A site-directed mutagenesis method utilising large double-stranded DNA templates for the simultaneous introduction of multiple changes and sequential multiple rounds of mutation: Application to the study of whole viral genomes


Liu, L; Lomonossoff, G; (2006) A site-directed mutagenesis method utilising large double-stranded DNA templates for the simultaneous introduction of multiple changes and sequential multiple rounds of mutation: Application to the study of whole viral genomes. Journal of virological methods, 137 (1). pp. 63-71. ISSN 0166-0934 DOI: https://doi.org/10.1016/j.jviromet.2006.05.034

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Abstract

A new technique for conducting site-directed mutagenesis was developed. This method allows the colour selection of mutants through the simultaneous activation or deactivation of the a-peptide of P-galactosidase. Double-stranded DNA plasmids containing large inserts (at least 6.4 kbp in the present experiments) can be used as the mutational template. The method can efficiently create mutations at multiple sites simultaneously and can be used to perform multiple rounds of mutation on the same construct. The utility of the method for the analysis of viral genomes was demonstrated by applying it to the mutagenesis of a full-length cDNA copy of RNA-1 of Cowpea mosaic virus (CPMV). Six single-site mutants were initially produced which gave a variety of phenotypes when inoculated on to plants. To confirm that the phenotypes were directly caused by the introduced mutations, a second round of mutagenesis was used to create revertants of two of the mutants. In both cases, the revertants had a wild-type phenotype, demonstrating that the original phenotype was, indeed, the result of the introduced mutation. Overall, the results show that the present technique is a powerful method for site-directed mutagenesis of large DNA fragments, such as whole viral genomes, for functional studies. (c) 2006 Elsevier B.V. All rights reserved.

Item Type: Article
Keywords: site-directed mutagenesis, beta-galactosidase, colour selection, multiple rounds of mutation, Cowpea mosaic virus, COWPEA-MOSAIC-VIRUS, MIDDLE-COMPONENT RNA, ESCHERICHIA-COLI, AGROBACTERIUM-TUMEFACIENS, DPNI DIGESTION, TI-PLASMID, ONE-STEP, POLYMERASE, SELECTION, PROTEIN
Faculty and Department: Faculty of Infectious and Tropical Diseases > Dept of Immunology and Infection
PubMed ID: 16857273
Web of Science ID: 240531000010
URI: http://researchonline.lshtm.ac.uk/id/eprint/10606

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