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KMID : 1007519990080050281
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Food Science and Biotechnology
1999 Volume.8 No. 5 p.281 ~ p.284
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Inhibition of Second - Site Mutations Arising from PCR - Based Megaprimer Mutagenesis
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Kim Tae-Rak
Carl A. Batt
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Abstract
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PCR-based site-directed mutagenesis is commonly used for various aims. The megaprimer method is simple and convenient, however, because of the terminal extension activity of Taq DNA polymerase, undesirable second-site mutations are generated. This results from the non-templated addition of a nucleotide (mainly an adenine) to the 3´-ends of `megaprimer´ PCR products. The added 3´ nucleotide decreases the utility of the megaprimer method by introducing second-site mutations. Phosphorylated 5´ terminus of the PCR primer may affect the nucleotide addition by the Taq DNA polymerase due to electrostatic repulsion between the phosphate group of the template and nucleotides. 5´Phosphorylation of the mutant primer can therefore inhibit secondary mutations better than widely used methods using a primer design or modified PCR cycling conditions. As a consequence, this can be used to improve the efficiency of PCR-based megaprimer methods for site-directed mutagenesis.
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