Differential Sedimentation of Linear versus Circular DNAs Using Divalent Metal Cations

Molecular biologists routinely purify and concentrate DNA and RNA molecules by precipitation with ethanol, a method that does not discriminate between the different structural forms of each nucleic acid. In the current project we have assessed the ability of monovalent Group I and divalent Group II metal cations to selectively precipitate DNAs with different shapes and sizes out of aqueous solutions. Particular emphasis was placed on separation of circular plasmid DNAs from contaminating linear chromosomal DNA fragments. This was done by mixing Group 1 or Group 2 metals with DNA at various pH values then spinning the solutions in a microcentrifuge. The absorbance was then measured at 260 nm. Group 1 metal ions proved to be ineffective at sedimenting large chromosomal DNA fragments. Group 2 metal cations (Mg 2+ , Ca 2+ , Sr 2+ and Ba 2+ ) sedimented linear DNAs strongly, but only at high pH values (10–12). Ca 2+ was chosen for further experiments because it is not a cofactor for nucleases like Mg 2+ and it is found naturally within cells, unlike Sr 2+ and Ba 2+ . Preliminary studies involving manipulation of parameters such as metal and buffer concentrations identified conditions that allowed chromosomal DNA to be sedimented approximately 10 times more efficiently than supercoiled plasmid DNA. Current experiments are seeking to maximize this difference in precipitation efficiencies, with a focus on sedimenting linear chromosomal DNA while leaving supercoiled plasmid DNA in solution. Support or Funding Information Texas State University