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DNA purification by triple-helix affinity precipitation

Titelangaben

Costioli, Matteo D. ; Fisch, Igor ; Garret-Flaudy, Frédéric ; Hilbrig, Frank ; Freitag, Ruth:
DNA purification by triple-helix affinity precipitation.
In: Biotechnology and Bioengineering. Bd. 81 (März 2003) Heft 5 . - S. 535-545.
ISSN 1097-0290
DOI: https://doi.org/10.1002/bit.10497

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Link zum Volltext (externe URL): Volltext

Abstract

Recent advances in DNA-based medicine (gene therapy, genetic vaccination) have intensified the necessity for pharmaceutical-grade plasmid DNA purification at comparatively large scales. In this contribution triple-helix affinity precipitation is introduced for this purpose. A short, single-stranded oligonucleotide sequence (namely (CTT)7), which is capable of recognizing a complementary sequence in the double-stranded target (plasmid) DNA, is linked to a thermoresponsive N-isopropylacrylamide oligomer to form a so-called affinity macroligand (AML). At 4°C, i.e., below its critical solution temperature, the AML binds specifically to the target molecule in solution; by raising the temperature to 40°C, i.e., beyond the critical solution temperature of the AML, the complex can be precipitated quantitatively. After redissolution of the complex at lower temperature, the target DNA can be released by a pH shift to slightly alkaline conditions (pH 9.0). Yields of highly pure (plasmid) DNA were routinely between 70 and 90. Non-specific co- precipitation of either the target molecule by the non-activated AML precursor or of contaminants by the AML were below 7 and presumably due to physical entrapment of these molecules in the wet precipitate. Ligand efficiencies were at least 1 order of magnitude higher than in triple-helix affinity chromatography.

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Publikationsform: Artikel in einer Zeitschrift
Begutachteter Beitrag: Ja
Keywords: downstream processing; lower critical solution temperature; plasmid DNA; PNIPAM; precipitation; thermoresponsive polymer; triple-helix affinity
Institutionen der Universität: Fakultäten > Fakultät für Ingenieurwissenschaften > Lehrstuhl Bioprozesstechnik > Lehrstuhl Bioprozesstechnik - Univ.-Prof. Dr. Ruth Freitag
Fakultäten
Fakultäten > Fakultät für Ingenieurwissenschaften
Fakultäten > Fakultät für Ingenieurwissenschaften > Lehrstuhl Bioprozesstechnik
Titel an der UBT entstanden: Nein
Themengebiete aus DDC: 500 Naturwissenschaften und Mathematik
500 Naturwissenschaften und Mathematik > 500 Naturwissenschaften
600 Technik, Medizin, angewandte Wissenschaften
600 Technik, Medizin, angewandte Wissenschaften > 620 Ingenieurwissenschaften
Eingestellt am: 25 Feb 2016 15:10
Letzte Änderung: 25 Feb 2016 15:10
URI: https://eref.uni-bayreuth.de/id/eprint/31066