title: Separation and recovery of nucleic acids with improved biological activity by acid-degradable polyacrylamide gel electrophoresis.
abstract: One of the fundamental challenges in studying biomacromolecules (e.g. nucleic acids and proteins) and their complexes in a biological system is isolating them in their structurally and functionally intact forms. Electrophoresis offers convenient and efficient separation and analysis of biomacromolecules but recovery of separated biomacromolecules is a significant challenge. In this study, DNAs of various sizes were separated by electrophoresis in an acid-degradable polyacrylamide gel. Almost 100% of the nucleic acids were recovered after the identified gel bands were hydrolyzed under a mildly acidic condition and purified using anion exchange resin. Further concentration by centrifugal filtration and a second purification using ion exchange column chromatography yielded 44-84% of DNA. The second conventional (non-degradable) gel electrophoresis confirmed that the nucleic acids recovered from acid-degradable gel bands preserved their electrophoretic properties through acidic gel hydrolysis, purification, and concentration processes. The plasmid DNA recovered from acid-degradable gel transfected cells significantly more efficiently than the starting plasmid DNA (i.e. improved biological activity via acid-degradable PAGE). Separation of other types of nucleic acids such as small interfering RNA using this convenient and efficient technique was also demonstrated.
authors.affiliation: Department of Biomedical Engineering, University of California, Irvine, CA, USA.
authors.forename: Yoon Kyung
authors.initials: YK
authors.lastname: Kim
authors.forename: Young Jik
authors.initials: YJ
authors.lastname: Kwon
mesh_terms: D000818:Animals; D004247:DNA / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D049452:Green Fluorescent Proteins; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008856:Microscopy, Fluorescence; D041681:NIH 3T3 Cells; D009696:Nucleic Acids / Q000302:isolation & purification*; D010957:Plasmids / Q000302:isolation & purification; D034741:RNA, Small Interfering / Q000302:isolation & purification; D014162:Transfection
chemical_list: D009696:Nucleic Acids; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D004247:DNA