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US 11,597,958 B2Granted
DNA assembly method and its application
AssigneeNanjing Zhongkeyouzi Institute of Biotechnology Co., Ltd
InventorsShuwen Liu, Tingyi Wen, Yun Zhang, Aihua Deng
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Abstract
The invention relates to a plasmid, a DNA assembly method and its application recombinant strain. The plasmid has single adjacent Type IIP and Type IIS RE recognition sites. The plasmid combines the properties of Type IIP and Type IIS REs to achieve recursive cycling, SCAR-free and repeat sequence assembly.
Claims7
A DNA assembly method, comprising:
- (1) performing a single-ended ligation of a gene to be inserted to a DNA fragment containing adjacent Type IIP and Type IIS RE recognition sites to obtain a target gene;
- (2) cleaving the target gene using the corresponding Type IIP RE to obtain a donor DNA with a sticky end;
- (3) cleaving a plasmid using the corresponding Type IIP and Type IIS REs to obtain an acceptor DNA, wherein the plasmid comprises the same Type IIP and Type IIS RE recognition sites as the target gene;
- and
- (4) ligating the sticky end of donor DNA to the Type IIP RE cleaved complementary sticky end of acceptor DNA and ligating the un-cleaved end of donor DNA to the Type IIS RE cleaved end of acceptor DNA.
2 more elements
A DNA assembly method, comprising:
- (1) performing a single-ended ligation of a gene to be inserted to a DNA fragment containing adjacent Type IIP and Type IIS RE recognition sites to obtain a target gene;
- (2) cleaving the target gene using the corresponding Type IIP RE to obtain a donor DNA with a sticky end;
- (3) cleaving a plasmid using the corresponding Type IIP and Type IIS REs to obtain an acceptor DNA, wherein the plasmid comprises the same Type IIP and Type IIS RE recognition sites as the target gene, wherein the Type IIP RE cleaves to produce sticky ends with two to four bases;
- and
- (4) ligating the sticky end of donor DNA to the Type IIP RE cleaved complementary sticky end of acceptor DNA and ligating the un-cleaved end of donor DNA to the Type IIS RE cleaved end of acceptor DNA.
2 more elements
A DNA assembly method, comprising:
- (1) performing a single-ended ligation of a gene to be inserted to a DNA fragment containing adjacent Type IIP and Type IIS RE recognition sites to obtain a target gene;
- (2) cleaving the target gene using the corresponding Type IIP RE to obtain a donor DNA with a sticky end;
- (3) cleaving a plasmid using the corresponding Type IIP and Type IIS REs to obtain an acceptor DNA, wherein the plasmid comprises the same Type IIP and Type IIS RE recognition sites as the target gene, wherein the Type IIS RE cleaves to produce single-base sticky ends, or cleaves to produce blunt ends;
- and
- (4) ligating the sticky end of donor DNA to the Type IIP RE cleaved complementary sticky end of acceptor DNA and ligating the un-cleaved end of donor DNA to the Type IIS RE cleaved end of acceptor DNA.
2 more elements
A DNA assembly method, comprising:
- (1) performing a single-ended ligation of a gene to be inserted to a DNA fragment containing adjacent Type IIP and Type IIS RE recognition sites to obtain a target gene;
- (2) cleaving the target gene using the corresponding Type IIP RE to obtain a donor DNA with a sticky end;
- (3) cleaving a plasmid using the corresponding Type IIP and Type IIS REs to obtain an acceptor DNA, wherein the plasmid comprises the same Type IIP and Type IIS RE recognition sites as the target gene;
- wherein the Type IIS RE is BmrI, BciVI, HphI or MlyI;
- and
- (4) ligating the sticky end of donor DNA to the Type IIP RE cleaved complementary sticky end of acceptor DNA and ligating the un-cleaved end of donor DNA to the Type IIS RE cleaved end of acceptor DNA.
3 more elements
Dependencies are inferred from the claim text.