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S1-nuclease enhancement of the ethidium bromide binding assay of drug-induced DNA interstrand crosslinking in human brain tumor cells.

Publication ,  Journal Article
Sriram, R; Ali-Osman, F
Published in: Anal Biochem
June 1990

A modification, using S1-nuclease, of a simple and sensitive fluorometric assay with ethidium bromide was developed for the measurement of cellular DNA interstrand crosslinking induced by bifunctional alkylators. Cells are lysed and treated with proteinase K and sodium dodecyl sulfate followed by extensive dialysis to yield intact high-molecular-weight DNA, free of contaminating proteins, on which the crosslink assay is then performed. The assay depends on the differential binding of ethidium bromide to single- and double-stranded DNA. Because of the higher ethidium bromide binding capacity of double-stranded DNA, the fluorescence retained after a heating/cooling cycle is directly proportional to the drug-induced cellular DNA interstrand crosslinking. We demonstrate that the sensitivity of this assay can be increased up to fourfold by including an S1-nuclease digestion step. This modified technique is simple and suited to the quantitation of low levels of DNA-interstrand crosslinking in cells.

Duke Scholars

Published In

Anal Biochem

DOI

ISSN

0003-2697

Publication Date

June 1990

Volume

187

Issue

2

Start / End Page

345 / 348

Location

United States

Related Subject Headings

  • Tumor Cells, Cultured
  • Single-Strand Specific DNA and RNA Endonucleases
  • Sensitivity and Specificity
  • Nucleic Acid Renaturation
  • Nucleic Acid Denaturation
  • Humans
  • Fluorescence
  • Ethidium
  • DNA, Single-Stranded
  • Cross-Linking Reagents
 

Citation

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Sriram, R., & Ali-Osman, F. (1990). S1-nuclease enhancement of the ethidium bromide binding assay of drug-induced DNA interstrand crosslinking in human brain tumor cells. Anal Biochem, 187(2), 345–348. https://doi.org/10.1016/0003-2697(90)90467-n
Sriram, R., and F. Ali-Osman. “S1-nuclease enhancement of the ethidium bromide binding assay of drug-induced DNA interstrand crosslinking in human brain tumor cells.Anal Biochem 187, no. 2 (June 1990): 345–48. https://doi.org/10.1016/0003-2697(90)90467-n.
Sriram, R., and F. Ali-Osman. “S1-nuclease enhancement of the ethidium bromide binding assay of drug-induced DNA interstrand crosslinking in human brain tumor cells.Anal Biochem, vol. 187, no. 2, June 1990, pp. 345–48. Pubmed, doi:10.1016/0003-2697(90)90467-n.
Journal cover image

Published In

Anal Biochem

DOI

ISSN

0003-2697

Publication Date

June 1990

Volume

187

Issue

2

Start / End Page

345 / 348

Location

United States

Related Subject Headings

  • Tumor Cells, Cultured
  • Single-Strand Specific DNA and RNA Endonucleases
  • Sensitivity and Specificity
  • Nucleic Acid Renaturation
  • Nucleic Acid Denaturation
  • Humans
  • Fluorescence
  • Ethidium
  • DNA, Single-Stranded
  • Cross-Linking Reagents