Quantitation of supercoiled circular content in plasmid DNA solutions using a fluorescence-based method.
A method for quantifying the proportion of supercoiled circular (SC) forms in DNA solutions is described. The method (SCFluo) takes advantage of the reversible denaturation property of SC forms and the high specificity of the PicoGreen fluorochrome for double-stranded (ds)DNA. Fluorescence values of forms capable of reversible denaturation after a 5 min heating, 2 min cooling step are normalised to fluorescence values of total dsDNA present in the preparation. For samples with a SC content >20-30%, good regression fits were obtained when values derived from densitometric scanning of an agarose gel and those derived from the SCFluo method were compared. The method represents an attractive alternative to currently established methods because it is simple, rapid and quantitative. During large-scale processing and long-term storage, enzymatic, chemical and shear degradation may substantially decrease the SC content of plasmid DNA preparations. Regulations for pharmaceutical grade products for use in gene therapy and DNA vaccination may require >90% of the plasmid to be in the SC form. In the present study the SC content of 6.9, 13 and 20 kb plasmid preparations that had been subjected to chemical and shear degradation was successfully quantified using the new method.
Levy
M S
MS
The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK. myriom.levy@ucl.ac.uk
Lotfian
P
P
O'Kennedy
R
R
Lo-Yim
M Y
MY
Shamlou
P A
PA
D004278:DNA, Superhelical / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli; D005069:Evaluation Studies as Topic; D005456:Fluorescent Dyes; D009691:Nucleic Acid Denaturation; D009930:Organic Chemicals; D010957:Plasmids / Q000235:genetics; D012680:Sensitivity and Specificity; D012996:Solutions
D004278:DNA, Superhelical; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C099476:PicoGreen; D012996:Solutions