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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-base-

pair changes, deletions, and insertions. Many of these

procedures require single-stranded DNA as template to

achieve high levels of efficient mutagenesis [1], though

recently, the use of double-stranded DNA and PCR-

based 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 prod-

uct, which is then used to transform Escherichia coli

cells. This method allows high-efficiency 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 sufficient

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 de-

scribe this simplified protocol in this report, whichachieves 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 neces-

sarily adjacent to each other) lay in the middle of the

oligonucleotide with sufficient flanking residues (912

basepairs) to allow a Tm close to 78 C. The formula

used for calculation of the Tm is Tm 81:5

0:41%GC 675=N % mismatch, where N is theprimer length.

Since only a single primer is used during the muta-

genesis reaction, the primer can be of sufficient 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 suc-

cessfully 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 GC-

rich 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 pri-

mer, 20 pmol; thermostable polymerase buffer (10),5ll; dNTPs, 0.6 ll of a solution containing 25 mM of

each dNTP; and polymerase, 2.5 U.

We have used proof-reading thermostable polyme-

rases, such as Pfu and Turbo Pfu (Strategene, USA), for

the synthesis of long plasmids. There will be no ampli-

fication 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 puri-fication 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 modifications 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 ex-

tension step at 68 C, where the extension time in min is

2 length of plasmid in kb, are performed. At the end ofthis step, the tube is held at 68 C for 20 min and then at

4 C indefinitely if required.

Analytical Biochemistry 319 (2003) 335336

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ANALYTICAL

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* Corresponding author. Fax: +91-80-3600999.

E-mail address: sandhya@mrdg.iisc.ernet.in (S.S. Visweswariah).

0003-2697/03/$ - see front matter 2003 Elsevier Science (USA). All rights reserved.

doi:10.1016/S0003-2697(03)00286-0

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DpnI is then added directly to the reaction tube (1 ll

equivalent to 10 U). The buffer conditions in the tube aresuch 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 find that

46 h is adequate. Following the digestion of templateDNA, 10ll of the reaction mixture is directly trans-

formed into chemically competent DH10B cells (effi-

ciency > 106=lg plasmid DNA) and transformants arechecked 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 compo-

nents 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 aver-

age, we have achieved mutation efficiencies 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 tempera-

tures as low as 40 C, with some problematic primers,

and in fact, lowering of the temperature does not appear

to appreciably reduce the efficiency of mutagenesis. Theselection of the host strain used for transformation ap-

pears to be important, and our most efficient mutagen-esis 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 ge-

notypes of the two strains. Our approach requires the

efficient 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 inother 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 ef-

ficient mutagenesis of DNA using only a single muta-

genic 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 incorpo-

ration of two mutations in a single oligonucleotide, as

long as the Tm of the oligonucleotide is high. We rou-

tinely adopt this procedure in the laboratory at present

with high success.

Acknowledgments

Financial assistance from the Department of Bio-

technology, Government of India is acknowledged.

References

[1] T.A. Kunkel, J.D. Roberts, R.A. Zakour, Rapid and efficient

site-specific mutagenesis without phenotypic selection, Methods

Enzymol. 154 (1987) 357382.

[2] W.P. Deng, J.A. Nickoloff, 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.

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.

336 Notes & Tips / Analytical Biochemistry 319 (2003) 335336