US 12,338,432 B2
Sperm cell isolation and sperm-associated DNA purification
Nicholas Alan Courtney, Madison, WI (US); Margaret Mary Ewing, Middleton, WI (US); Robert Stewart McLaren, Oregon, WI (US); and David Harrison Warshauer, Verona, WI (US)
Assigned to Promega Corporation, Madison, WI (US)
Filed by Promega Corporation, Madison, WI (US)
Filed on Mar. 11, 2021, as Appl. No. 17/199,173.
Claims priority of provisional application 62/989,377, filed on Mar. 13, 2020.
Prior Publication US 2021/0284989 A1, Sep. 16, 2021
Int. Cl. C12N 15/10 (2006.01); C12Q 1/6806 (2018.01); C12Q 1/6888 (2018.01); G16B 50/00 (2019.01)
CPC C12N 15/101 (2013.01) [C12Q 1/6806 (2013.01); C12Q 1/6888 (2013.01); G16B 50/00 (2019.02)] 14 Claims
OG exemplary drawing
 
1. A method comprising:
(a) contacting a sample comprising sperm cells with a digestion agent comprising a detergent and proteinase K to a concentration of 0.1 mg/ml to 1 mg/ml proteinase K under conditions capable of efficient lysis of epithelial cells but wherein a portion of the sperm cells remain unlysed;
(b) removing the digestion agent from the sample, inactivating the digestion agent, or allowing the digestion agent to become inactivated;
(c) adding a reducing agent to the sample, wherein the reducing agent breaks disulfide bonds present on the sperm cells, but wherein a portion of the sperm cells remain unlysed;
(d) contacting the sample with a cellulose resin under conditions that the unlysed sperm cells bind to the cellulose resin;
(e) washing the cellulose resin with a wash buffer; and
(f) contacting the cellulose resin with an elution buffer capable of lysing the cellulose-resin-bound sperm cells and eluting sperm-associated DNA from the cellulose-resin-bound sperm cells.