A dot-blot method for quantification of apurinic/apyrimidinic sites in DNA using an avidin plate and liposomes encapsulating a fluorescence dye

Hiroyuki Yanagisawa, Ayumi Hirano, Masao Sugawara

Research output: Contribution to journalArticlepeer-review

9 Citations (Scopus)

Abstract

A dot-blot method for quantification of apurinic/apyrimidinic (AP) sites in genomic DNA (calf thymus DNA) is described using an avidin-modified glass slip and biotinylated liposomes containing sulforhodamine B as a fluorescence marker. Aldehyde reactive probe (ARP)-tagged DNA was found to be strongly adsorbed on an avidin slip, even if treated with ethanolamine and biotin, with an efficiency of 51% due to the positive surface charge of avidin, and unbound ARP was easily washed out of the surface with Milli-Q water. In the assay protocol, calf thymus DNA containing AP sites is reacted with ARP in solution and immobilized on an ethanolamine- and biotin-treated avidin slip (EAB-avidin slip), followed by incubation with streptavidin. The AP sites were finally quantified with biotinylated liposomes containing 1.5 mM sulforhodamine B as a fluorescence marker. The mean fluorescence intensity over the surface of the slip was an analytically relevant measure of the amount of AP sites in calf thymus DNA. By using the dot-blot assay, 1-5 AP sites per 104 nucleotides in 5 and 100 ng of DNA were quantified. The current dot-blot method has potential for quantification of AP sites in genomic DNA at a level of several nanograms.

Original languageEnglish
Pages (from-to)358-367
Number of pages10
JournalAnalytical Biochemistry
Volume332
Issue number2
DOIs
Publication statusPublished - 2004 Sep 15
Externally publishedYes

Keywords

  • AP sites
  • ARP
  • Dot-blot method
  • Fluorometric imaging method
  • Liposome
  • Sulforhodamine B

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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