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 (2003) Heft 5 . - S. 535-545.
ISSN 1097-0290
DOI: https://doi.org/10.1002/bit.10497

Volltext

Link zum Volltext (externe URL):

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.