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High-Efficiency Site-Directed Mutagenesis of Large Plasmid Vectors


QuikChange XL: a highly versatile kit for site-directed mutagenesis of large plasmids

Michael Borns Peter Vaillancourt Holly Hogrefe Jeff Braman
Stratagene

The QuikChange site-directed mutagenesis kit* characteristically generates mutagenesis efficiencies exceeding 80%. We have created a new kit for mutagenesis of large plasmid templates. The QuikChange XL site-directed mutagenesis kit has been specifically formulated for mutagenesis of longer targets, and is packaged with XL10-Gold ultra competent cells**, which are optimized for the transformation of larger DNA molecules. We demonstrate the improved performance of the QuikChangeXL kit by mutagenizing a 10.1-kb retroviral vector that was otherwise difficult to mutagenize.

Site-directed mutagenesis is routinely employed in numerous research endeavors. The procedure can be laborious and time-consuming, requiring either single-stranded DNA templates1, subcloning into specialized vectors2, plasmid vectors containing unique restriction sites, and/or multiple transformations into different host strains3. In contrast, the QuikChange site-directed mutagenesis kit4 features a highly efficient procedure that is significantly streamlined when compared to other mutagenesis methods. With this kit, site-specific mutation can take place in virtually any double-stranded plasmid, eliminating the need for subcloning into M13-based bacteriophage vectors and ssDNA rescue. In addition, the kits procedure does not require specialized vectors, unique restriction sites, or multiple transformations. The protocol can be completed in just one day and generates mutagenesis efficiencies exceeding 80%.

Another important feature of the kit is the use of high-fidelity PfuTurbo DNA polymerase.*** This key component permits template DNA replicatio n with an error rate that is significantly lower than conventional thermostable enzymes5 minimizing undesirable second-site mutations.

Improving Mutagenesis of Large Plasmids

In the new QuikChange XL kit, modifications have been made to the QuikChange method to improve the efficiency of mutagenizing large or difficult plasmid vectors. In addition, XL10-Gold ultracompetent cells have been included to ensure the highest transformation efficiencies possible (Figure 1). The transformation efficiency of XL10-Gold cells is 5-fold higher than the transformation efficiency of XL1-Blue cells employed in the standard QuikChange kit6. Moreover, XL10-Gold cells contain the Hte phenotype, which increases transformation efficiency of larger DNA plasmids.

The performance of the QuikChange XL kit is demonstrated in mutagenesis studies employing a 10.1-kb plasmid. In this study, primers were designed to repair a frameshift mutation by inserting the missing nucleotide in the open reading frame (ORF) of the Moloney Murine Leukemia Virus (MMLV) gag-pol fusion protein. QuikChange reactions were performed with components of the standard kit or the QuikChange XL kit. Electrophoresis of Dpn I-digested reaction products showed that bands of the appropriate size (10.1-kb) were only evident in reactions performed with components of the QuikChange XL kit (Figure 2, lane 1). Moreover, transformation of XL10-Gold cells with a QuikChange XL reaction produced 32 colonies. In contrast, no colonies were produced with the standard QuikChange kit (Table 1). Nine out of 10 colonies produced a plasmid with the correct base insertion, as evidenced by DNA sequence analysis, and restoration of gag-pol gene function was verified by assaying reverse transcriptase activity and production of infectious virus (data not shown).

DNA Template
(kb)

Extension Time
(minutes/kb)

Number of Transformantsa

QuikChange kit

QuikChange XL kit

10.1

1

0

32

aReactions were carried out as described in the text and kit manuals. Values represent mean number of transformants from 3 plates.

Small-to-moderately sized plasmid DNA templates can be efficiently mutagenized with either QuikChange kit4. For such targets, the QuikChange XL kit permits the use of reduced reaction times. For example, the 4.5-kb kit control was efficiently replicated with the QuikChange XL kit using 0.5 min/kb extension times rather the 2 minute/kb extension times recommended for the standard QuikChange kit (data not shown). In these experiments, the average mutation frequency for the kit control was 98%, indicating that QuikChange XL kit retains the high mutagenesis efficiency of the QuikChange site-directed mutagenesis method.

Conclusions

We have introduced the QuikChange XL site-directed mutagenesis kit to improve the efficiency of mutagenizing long or difficult-to-mutagenize plasmid DNA templates using this single day mutagenesis method. The QuikChange XL kit features components specifically designed for more efficient DNA replication and bacterial t ransformation, which are important when mutagenizing large templates. The QuikChange XL kit retains the simplicity and ease-of-use of the parent kit, while ensuring high-efficiency mutagenesis of a broad range of plasmid DNA templates.

REFERENCES
  1. Kunkel, T.A. (1985) Proc. Nat. Acad. Sci. USA 82: 488-492.
  2. Bohnsack, R.N. (1996) In Vitro Mutagenesis Protocols 57: 1-12.
  3. Zhu, L. (1996) In Vitro Mutagenesis Protocols 57: 13-29.
  4. Papworth, C., et al. (1996) Strategies 9: 3-4.
  5. Hogrefe, H., et al. (1997) Strategies 10: 93-96.
  6. Jerpseth, A., et al. (1997) Strategies 10: 37-38.

* U.S. Patent Nos. 5,789,166 and 5,932,419 and patents pending
** U.S. Patent Nos. 5,512,468 and 5,707,841 and patents pending
*** U.S. Patent No. 5,545,552 and 5,866,395 and 5,948,663 and patents pending


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