Mutations Quickly And Efficiently

Generate Multiple Mutations Quickly and Efficiently

• Simultaneous Mutagenesis of up to Five Sites
• Easy One-Day, Three-Step Procedure
• Unique Method Delivers High Accuracy and Efficiency

Introducing multiple site-directed mutations is often accomplished by sequentially mutagenizing a gene of interest, requiring numerous mutation experiments which take many days to complete. The QuikChange Multi kit allows this series of experiments to be performed in a single day!

The QuikChange™ Multi site-directed mutagenesis kit*^δδ offers a rapid and reliable method for site-directed mutagenesis of plasmid DNA at up to five different sites simultaneously. The kit features a one-day, three-step procedure and a single mutagenic oligonucleotide is required to mutagenize each site, using a double-stranded DNA template.

Mutagenesis: An Important Tool for Proteomics

In vitro site-directed mutagenesis is an invaluable technique for studying gene and protein structure-function relationships and for modifying vector sequences to facilitate cloning and expression strategies. Several approaches to this technique have been published, but these methods generally require the use of single-stranded DNA (ss-DNA) template and are labor intensive and technically difficult. Stratagene’s QuikChange site-directed mutagenesis technology** eliminates the need for subcloning into M13-based bacteriophage vectors and for ss-DNA rescue, using simple protocols to direct site-specific mutations in double-stranded plasmid DNA. The QuikChange Multi site-directed mutagenesis kit features a three-day procedure and offers the additional advantage of allowing mutagenesis at multiple sites in a single round. There are no specialized vectors or unique restriction sites required—virtually any plasmid of up to 8 kb is a suitable template.

Easy, One-Day Protocol

The QuikChange Multi kit features a fast and easy method to introduce multiple mutations simultaneously (Figure 1). Components of the thermal cycling reaction include a supercoiled double-stranded DNA template, two or more synthetic phosphorylated oligonucleotide primers containing the desired mutations, and the provided enzyme blend featuring PfuTurbo® DNA polymerase***. Primers containing the mutagenic sites are synthesized to only one strand of the ds template. PfuTurbo DNA polymerase extends the mutagenic primers with the highest possible fidelity under non-strand displacing conditions, generating ds-DNA molecules with one strand bearing multiple mutations and nicks. The nicks are then sealed by the unique enzyme blend. The thermal cycling reaction products are treated with the restriction endonuclease Dpn I to digest the parental DNA template.

High Transformation Efficiency

The reaction mixture, enriched for multiple mutated single-stranded DNA, is transformed into XL10-Gold® ultracompetent cells****, where the mutant closed circle ss-DNA is converted into duplex form in vivo. Double-stranded plasmid DNA may then be prepared from the transformants and analyzed by appropriate methods to identify clones bearing each of the desired mutations. The mutagenic efficiency is approximately 50 to 60% using the control system with three mutagenic primers. Table 1 demonstrates the efficiencies obtained from one to five mutagenic primers.

Figure 1

Overview of the QuikChange™ Multi Site-Directed Mutagenesis Method

Unique Application

Because only one primer is used to generate each mutation, the QuikChange Multi site-directed mutagenesis system is well suited for creating mutant collections using one or more degenerate oligonucleotides. The high transformation efficiency results in large collections of mutants (>10⁴). This site-directed random mutagenesis presents a unique potential to more rapidly generate mutants of interest.

Mutate Up to Five Different Sites in a Single Round

The highly efficient QuikChange Multi site-directed mutagenesis kit makes it easy to mutagenize up to five different sites in plasmid with a single round. The one-day, three-step procedure does not require specialized vectors or unique restriction sites—virtually any plasmid is a suitable template. Contributing Scientist Stratagene: Holly Hogrefe

Contributing Scientist
Stratagene: Holly Hogrefe

* Patent pending
**U.S. Patent Nos. 5,789,166 and 5,923,419 and patent pending
*** U.S. Patent Nos. 6,183,997 and 5,948,663 and 5,866,395 and 5,545,552 and patents pending
**** U.S. Patent Nos. 5,512,468 and 5,707,841 and patents pending