HPLC preparation of highly purified single-stranded M13 DNA.
Closed-circular, single-stranded viral DNAs are widely employed in DNA cloning and sequencing experiments. Because of their well-defined structure and sequence, closed-circular, single-stranded DNAs have also been used for ligand binding experiments and light scattering measurements. However, there is a high molecular weight impurity observed in light scattering experiments, which sometimes contaminates single-stranded DNA purified from phage that has been precipitated in polyethylene glycol, average molecular weight 8000, and purified by standard phenol-chloroform extraction. Three methods have been examined that remove this impurity from closed-circular, single-stranded M13mp19 DNA (SS M13 DNA). One employs a commercial peparation. This procedure yields pure but degraded SS M13 DNA, as shown by light scattering measurements and HPLC. Another employs a Whatman DE52 (diethylamino cellulose) column. This procedure yields intact DNA, but in poor yield (less than 20% of that obtained by phenol-chloroform extraction). The last was the most successful. This employs HPLC with a Waters AP-1 column with DEAE 8HR bedding. This procedure, which provides DNA in high yield (80%-90% column recovery) with an intact structure, is an efficient method for the isolation of high-purity, closed-circular, single-stranded viral DNA suitable for physical investigations and ligand binding measurements.
Milliman
A
A
Department of Chemistry, University of Illinois, 60607-7061, USA.
Huang
C R
CR
Kim
Y
Y
LeBreton
P R
PR
D016477:Artifacts; D017104:Bacteriophage M13 / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004277:DNA, Single-Stranded / Q000302:isolation & purification*; D004279:DNA, Viral / Q000302:isolation & purification*; D004983:Ethanolamines; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008027:Light; D009691:Nucleic Acid Denaturation; D011092:Polyethylene Glycols; D012542:Scattering, Radiation; D013056:Spectrophotometry, Ultraviolet
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004983:Ethanolamines; D005452:Fluoresceins; D005456:Fluorescent Dyes; C039083:5-((4,6-dichloro-1,3,5-triazin-2-yl)amino)fluorescein; D011092:Polyethylene Glycols; C007369:2-diethylaminoethanol