This invention relates to the general field of bio-chemical separations. Current methods for separating nucleic acids either lack sufficient selectivity or large-capacity production as required for gene therapy or DNA vaccines or other applications. There is a need for more selective methods of separating nucleic acids that results in a relatively uncontaminated product in larger volumes and yields than achieved with currently available separation methods.
This method is used for separating nucleic acids and other similar constructs. It involves selective introduction, enhancement, or stabilization of affinity handles such as single-strandedness in the desired nucleic acids as compared to the usual structure (e.g. double-strandedness) of the nucleic acids. The undesired (or desired) nucleic acids are separated from the nucleic acids due to capture by methods including immobilized metal affinity chromatography, immobilized single-stranded DNA binding (SSB) protein, and immobilized oligonucleotides. The invention is useful for removing contaminating genomic DNA from plasmid DNA; removing genomic DNA from plasmids, BACs, and similar constructs; selectively separating oligonucleotides and similar DNA fragments from their partner strands; purifying aptameters, (deoxy)-ribozymes, and other highly structured nucleic acids; separating restriction fragments without using agarose gels; and manufacturing recombinant Taq polymerase or similar products that are free of host genomic DNA contamination.
The novel separation technique described herein addresses these shortcomings. This method, which extends to a number of alternative structures and chemistries for nucleic acids (for example, alternative backbone structures), involves the selective introduction, enhancement, and stabilization of structural “affinity handles” such as singlestrandedness in the nucleic acids as compared to the usual structure such as double-strandedness of the nucleic acids. The exposed bases of single-stranded nucleic acids allow capture of the nucleic acids by techniques that are selective for single-stranded regions of nucleic acids, such as immobilized metal affinity chromatography (IMAC), hydrophobic interaction chromatography (HIC), reversedphase chromatography (RFC), immobilized single-stranded DNA binding (SSB) protein, immobilized nucleic acids (polydT, poly-dU, or specific sequences), or the use of peptide nucleic acids (PNAs).
This method can selectively and efficiently capture nucleic acids containing exposed purine bases and separate them from the desired product. Unlike anion exchangers, only the trace contaminant is bound, and the effective capacity for the desired product is high. This technique can be used to remove unwanted nucleic acid sequences that are already single-stranded, such as irreversibly denatured plasmids that have lost their supercoiled nature or other nucleic acid fragments, and enhance the purification of the favored nucleic acid product or other product. This novel separation method can be easily scaled up for production of larger yields.
This work was done by Richard Willson and Luis Cano of the University of Houston Office of Intellectual Property Management for Johnson Space Center. MSC-26074-1