US 11,952,570 B2
Methods for rapid separation and purification of DNA topological forms
Massa Shoura, Stanford, CA (US); Stephen Levene, Redwood City, CA (US); and David Girata, Redwood City, CA (US)
Assigned to The Board of Trustees of the Leland Stanford Junior University, Stanford, CA (US)
Filed by The Board of Trustees of the Leland Stanford Junior University, Stanford, CA (US); and BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM, Austin, TX (US)
Filed on Jan. 18, 2023, as Appl. No. 18/098,615.
Application 18/098,615 is a continuation of application No. PCT/US2021/042594, filed on Jul. 21, 2021.
Application PCT/US2021/042594 is a continuation of application No. 63/064,261, filed on Aug. 11, 2020.
Claims priority of provisional application 63/055,029, filed on Jul. 22, 2020.
Prior Publication US 2023/0151349 A1, May 18, 2023
Int. Cl. C12N 15/10 (2006.01)
CPC C12N 15/1006 (2013.01) [C12N 15/1003 (2013.01)] 21 Claims
 
1. 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.