C - Chemistry – Metallurgy – 12 – N
Patent
C - Chemistry, Metallurgy
12
N
C12N 15/69 (2006.01)
Patent
CA 2375821
Methods that facilitates 1) cloning of large inserts into BAC plasmids, 2) isolation of large amounts of BAC DNA (by increasing plasmid copy number), and 3) increasing heterologous expression from BAC plasmid inserts (by increasing plasmid copy number and/or introducing promoters into the insert). A vector for increasing the copy number of plasmids, comprising a transposable element containing a moderate or high copy number origin of replication capable of in vitro transposition into a target plasmid is provided. The target plasmid is a single or low copy plasmid, e.g. a BAC vector, that is useful for cloning large pieces of DNA. The transposon plasmid may contain any moderate or high copy origin of replication that is compatible with a bacterial host such as E. coli. Thus, an exemplary ori is the colE1 ori from pBR322.
L'invention concerne des procédés qui facilitent 1) le clonage de gros inserts dans des plasmides de BAC et 2) l'isolement d'importantes quantités d'ADN de BAC (grâce à l'augmentation du nombre de copies des plasmides et/ou à l'introduction de promoteurs dans l'insert). L'invention concerne également un vecteur permettant d'augmenter le nombre de copies des plasmides qui comprend un élément transposable contenant un nombre d'origine moyen ou élevé de copies des originaux de réplication, capables de la transposition in vitro dans un plasmide cible. Le plasmide cible se présente comme un plasmide unique ou à faible nombre de copies, p.ex. un vecteur de BAC, utile pour le clonage de grosses parties de l'ADN. Le plasmide de transposon peu contenir n'importe quel nombre moyen ou élevé de réplications qui doivent être compatibles avec un hôte bactérien tel que E. coli; ainsi, un ori de l'exemple est un ori colE1 à partir de pBR322.
August Paul
Grossman Trudy
Macneil Ian
Aventis Pharmaceuticals Inc.
Bereskin & Parr Llp/s.e.n.c.r.l.,s.r.l.
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