Abstract
Protocols for site-directed mutagenesis are widely used in molecular biology and include many polymerase chain reaction (PCR)-based methods that have been developed in order to achieve efficient mutagenesis of a target DNA sequence (1–4). However, some of these methods described require two or more specific-oligonucleotides for each round of mutagenesis, making the cost of such procedures expensive. This chapter describes an efficient and economic PCR-based site-directed mutagenesis method, which is designed to introduce a series of mutations into DNA cloned into pUC vectors (pUC 18, 19, 118, 119). The protocol uses a combination of a primer designed for introducing a mutation at the target sequence, with primers that may be reused for each mutagenesis reaction (Fig. 1). By using this method, a series of site-directed mutations may be undertaken that only require a single primer for each desired change, and furthermore, no reiterative transformation steps are necessary.
Principle of PCR in vitro mutagenesis. [1] First round PCR of target DNA following cloning of sequence into multicloning site of one of the pUC series of vectors. One of the MUT primers is chosen to destroy a restriction site, based on both the direction of R1 primer (primer for introducing a mutation) and the restriction site used for cloning the target DNA sequence. The first round PCR is carried out by the combination of R1 primer and M13 primer RV (or M13 primer M4), MUT primer, and M13 primer M4 (or M13 primer RV) separately in two tubes. [2] After DNA purification to remove excess primers, the amplified products are mixed, heat denatured, and annealed. [3] Taq DNA polymerase is added to complete the DNA heteroduplex. [4] The second round PCR is performed using M13 primer M4 and M13 primer RV flanking oligonucleotides, which will result in two types of amplified products, (a) and (b). [5] The amplified products are digested with two restriction enzymes, one of which shall recognize the site (X) that had been destroyed by the MUT primer and the other which recognizes the appropriate site (Y) within the multicloning site. [6] Reclone the digested fragment into the vector digested with the same two restriction enzymes. Only the fragment (a) that contains the mutation introduced by R1 primer sequence will be recloned.
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Notes
- 1.
* Different M13 primers must be used in a and b.
References
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© 1996 Humana Press Inc.
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Shimada, A. (1996). PCR-Based Site-Directed Mutagenesis. In: Trower, M.K. (eds) In Vitro Mutagenesis Protocols. Methods In Molecular Medicine™, vol 57. Humana Press. https://doi.org/10.1385/0-89603-332-5:157
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DOI: https://doi.org/10.1385/0-89603-332-5:157
Publisher Name: Humana Press
Print ISBN: 978-0-89603-332-0
Online ISBN: 978-1-59259-544-0
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