EP 2625275 A1 20130814 - METHODS FOR SEMI-SYNTHETICALLY PRODUCING HIGHLY PURE MINICIRCLE DNA VECTORS FROM PLASMIDS
Title (en)
METHODS FOR SEMI-SYNTHETICALLY PRODUCING HIGHLY PURE MINICIRCLE DNA VECTORS FROM PLASMIDS
Title (de)
VERFAHREN ZUR SEMI-SYNTHETISCHEN HERSTELLUNG HOCHREINER "MINICIRCLE" DNA-VEKTOREN AUS PLASMIDEN
Title (fr)
PROCÉDÉ DE PRODUCTION PAR SEMI-SYNTHÈSE DE VECTEURS D'ADN "MINICERCLES" À HAUT DEGRÉ DE PURETÉ À PARTIR DE PLASMIDES
Publication
Application
Priority
- EP 10186568 A 20101005
- EP 2011067280 W 20111004
- EP 11767977 A 20111004
Abstract (en)
[origin: EP2439276A1] Producing circular DNA-vectors (c-DNA-v) in super-helical form comprises: (a) splitting parental plasmids with restriction enzymes to obtain linear DNA vector fragments with the sequence of the DNA vector; (b) separating the linear DNA vector fragments from other products of the splitting reaction; (c) ligating the linear DNA vector fragment to obtain c-DNA-v in relaxed form; (d) separating the c-DNA-v from other products of the ligation; (e) twisting the c-DNA-v of step (d) with a gyrase to obtain c-DNA-v in super-helical form; and (f) optionally purifying the c-DNA-v in super-helical form. Producing circular DNA-vectors (c-DNA-v) in super-helical form comprises: (a) splitting parental plasmids with one or more restriction enzymes to obtain linear DNA vector fragments with the sequence of the DNA vector, where the parental plasmid contains the sequence of the DNA-vectors and heterologous sequences; (b) separating the linear DNA vector fragments from other products of the splitting reaction; (c) ligating the linear DNA vector fragment to obtain c-DNA-v in relaxed form; (d) separating the c-DNA-v from other products of the ligation; (e) twisting the c-DNA-v of step (d) with a gyrase to obtain c-DNA-v in super-helical form; and (f) optionally purifying the c-DNA-v in super-helical form for the separation of byproducts. Independent claims are included for: (1) a reagent kit for producing a circular DNA vector in super-helical form, comprising a ligase, a gyrase, and optionally one or more restriction enzymes; (2) preparation of a minicircle DNA vector in super-helical form, characterized by the absence of byproducts after PCR, preferably linear or circular miniplasmid and/or parental plasmid; and (3) a method for producing super-helical minicircle DNA vectors without the use of location (sequence) specific recombinases such as flippase recombination enzyme.
IPC 8 full level
C12N 15/64 (2006.01); C12N 15/79 (2006.01)
CPC (source: EP US)
C12N 15/10 (2013.01 - EP US); C12N 15/64 (2013.01 - EP US); C12P 19/34 (2013.01 - US)
Citation (search report)
See references of WO 2012045722A1
Designated contracting state (EPC)
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
DOCDB simple family (publication)
EP 2439276 A1 20120411; AU 2011311637 A1 20130418; AU 2011311637 A8 20130502; BR 112013009244 A2 20160726; CA 2813664 A1 20120412; EP 2625275 A1 20130814; IL 225518 A0 20130627; SG 189272 A1 20130531; US 2013203121 A1 20130808; WO 2012045722 A1 20120412
DOCDB simple family (application)
EP 10186568 A 20101005; AU 2011311637 A 20111004; BR 112013009244 A 20111004; CA 2813664 A 20111004; EP 11767977 A 20111004; EP 2011067280 W 20111004; IL 22551813 A 20130402; SG 2013025317 A 20111004; US 201113877586 A 20111004