US 12,065,689 B2
Methods for the identification and characterization of double-strand break sites and compositions and uses thereof
Stephane Deschamps, West Des Moines, IA (US); Virginijus Siksnys, Vilnius (LT); Joshua K Young, Johnston, IA (US); and Mindaugas Zaremba, Vilnius (LT)
Assigned to PIONEER HI-BRED INTERNATIONAL, INC, Johnston, IA (US); and VILNIUS UNIVERSITY, Vilnius (LT)
Appl. No. 17/054,637
Filed by PIONEER HI-BRED INTERNATIONAL, INC., Johnston, IA (US); and VILNIUS UNIVERSITY, Vilnius (LT)
PCT Filed May 10, 2019, PCT No. PCT/US2019/031719
§ 371(c)(1), (2) Date Nov. 11, 2020,
PCT Pub. No. WO2019/217816, PCT Pub. Date Nov. 14, 2019.
Claims priority of provisional application 62/670,366, filed on May 11, 2018.
Prior Publication US 2021/0147909 A1, May 20, 2021
Int. Cl. C12Q 1/6806 (2018.01); C12Q 1/6869 (2018.01)
CPC C12Q 1/6806 (2013.01) [C12Q 1/6869 (2013.01); C12Q 2521/301 (2013.01); C12Q 2521/525 (2013.01); C12Q 2525/191 (2013.01); C12Q 2535/122 (2013.01)] 64 Claims
 
1. A method for characterizing a double-strand-break-inducing agent cleavage site of an isolated, purified polynucleotide, comprising:
(a) adding phosphatase to the isolated, purified polynucleotide,
(b) contacting the phosphatase-treated polynucleotide from (a) with a double-strand-break-inducing agent to create a library of polynucleotides,
(c) optionally adding an adenine to the 3′ ends of the polynucleotides of the library, and
(d) ligating an adapter to the polynucleotides of the library, wherein the adapter comprises a nucleotide that is complementary to a terminal unpaired nucleotide of the polynucleotides of (b) or (c);
further comprising sequencing said library of polynucleotides, identifying at least one double-strand-break site, and assessing at least one qualitative characteristic or quantitative characteristic; wherein the phosphatase addition results in an increased number of double-strand-break sequence reads compared to a control method not comprising the addition of phosphatase.