Rapid analysis of a plasmid by hydrophobic-interaction chromatography with a non-porous resin.
A HPLC technique has been developed, based on hydrophobic-interaction Chromatography with a non-porous packing (TSKgel Butyl-NPR, Tosoh Biosep LLC), that allows separation of the open circular (nicked) and supercoiled forms of five DNA plasmids, ranging in size from 4 to 30 kilo base pairs (kb). The identity of the bands was determined through light scattering and gel electrophoresis. Several buffers, gradients, flow-rates and temperatures were evaluated in determining the optimum operating conditions for the separation. For all plasmids a reversed ammonium sulfate in phosphate buffer (pH 7.1) gradient was established. The chromatographic resolution between the supercoiled and nicked peaks was found to be a function of flow-rate and temperature. The resolution and the elution order did not vary with plasmid size, with the open-circular form always being eluted before the supercoiled form. Hydrophobic-interaction chromatography is a useful alternative to ion exchange or size exclusion for the chromatography of large plasmids, up to 30 kb.
Iuliano
S
S
Tosoh Biosep LLC, Montgomeryville, PA 18936, USA.
Fisher
J R
JR
Chen
M
M
Kelly
W J
WJ
D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D004587:Electrophoresis, Agar Gel; D008027:Light; D010957:Plasmids; D012542:Scattering, Radiation