Methods for rapid separation and purification of DNA topological forms
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorsMassa Shoura, Stephen Levene, David Girata
Life of this patent
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Abstract
Methods are provided for the isolation and analysis of circular DNA from complex samples, based on the topology of the DNA molecule. A sample comprising DNA species is combined with a chaotropic dense salt solution. A fraction containing the circular DNA of interest is isolated and dialyzed to remove excess salt. In some embodiments salt gradients are generated by ultracentrifugation in the absence of intercalating dyes, e.g. ethidium bromide; and in the absence of protease digestion. The circular DNA thus isolated is substantially pure, e.g. greater than about 75%, greater than about 80%, greater than about 90%, greater than about 95% of DNA in the isolated fraction is comprised of circular DNA.
Claims21
A method for topology-dependent, rapid DNA purification of circular DNA greater than 50 kbp in size;
- the method comprising:
- a) embedding cells in a polymer matrix;
- b) dissolving the polymer matrix in a chaotropic dense salt solution to lyse the cells in situ, thereby providing a DNA sample comprising circular DNA greater than 50 kbp in size in multiple topological states in the absence of intercalating dye and in the absence of protein-dependent digests;
- c) performing ultracentrifugation on the DNA sample in the chaotropic dense salt solution in the absence of intercalating dye;
- d) isolating fractions of DNA from a gradient of the DNA sample created by the ultracentrifugation;
- e) removing excess salt from the DNA fractions to generate a purified population of circular DNA greater than 50 kbp in size.
Dependencies are inferred from the claim text.