Begum Dilara, Piffanelli Pietro.
2003. A new BAC vector containing an inducible high-copy origin of replication.
Résumé : The BAC vector system based on F plasmid allows stable maintenance of large genomic DNA fragments as single copy plasmids. The disadvantage of low DNA yield from single copy BACs is a bottleneck to many high throughput processes such as BAC end sequencing, fingerprinting and shotgun library construction. A new BAC cloning vector, pCC1 has been developed that contains both a single copy origin of replication and the inducible oriV multiple copy origin of replication. When transformed into a specially engineered E. coli host containing a tightly controlled trfA gene, whose gene product is required for initiation of replication from oriV, pCC1 BAC clones are maintained at single copy and then can be induced on-demand to 10-20 copies per cell. This results in higher yields of BAC DNA with less chromosomal contamination. In addition, high copy induction can be incorporated into existing single-copy BAC clones in an in-vitro insertion reaction by using tn5 transposon-based EZTN-oriV-kan-2 system. Each insertion reaction results in a large population of subclones, each clone harboring a single randomly inserted transposon containing oriV. The insertion clones are maintained as single copy and can be induced to high copy and then sequence bidirectionally from the primer binding sites contained at the ends of the inserted transposon. This process can be easily adopted for high throughput sequencing as an alternative to shotgun library sequencing strategy. (Texte intégral)
Mots-clés Agrovoc : banque de gènes, séquence d'acides aminés
Mots-clés complémentaires : Bac
Classification Agris : F30 - Génétique et amélioration des plantes
Auteurs et affiliations
- Begum Dilara, EPICENTRE (USA)
- Piffanelli Pietro, CIRAD-AMIS-BIOTROP (FRA)
Autres liens de la publication
Source : Cirad - Agritrop (https://agritrop.cirad.fr/511765/)
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