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US 7,622,252 B2Granted
Generation of minicircle DNA with physiological supercoiling
AssigneesBaylor College of Medicine, The Regents of the University of California
InventorsE. Lynn Zechiedrich, Jonathan Fogg, John Perona
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Abstract
Methods for the production of supercoiled DNA minicircles and their use as substrates are described herein.
Claims16
A method for producing supercoiled DNA minicircles, comprising:
- a) engineering a plasmid DNA molecule comprising site-specific recombination sites;
- b) transforming the plasmid into a cell suitable for site-specific recombination to occur, under conditions such that topoisomerase IV decatenation activity is inhibited, thereby producing a plurality of catenated DNA circles, wherein at least one of the circles in each catenane is a supercoiled DNA minicircle of less than about 1 kb in size;
- c) decatenating the catenated site-specific recombination products, thereby releasing the supercoiled DNA minicircles from the catenanes;
- and
- d) isolating the supercoiled DNA minicircles.
2 more elements
A method for producing a population of supercoiled DNA minicircles
- a) engineering a plasmid DNA molecule comprising site-specific recombination sites;
- b) transforming the plasmid into a cell suitable for site-specific recombination to occur, under conditions such that topoisomerase IV decatenation activity is inhibited, thereby producing a plurality of catenated DNA circles, wherein at least one of the circles in each catenane is a supercoiled DNA minicircle of less than about 1 kb in size;
- c) recovering the supercoiled DNA minicircles from the cell;
- d) treating the supercoiled DNA minicircles with an enzyme that introduces nicks into the DNA minicircle;
- and
- e) ligating the nicked minicircle in the presence of a DNA intercalator that introduces supercoils,
- thereby producing a population of supercoiled DNA minicircles.
4 more elements
Dependencies are inferred from the claim text.