Separation and recovery of nucleic acids with improved biological activity by acid-degradable polyacrylamide gel electrophoresis.
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.
Kim
Yoon Kyung
YK
Department of Biomedical Engineering, University of California, Irvine, CA, USA.
Kwon
Young Jik
YJ
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
D009696:Nucleic Acids; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D004247:DNA