US 12,435,332 B2
Ordered assembly of multiple DNA fragments
Gregory Lohman, Cambridge, MA (US); John M. Pryor, Gerogetown, MA (US); Rebecca Kucera, Hamilton, MA (US); Vladimir Potapov, Auburndale, MA (US); Katharina Bilotti, Burlington, MA (US); and Richard D. Morgan, Middleton, MA (US)
Assigned to New England Biolabs, Inc., Ipswich, MA (US)
Filed by New England Biolabs, Inc., Ipswich, MA (US)
Filed on Dec. 15, 2021, as Appl. No. 17/644,516.
Application 17/644,516 is a continuation in part of application No. 17/286,066, previously published as PCT/US2019/056670, filed on Oct. 17, 2019.
Claims priority of provisional application 63/213,859, filed on Jun. 23, 2021.
Claims priority of provisional application 63/213,807, filed on Jun. 23, 2021.
Claims priority of provisional application 63/125,530, filed on Dec. 15, 2020.
Claims priority of provisional application 62/909,641, filed on Oct. 2, 2019.
Claims priority of provisional application 62/820,435, filed on Mar. 19, 2019.
Claims priority of provisional application 62/747,874, filed on Oct. 19, 2018.
Prior Publication US 2022/0098577 A1, Mar. 31, 2022
Int. Cl. C12N 15/10 (2006.01); C12Q 1/6869 (2018.01)
CPC C12N 15/1065 (2013.01) [C12N 15/1044 (2013.01); C12Q 1/6869 (2013.01)] 12 Claims
 
1. A method comprising:
(a) obtaining a reaction mixture comprising:
(i) a synthetic self-complementary oligonucleotide comprising a double-stranded region and a loop, wherein the double-stranded region contains a binding sequence for PacCI and wherein the oligonucleotide comprises unligatable 3′ and 5′ ends and cannot be cleaved by PaqCI;
(ii) PaqCI;
(iii) a ligase; and
(iv) a library of DNA substrates each having at least one PaqCI recognition sequence and a cleavage site;
(b) cleaving the library of DNA substrates with PaqCI to generate fragments that have 4-base overhangs; and
(c) ligating complementary 4-base overhangs together to produce an ordered assembly of the fragments.