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...

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Bibliographic Details
Published inBioTechniques Vol. 18; no. 2; p. 308
Main Authors Milliman, A, Huang, C R, Kim, Y, LeBreton, P R
Format Journal Article
LanguageEnglish
Published England 01.02.1995
Subjects
Bacteriophage M13 - genetics
Chromatography, High Pressure Liquid - methods
DNA, Single-Stranded - isolation & purification
DNA, Viral - isolation & purification
Ethanolamines
Fluoresceins
Fluorescent Dyes
Light
Nucleic Acid Denaturation
Polyethylene Glycols
Scattering, Radiation
Spectrophotometry, Ultraviolet
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Summary: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.
ISSN:0736-6205