Quantitation of supercoiled DNA cleavage in nonradioactive DNA: Application to lonizing radiation and synthetic endonuclease cleavage

Betsy M. Sutherland, Paula V. Bennett, Kim Conlon, Gary A. Epling, John C. Sutherland

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Quantitation of the conversion of nonradioactive supercoiled DNA to its open circular or linear forms on ethidium-stained electrophoretic gels has been difficult because of differential binding of ethidium to supercoiled DNA vs other forms under different conditions and the nonlinear response of photographic film. We have developed methods for adding a linear DNA as an internal fluorescence standard to "normalize" the quantity of DNA loaded into each lane of a gel. Inclusion of a linear normalizing DNA in samples before partitioning for individual supercoil cleavage reactions allows the quantitation of the resultant species, is technically easy, and does not require quantitative application of the sample to the gel. If the presence of a normalizing DNA during supercoil cleavage is undesirable, the addition of a normalizing plasmid to each sample after supercoil cleavage (but before electrophoresis) or the quantitative application of samples containing test DNA alone to the gel gives similar data, but with increased variability. We use the normalizing DNA method in cleavage by a physical agent (ionizing radiation) and in a more complex situation, by a protein-based, light-dependent synthetic endonuclease. We show how the fraction of intact supercoiled DNA can be calculated from measurement of the cleaved and normalizing species only. The method also can be used in reactions involving the depletion of one DNA species, whether supercoiled or not, such as protein-DNA interactions as detected by gel retardation assays.

Original languageEnglish (US)
Pages (from-to)80-86
Number of pages7
JournalAnalytical Biochemistry
Volume201
Issue number1
DOIs
StatePublished - Feb 14 1992
Externally publishedYes

Fingerprint

Superhelical DNA
DNA Cleavage
Endonucleases
Radiation
DNA
Gels
Ethidium
Photographic films
Ionizing radiation
Electrophoretic Mobility Shift Assay
Motion Pictures
Ionizing Radiation
Electrophoresis
Assays
Proteins
Plasmids
Fluorescence
Light

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Quantitation of supercoiled DNA cleavage in nonradioactive DNA : Application to lonizing radiation and synthetic endonuclease cleavage. / Sutherland, Betsy M.; Bennett, Paula V.; Conlon, Kim; Epling, Gary A.; Sutherland, John C.

In: Analytical Biochemistry, Vol. 201, No. 1, 14.02.1992, p. 80-86.

Research output: Contribution to journalArticle

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