Copy-Paste Mutagenesis: A Method for Large-Scale Alteration of Viral Genomes
Abstract
The cloning of the large DNA genomes of herpesviruses, poxviruses, and baculoviruses as bacterial artificial chromosomes (BAC) in Escherichia coli has opened a new era in viral genetics. Several methods of lambda Red-mediated genome engineering (recombineering) in E. coli have been described, which are now commonly used to generate recombinant viral genomes. These methods are very efficient at introducing deletions, small insertions, and point mutations. Here we present Copy-Paste mutagenesis, an efficient and versatile strategy for scarless large-scale alteration of viral genomes. It combines gap repair and en passant mutagenesis procedures and relies on positive selection in all crucial steps. We demonstrate that this method can be used to generate chimeric strains of human cytomegalovirus (HCMV), the largest human DNA virus. Large (~15 kbp) genome fragments of HCMV strain TB40/E were tagged with an excisable marker and cloned (copied) in a low-copy plasmid vector by gap repair recombination. The cloned fragment was then excised and inserted (pasted) into the HCMV AD169 genome with subsequent scarless removal of the marker by en passant mutagenesis. We have done four consecutive rounds of this procedure, thereby generating an...Continue Reading
References
Citations
Methods Mentioned
Related Concepts
Related Feeds
Artificial Chromosomes
Artificial chromosomes are genetically engineered chromosomes derived from the DNA of a species. Discover the latest research on artificial chromosomes here.