A ligation-independent cloning method using nicking DNA endonuclease
Using nicking DNA endonuclease (NiDE), we developed a novel technique to clone DNA fragments into plasmids. We created a NiDE cassette consisting of two inverted NiDE substrate sites sandwiching an asymmetric four-base sequence, and NiDE cleavage resulted in 14-base single-stranded termini at both e...
| Veröffentlicht in: | BioTechniques. - 1991. - 49(2010), 5 vom: 15. Nov., Seite 817-21 |
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| 1. Verfasser: | |
| Weitere Verfasser: | , , |
| Format: | Online-Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
2010
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| Zugriff auf das übergeordnete Werk: | BioTechniques |
| Schlagworte: | Journal Article Research Support, Non-U.S. Gov't Bacterial Proteins DNA, Complementary Luminescent Proteins yellow fluorescent protein, Bacteria DNA 9007-49-2 Deoxyribonuclease I EC 3.1.21.1 |
| Zusammenfassung: | Using nicking DNA endonuclease (NiDE), we developed a novel technique to clone DNA fragments into plasmids. We created a NiDE cassette consisting of two inverted NiDE substrate sites sandwiching an asymmetric four-base sequence, and NiDE cleavage resulted in 14-base single-stranded termini at both ends of the vector and insert. This method can therefore be used as a ligation-independent cloning strategy to generate recombinant constructs rapidly. In addition, we designed and constructed a simple and specific vector from an Escherichia coli plasmid back-bone to complement this cloning method. By cloning cDNAs into this modified vector, we confirmed the predicted feasibility and applicability of this cloning method |
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| Beschreibung: | Date Completed 24.02.2011 Date Revised 24.11.2010 published: Print Citation Status MEDLINE |
| ISSN: | 1940-9818 |
| DOI: | 10.2144/000113520 |