Method for genetic selection of high-plasmid producing e....

C - Chemistry – Metallurgy – 12 – Q

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C12Q 1/68 (2006.01)

Patent

CA 2646218

The present invention relates to methods of selecting for highly productive clones of E. coli for the production of plasmid DNA comprising measuring the frequency of IS1 transposon insertional mutagenesis within either the plasmid or genomic DNA of transformed clonal subtypes. An increase in IS1 insertional mutagenesis is correlated with clonal subtypes likely to exhibit a low specific productivity. The PCR-based, genetic selection assays disclosed herein are amenable to high throughput analysis, reducing the time to identify highly productive clones capable of cultivating large quantities of plasmid DNA on an industrial scale.

La présente invention concerne des méthodes servant à sélectionner des clones extrêmement producteurs d'E. coli de manière à produire de l'ADN plasmidique. Ces méthodes consistent à mesurer la fréquence de la mutagenèse insertionnelle du transposon IS1 à l'intérieur de l'ADN plasmidique ou génomique de sous-types clonaux transformés. Une augmentation de la mutagenèse insertionnelle IS1 est corrélée à des sous-types clonaux susceptibles de présenter une faible productivité spécifique. Selon l'invention, les dosages de sélection génétique, basés sur PCR peuvent faire l'objet d'une analyse à rendement élevé, ce qui permet de diminuer le temps nécessaire à l'identification de clones extrêmement producteurs capables de cultiver des quantités importantes d'ADN plasmidique sur une échelle industrielle.

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