US 12,241,057 B2
Methods for nucleic acid assembly and high throughput sequencing
Michael E. Hudson, Boston, MA (US); Li-Yun A. Kung, Boston, MA (US); Daniel Schindler, Boston, MA (US); Stephen Archer, Boston, MA (US); and Ishtiaq Saaem, Boston, MA (US)
Assigned to Gen9, Inc., Boston, MA (US)
Filed by Gen9, Inc., Boston, MA (US)
Filed on Jun. 28, 2021, as Appl. No. 17/361,091.
Application 17/361,091 is a continuation of application No. 16/714,208, filed on Dec. 13, 2019, granted, now 11,072,789.
Application 16/714,208 is a continuation of application No. 14/408,103, abandoned, previously published as PCT/US2013/047370, filed on Jun. 24, 2013.
Claims priority of provisional application 61/731,627, filed on Nov. 30, 2012.
Claims priority of provisional application 61/664,118, filed on Jun. 25, 2012.
Prior Publication US 2021/0395724 A1, Dec. 23, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/686 (2018.01); C12N 15/10 (2006.01); C12N 15/66 (2006.01)
CPC C12N 15/1031 (2013.01) [C12N 15/1093 (2013.01); C12N 15/66 (2013.01); C12Q 1/686 (2013.01)] 26 Claims
 
1. A method of producing a nucleic acid having a predefined sequence, the method comprising:
(a) providing a first pool of double-stranded oligonucleotides, wherein the double-stranded oligonucleotides comprise:
(i) internal sequences identical to different portions of a first target nucleic acid, wherein the internal sequences comprise an overlapping region with another double-stranded oligonucleotide in the first pool; and
(ii) 5′ flanking sequences and/or 3′ flanking sequences, each of the flanking sequences comprising a common primer recognition site and a first restriction enzyme recognition site, the first restriction enzyme recognition site being oriented so that digestion with a first restriction enzyme that recognizes the first restriction enzyme recognition site will remove the flanking sequences and expose the internal sequence; and
(b) exposing the first pool of double-stranded oligonucleotides to a ligase and the first restriction enzyme under conditions suitable to promote concurrent restriction enzyme digestion and ligation, thereby generating the first target nucleic acid;
wherein the first target nucleic acid comprises:
(i) an internal sequence identical to a portion of a final target nucleic acid; and
(ii) a 5′ flanking sequence and/or a 3′ flanking sequence, each of the flanking sequences comprising a second restriction enzyme recognition site, wherein the first restriction enzyme recognition site and the second restriction enzyme recognition site have different sequences.