DNA purification by triple-helix affinity precipitation.
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 degrees C, i.e., below its critical solution temperature, the AML binds specifically to the target molecule in solution; by raising the temperature to 40 degrees 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.
Costioli
Matteo D
MD
Center of Biotechnology, Swiss Federal Institute of Technology Lausanne, 1015 Ecublens, Switzerland.
Fisch
Igor
I
Garret-Flaudy
Frédéric
F
Hilbrig
Frank
F
Freitag
Ruth
R
D000345:Affinity Labels / Q000737:chemistry*; D011232:Chemical Precipitation; D004247:DNA / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics; D010957:Plasmids / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D011786:Quality Control; D013696:Temperature
D000345:Affinity Labels; D009838:Oligodeoxyribonucleotides; C051802:triplex DNA; D004247:DNA