Protein Engineering vol.5 no.5 pp.467-468, 1992

PROTOCOL

Efficient deletion mutagenesis by PCR

Zumrut B.Ogel and Michael J.McPherson1 Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK 'To whom correspondence should be addressed

Key words: deletion mutagenesis/PCR

© Oxford University Press

Experimental procedure The PCR 1 in a volume of 50 fi\ contained 1 pmol of deletion primer, 100 pmol of primer a, 10 ng of wild-type DNA, 0.25 mM of dNTPs, 2.5 U of Taq polymerase and 5 /*1 of 10 X PCR buffer (Promega Corp., Madison, WI). After overlaying with 30 /il light mineral oil the reaction was subjected to 35 cycles of (95°C for 1 min, 55°C for 1 min and 72°C for 30 s). The DNA product (150 bp) was recovered from a 1% agarose gel by first soaking the gel slice in double distilled water for 20 min before centrifuging (13 000 g, 10 min) in a Spin-X filter unit (Costar). The purified megaprimer (10-20 ng) and 100 pmol each of primers a and b were included in PCR2 together with 10 ng of restriction fragment template (800 bp) with buffer, nucleotide and Taq polymerase conditions as for PCR1. The reaction was performed for 35 cycles of (95°C for 1 min, 55°C for 1 min and 72°C for 1 min). The resultant mutant fragment was cloned to construct the mutant gao A gene. The presence of the desired deletion and mismatch mutation was confirmed by DNA sequencing. Acknowledgements ZBO is supported by a PhD scholarship from the Middle East Technical University, Ankara, Turkey.

References Higuchi.R., Krummel.B. and Saiki,R.K. (1988) Nucleic Acids Res., 16, 7351-7367. Horton.R.M. and Pcase.L.R. (1991) In McPherson.M.J. (ed.), Directed Mutagenesis: a Practical Approach. IRL Press, Oxford, pp. 217—247. Ito.N., Phillips.S.E.V., Stevens.C, Ogel.Z., McPherson.M.J., Keen,J.N., Yadav.K.D.S. and Knowles.P.F. (1991) Nature, 350, 87-90. McPherson.M.J., Ogel.Z.B., Stevens,C, Yadav,K.D.S., KeenJ.N. and Knowles,P.F. (1992)/ Bid. Chem., 267, 8146-8152. Pemn.S. and Gilliland.G. (1990) Nucleic Acids Res., 18, 7433-7438. Sarkar.G. and Sommers.S.S. (1990) BioTechniques, 8, 404-407. Received on December 2, 1991; revised and accepted on May 13, 1992

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The PCR is of particular use in the construction of precisely engineered DNA molecules for applications such as site-directed mutagenesis or gene recombinations. One important class of mutational variants are those with large deletions which are often difficult to generate by traditional mutagenesis techniques. Such deletions can be generated using the PCR overlap extension procedure (Higuchi et al., 1988; Horton and Pease, 1991) which requires two mutagenic primers. An alternative one-sided (megaprimer) procedure uses only one mutagenic primer but results in lower mutant yield (Horton and Pease, 1991) and is of limited use in constructing deletions. Here we describe a megaprimer approach for generating deletions with high efficiency by a two step PCR procedure. A restricted fragment of DNA is used as a template in the second PCR and offers several advantages which are described below. This approach was used to delete a 460 bp region of the gao A gene encoding the N-terminal domain of galactose oxidase of Dactylium dendroides (Ito et al., 1991; McPherson et al., 1992). The overall procedure (Figure 1A) involves two PCR amplifications which use the mutagenic primer and two short flanking primers that may be either gene- or vector-specific primers depending on availability and subsequent cloning strategy. In the first PCR (PCR1), the deletion primer (d; Figure IB) and upstream primer (a) are used to amplify a DNA fragment (megaprimer; Sarkar and Sommers, 1990) which may be either double or single stranded if produced by asymmetric PCR (Perrin and Gilliland, 1990). The 3' end of this megaprimer derived from the 5' half of the deletion primer (see Figure 1A and B) is complementary to the downstream deletion end-point. In the second PCR (PCR2), primers a and b and the megaprimer are used in the presence of a wild-type DNA fragment which, importantly, lacks the sequence corresponding to the template region amplified during PCR1. During the initial cycles of PCR2 the megaprimer, primer b and primer a sequentially synthesize the mutant fragment (see Figure 1A) which is then amplified by primers a and b in the subsequent cycles of the PCR. The second strand of the megaprimer fragment is non-productive in PCR2. A key feature of our approach is the use of a limited wild-type template in PCR2 which increases the efficiency of mutagenesis in two ways: (i) it facilitates amplification of the mutant fragment by primers a and b, without the possibility of wild-type DNA co-amplification; and (ii) it eliminates a potential binding site for the megaprimer which could otherwise preferentially anneal to the template region of PCR 1, thereby sequestering megaprimer from the correct binding site at the downstream deletion endpoint (see Figure 1C). The limited template for PCR2 is an

essentially arbitrary fragment prepared simply by digesting wild-type DNA at any restriction site within the region to be deleted (see Figure 1A). Alternatively, it can be a PCR product amplified by primer b in combination with, for example, a convenient progressive sequencing primer that binds within the region to be deleted. To facilitate subsequent manipulation and analysis, the deletion primer d can be designed to incorporate additional point mutations. In our case two centrally placed base mismatches introduced an Apal site at the deletion junction.

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Efficient deletion mutagenesis by PCR.

Protein Engineering vol.5 no.5 pp.467-468, 1992 PROTOCOL Efficient deletion mutagenesis by PCR Zumrut B.Ogel and Michael J.McPherson1 Department of...
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