US 12,331,340 B1
Efficient synthesis and assembly method for large fragment DNA based on programmable nuclease Argonaute
Lixin Ma, Wuhan (CN); Wanping Chen, Wuhan (CN); Jiakai Cui, Wuhan (CN); Miaomiao Chen, Wuhan (CN); Fei Wang, Wuhan (CN); Longyu Wang, Wuhan (CN); and Xiaochen Xie, Wuhan (CN)
Assigned to Hubei University, Wuhan (CN)
Filed by Hubei University, Wuhan (CN)
Filed on Oct. 14, 2024, as Appl. No. 18/915,035.
Application 18/915,035 is a continuation of application No. PCT/CN2024/095187, filed on May 24, 2024.
Claims priority of application No. 202311725165.0 (CN), filed on Dec. 14, 2023.
Int. Cl. C12P 19/34 (2006.01); C12N 1/20 (2006.01); C12N 9/22 (2006.01); C12N 15/11 (2006.01); C12N 15/70 (2006.01); C12N 15/90 (2006.01); C12R 1/19 (2006.01)
CPC C12P 19/34 (2013.01) [C12N 1/20 (2013.01); C12N 9/22 (2013.01); C12N 15/111 (2013.01); C12N 15/70 (2013.01); C12N 15/902 (2013.01); C12N 2310/20 (2017.05); C12R 2001/19 (2021.05)] 9 Claims
 
1. A synthesis and assembly method for fragment deoxyribonucleic acid (DNA) based on a programmable nuclease Argonaute, comprising:
S1, constructing plasmids as antibiotic resistance gene vectors, wherein each plasmid comprises a filler fragment and resistance gene fragments, with m base pairs (bp) of a homologous sequence between the resistance gene fragments;
S2, excising the filler fragment from each plasmid by using the programmable nuclease Argonaute and guide DNA (gDNA) to obtain linear vectors each with a 3′ overhang of m nucleotides (nt);
S3, dividing a target DNA into n small DNA fragments with 450-500 bp, and synthesizing the small DNA fragments and introducing m nt of sequences which are complementary to homologous sequences of the linear vectors to the small DNA fragments to obtain synthesized small DNA fragments;
S4, mixing and transforming the linear vectors obtained from the step S2 and the synthesized small DNA fragments obtained from the step S3 in competent cells of Escherichia coli to obtain recombined plasmids 1, 2, 3 to n; and
S5, assembling the recombined plasmids 1, 2, 3 to n;
wherein the amino acid sequence of the programmable nuclease Argonaute is shown in SEQ ID NO: 1.