Generation of anNco I restriction site for translational fusions using PCR-mediated,site-directed mutagenesis |
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Authors: | Alfredo F Galvez Benito O de Lumen |
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Institution: | (1) Department of Nutritional Sciences, University of California at Berkeley, 97420-3104 Berkeley, CA, USA |
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Abstract: | A polymerase chain reaction-based method of site-directed mutagenesis was used to introduce anNco I restriction site on the translation start site of a tomato peroxidase gene. This quick and efficient method utilized two
overlapping synthetic oligonucleotide primers containing the requisite base pair changes on the ATG translation start site
and two flanking primers in PCR. The resulting DNA amplified fragments were fused together byNco I digestion at the mutated ends followed by a T4 ligation reaction. A rapid alternative method utilizing the overlapping
fragments and the flanking primers in PCR can also be used for ligating the two fragments. Cloning and sequencing of the PCR-amplified
fragments provided additional evidence for the presence of the site-specific mutations. Unique restriction sites upstream
and downstream of the site-specific mutation allows for the easy transfer of this mutated region into the wild type peroxidase
gene. |
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Keywords: | site-directed mutagensis polymerase chain reaction translation start site peroxidase gene |
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