	US 11,674,168 B2
	Isolation and detection of DNA from plasma
Hatim Allawi, Middleton, WI (US); Graham P. Lidgard, Middleton, WI (US); Brian Aizenstein, Madison, WI (US); Tamara J. Sander, Mazomanie, WI (US); Maria Giakoumopoulos, Middleton, WI (US); Michael W. Kaiser, Stoughton, WI (US); Melissa M. Gray, Madison, WI (US); and Abram Michael Vaccaro, Rio, WI (US)
Assigned to Exact Sciences Corporation, Madison, WI (US)
Filed by Exact Sciences Corporation, Madison, WI (US)
Filed on Sep. 24, 2020, as Appl. No. 17/31,383.
Application 17/031,383 is a continuation of application No. 15/335,111, filed on Oct. 26, 2016, granted, now 10,822,638.
Claims priority of provisional application 62/249,097, filed on Oct. 30, 2015.
Prior Publication US 2021/0079451 A1, Mar. 18, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/6806 (2018.01); C12Q 1/686 (2018.01); C12N 15/10 (2006.01); C12Q 1/6837 (2018.01)

CPC C12Q 1/6806 (2013.01) [C12N 15/1006 (2013.01); C12Q 1/686 (2013.01); C12Q 1/6837 (2013.01); C12Q 2600/154 (2013.01); C12Q 2600/166 (2013.01)]

11 Claims

1. A method of processing a plasma sample, the method comprising:

a) combining a plasma sample with

i) protease;

ii) a first portion of guanidine thiocyanate;

iii) a first portion of non-ionic detergent; and

iv) bulk fish DNA diluent,

to form a first mixture wherein proteins are digested by said protease;

b) to the first mixture adding

v) silica particles;

vi) isopropyl alcohol;

vii) a second portion of guanidine thiocyanate; and

viii) a second portion of non-ionic detergent,

to form a second mixture under conditions wherein DNA is bound to said silica particles;

c) separating silica particles with bound DNA from the second mixture;

d) to the separated silica particles with bound DNA adding a first wash solution, said first wash solution comprising guanidine hydrochloride or guanidine thiocyanate, and ethyl alcohol;

e) separating the silica particles with bound DNA from said first wash solution;

f) to the separated silica particles with bound DNA adding a second wash solution, said second wash solution comprising a buffer and ethyl alcohol;

g) separating the washed silica particles with bound DNA from said second wash solution;

h) eluting DNA from said washed silica particles with bound DNA to produce an isolated DNA sample.