Quantitation of supercoiled circular content in plasmid DNA solutions using a fluorescence-based method.

Abstract:

: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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Levy MS,Lotfian P,O'Kennedy R,Lo-Yim MY,Shamlou PA

doi

10.1093/nar/28.12.e57

keywords:

subject

Has Abstract

pub_date

2000-06-15 00:00:00

pages

E57

issue

12

eissn

0305-1048

issn

1362-4962

journal_volume

28

pub_type

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