Preparative concentration and size fractionation of DNA by porous media using a combination of flow and low electric field strength.
The retention of DNA by porous media during chromatography with an applied axial electric field was investigated. DNA was retained by negative electric fields in columns packed with Sephadex G-75 and G-25. A negative field was defined as the electric field orientation in which the direction of electrophoresis was opposing the direction of buffer flow (positive electrode at the column inlet). A positive field was not effective at retaining the DNA. The electric field strength required to retain the DNA was dependent upon the buffer flow rate. The retention of DNA using Sephadex G-25, a gel filtration medium with a higher degree of cross-linking, required higher electric fields than the more porous Sephadex G-75. A dilute DNA solution was concentrated at the inlet of the chromatography bed by an electric field. Mixtures of DNA restriction fragments were used to determine size-dependent retention. DNA was size-fractionated by varying the electric field strength and flow rate. At a given electric field strength and flow rate, the lower molecular weight DNA fragments were not as strongly retained as the higher molecular weight fragments. Decreasing the flow rate or increasing the electric field strength resulted in increased retention of the lower molecular weight DNA fragments. In this manner, by selecting a specific set of conditions (packing material, flow rate, and electric field strength), the molecular weight of DNA fragments retained by the column can be adjusted. Efficient separation of high molecular weight DNA from bovine serum albumin, a protein with high electrophoretic mobility, was demonstrated using a field of 2 V/cm in a column packed with Sephadex G-75.
Cole
K D
KD
National Institute of Standards and Technology, Biotechnology Division, Gaithersburg, Maryland 20899, USA. cole@mailserver.nist.gov
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D002850:Chromatography, Gel / Q000379:methods*; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry / Q000302:isolation & purification; D004560:Electricity; D008297:Male; D008970:Molecular Weight; D012474:Salmon; D012710:Serum Albumin, Bovine; D012996:Solutions; D013737:Testis / Q000737:chemistry
D004279:DNA, Viral; D012996:Solutions; D012710:Serum Albumin, Bovine; D004247:DNA; D004262:DNA Restriction Enzymes