ANALYTICAL BIOCHEMISTRY

Analytical Biochemistry 319 (2003) 335336 www.elsevier.com/locate/yabio

Notes & Tips

Site-directed mutagenesis using a single mutagenic oligonucleotide and DpnI digestion of template DNA
Avinash R. Shenoy and Sandhya S. Visweswariah*
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India Received 4 April 2003

Site-directed mutagenesis is used extensively for the analysis of gene structure and function, and several methods are currently employed to obtain single-basepair changes, deletions, and insertions. Many of these procedures require single-stranded DNA as template to achieve high levels of ecient mutagenesis [1], though recently, the use of double-stranded DNA and PCRbased methods have gained popularity, given the ease of preparation of the template DNA [2,3]. PCR-based approaches require the synthesis of two complimentary oligonucleotides that contain the desired mutation(s) and these are used to prime the PCR on a plasmid DNA template. Subsequent digestion of the reaction mixture with DpnI removes the template DNA, leaving intact the newly synthesized double-stranded mutant PCR product, which is then used to transform Escherichia coli cells. This method allows high-eciency mutagenesis in a fairly short period of time [4]. We have utilized this approach for some of our studies, but have made the observation that a single mutagenic primer was sucient to generate mutant single-stranded DNA, which could then be transformed into E. coli DH10B cells to obtain plasmid DNA containing the desired mutation. We describe this simplied protocol in this report, which achieves successful mutation frequencies on par with that using two primers (Fig. 1). Primers used for the mutagenesis were designed such that the mutation (in some cases 2 basepairs not necessarily adjacent to each other) lay in the middle of the oligonucleotide with sucient anking residues (912 basepairs) to allow a Tm close to 78 C. The formula used for calculation of the Tm is Tm 81:5

* Corresponding author. Fax: +91-80-3600999. E-mail address: sandhya@mrdg.iisc.ernet.in (S.S. Visweswariah).

0:41%GC 675=N % mismatch, where N is the primer length. Since only a single primer is used during the mutagenesis reaction, the primer can be of sucient length to achieve a high Tm, since no self-annealed primers can form during the annealing and DNA synthesis steps. For example, we have used a 41-mer primer very successfully during mutagenesis of AT-rich sequences in the human guanylyl cyclase C cDNA to bring in 2 basepair changes separated by 11 bases. In addition, highly GCrich sequences, such as those present in Mycobacterium tuberculosis genes, were successfully mutated using shorter 25-mer primers. The conditions used for PCR are as follows, in a total volume of 50 ll: template DNA, 100 ng; mutagenic primer, 20 pmol; thermostable polymerase buer (10), 5 ll; dNTPs, 0.6 ll of a solution containing 25 mM of each dNTP; and polymerase, 2.5 U. We have used proof-reading thermostable polymerases, such as Pfu and Turbo Pfu (Strategene, USA), for the synthesis of long plasmids. There will be no amplication of DNA during the mutagenesis procedure, since only a single primer is used. Primers have been used as supplied by the manufacturer with no gel purication or phosphorylation required. The conditions for the synthesis of the mutant DNA strand is based on the annealing temperature of the primer and may require some modications to the conditions that we describe here. The tube is initially taken to 96 C for 2 min, and then 18 cycles consisting of 1 min at 96 C, an annealing step at a temperature suitable for the primer (can be 4055 C), and an extension step at 68 C, where the extension time in min is 2 length of plasmid in kb, are performed. At the end of this step, the tube is held at 68 C for 20 min and then at 4 C indenitely if required.

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Notes & Tips / Analytical Biochemistry 319 (2003) 335336

Fig. 1. Schematic of the mutagenesis protocol. PCR is carried out as described and a single-stranded nicked DNA molecule containing the desired mutation is left at the end of the reaction following digestion with DpnI. This is transformed into competent DH10B cells to obtain plasmid DNA with the desired mutation.

DpnI is then added directly to the reaction tube (1 ll equivalent to 10 U). The buer conditions in the tube are such that DpnI digestion can occur, and in the low concentrations of salt that are present in the tube, both methylated (parent) and hemimethylated DNA will be digested. Digestion with DpnI can be performed for long periods of time if necessary, though we usually nd that 46 h is adequate. Following the digestion of template DNA, 10 ll of the reaction mixture is directly transformed into chemically competent DH10B cells (eciency > 106 =lg plasmid DNA) and transformants are checked for the presence of the mutation by sequencing and restriction digestion if a suitable site was introduced during the mutagenesis reaction. A control reaction can be set up where all components of the reaction are added except the polymerase. This reaction on transformation should give no colonies on the plate, and with adequate DpnI digestion, this is found to be the case. Transformation of the mutagenesis reaction can give up to 100 colonies. We usually screen 5 colonies for the presence of the mutation, and on average, we have achieved mutation eciencies of 6090% using this method.

As mentioned above, we have used this approach to mutagenize genes which were more than 70% GC-rich, with no requirement for addition of dimethyl sulfoxide during the reaction. We have used annealing temperatures as low as 40 C, with some problematic primers, and in fact, lowering of the temperature does not appear to appreciably reduce the eciency of mutagenesis. The selection of the host strain used for transformation appears to be important, and our most ecient mutagenesis has been obtained when DH10B or DH5a cells were used. We did not achieve high mutation frequencies using TOP10 cells (Invitrogen) but cannot provide an explanation for this at this time, given the similar genotypes of the two strains. Our approach requires the ecient transformation of single-stranded DNA, and perhaps strain-to-strain variation is seen. In general, mutagenesis performed on a fragment of the gene of interest, followed by subcloning back into the full-length gene, would reduce the chance of random mutation in other regions of the template during the mutagenesis step. We usually sequence the entire fragment used for mutagenesis to check for mutations in sequences other than that brought in with the primer. In summary, we describe here a protocol for the efcient mutagenesis of DNA using only a single mutagenic oligonucleotide and incorporating the step of DpnI digestion to reduce the number of nonmutagenized colonies obtained after transformation. The procedure works well for GC-rich DNA and allows the incorporation of two mutations in a single oligonucleotide, as long as the Tm of the oligonucleotide is high. We routinely adopt this procedure in the laboratory at present with high success.

Acknowledgments Financial assistance from the Department of Biotechnology, Government of India is acknowledged.

References
[1] T.A. Kunkel, J.D. Roberts, R.A. Zakour, Rapid and ecient site-specic mutagenesis without phenotypic selection, Methods Enzymol. 154 (1987) 357382. [2] W.P. Deng, J.A. Nickolo, Site-directed mutagenesis of virtually any plasmid by eliminating a unique site, Anal. Biochem. 200 (1992) 8188. [3] S. Barik, Site-directed mutagenesis in vitro by megaprimer PCR, Methods Mol. Biol. 57 (1996) 203215. [4] S. Li, M.F. Wilkinson, Site-directed mutagenesis: a two-step method using PCR and DpnI, Biotechniques 23 (1997) 588590.