Fractional precipitation of plasmid DNA from lysate by CTAB.
Preparative-scale purification of plasmid DNA has been attempted by diverse methods, including precipitation with solvents, salts, and detergents and chromatography with ion-exchange, reversed-phase, and size-exclusion columns. Chromatographic methods such as hydrophobic interaction chromatography (HIC), reversed phase chromatography (RPC), and size exclusion chromatography (SEC) are the only effective means of eliminating the closely related relaxed and denatured forms of plasmid as well as endotoxin to acceptable levels. However, the anticipated costs of manufacturing-scale chromatography are high due to (a) large projected volumes of the high-dosage therapeutic molecule and (b) restricted loading of the large plasmid molecule in the pores of expensive resins. As an alternative to chromatography, we show herein that precipitation with the cationic detergent, cetyltrimethylammonium bromide (CTAB), is effective for selective precipitation of plasmid DNA from proteins, RNA, and endotoxin. Moreover, CTAB affords novel selectivity by removal of host genomic DNA and even the more closely related relaxed and denatured forms of plasmid as earlier, separate fractions. Finally, plasmid that has been precipitated by CTAB can be purified by selectively dissolving under conditions of controlled salt concentration. The selectivity mechanism is most likely based upon conformational differences among the several forms of DNA. As such, CTAB precipitation provides an ideal nonchromatographic capture step for the manufacture of plasmid DNA.
Lander
Russel J
RJ
Merck & Co., Inc., Merck Research Laboratories, West Point, Pennsylvania 19486, USA. russ_lander@merck.com
Winters
Michael A
MA
Meacle
Francis J
FJ
Buckland
Barry C
BC
Lee
Ann L
AL
D000077286:Cetrimonium; D002593:Cetrimonium Compounds; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000032:analysis / Q000302:isolation & purification; D003902:Detergents; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics*; D005590:Fractional Precipitation; D008070:Lipopolysaccharides / Q000302:isolation & purification; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics / Q000302:isolation & purification*; D012313:RNA / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D013237:Stereoisomerism
D002593:Cetrimonium Compounds; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D003902:Detergents; D008070:Lipopolysaccharides; C082161:lipopolysaccharide A; D012313:RNA; D004247:DNA; D000077286:Cetrimonium