Preparation of DNA topoisomers by RP-18 high-performance liquid chromatography.
A method for the separation of superhelical DNA on the basis of superhelical density by reverse-phase HPLC on RP-18 columns is described. The technique can be used to prepare superhelical DNA in milligram amounts and narrow topoisomer distributions in 0.1 mg amounts. We show example separations of the plasmids pUC18 (2687 bp) and pi AN13 (895 bp). While the best separation for pUC18 yields topoisomer distributions of two or three major components, the small plasmid can be separated into single topoisomer fractions. The basis of the separation is probably an interaction of partially opened bases with the hydrophobic column matrix. This hypothesis is supported by the elution behavior of DNA fragments on this column: DNA fragments with sticky ends, even at a length of several hundred base pairs, elute at much higher methanol concentrations than blunt-ended fragments.
Kapp
U
U
EMBL, Grenoble, France.
Langowski
J
J
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification*; D004278:DNA, Superhelical / Q000737:chemistry / Q000302:isolation & purification*; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry; D007536:Isomerism; D010957:Plasmids; D015183:Restriction Mapping
D004269:DNA, Bacterial; D004278:DNA, Superhelical; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; C040158:GTAC-specific type II deoxyribonucleases