Efficient purification of plasmid DNA for gene transfer using triple-helix affinity chromatography
Abstract
Plasmid DNA used for nonviral therapeutic gene transfer or nucleic acid vaccination has to be highly purified devoid of contaminating components such as bacterial proteins, endotoxins, or bacterial chromosomal DNA. We have developed a new affinity chromatography technique for plasmid DNA purification: triple-helix affinity chromatography (THAC). This technique is based on the sequence-specific interaction of an oligonucleotide forming a triple-helix with plasmid DNA. The oligonucleotide was covalently linked to a chromatographic matrix, thus providing a reusable affinity support. By inserting a suitable homopurine sequence in the plasmid DNA, it is possible to obtain a triple-helix interaction that will only be stable at mild acidic pH and that will dissociate in alkaline conditions. A crude lysate from a recombinant E. coli, or a pre-purified plasmid DNA, is thus applied at acidic pH on to a THAC column. After extensive washing of the column, purified plasmid DNA is eluted using an alkaline buffer. The binding conditions of the plasmid DNA on to the column have been optimized, as well as the hybridization sequence and the linker group between the matrix and the third strand oligonucleotide. The THAC technique makes it possible...Continue Reading
Citations
Protein-mediated isolation of plasmid DNA by a zinc finger-glutathione S-transferase affinity linker
Separation of supercoiled from open circular forms of plasmid DNA, and biological activity detection
Direct capture of plasmid DNA from non-clarified bacterial lysate using polycation-grafted monoliths
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