Comparison of an intercalating dye and an intercalant-enzyme conjugate for DNA detection in a microtiter-based assay

Beata Kolakowski, Fernando Battaglini, Yoon Suk Lee, Giannoula Klironomos, Susan R. Mikkelsen

Research output: Journal article publicationJournal articleAcademic researchpeer-review

7 Citations (Scopus)

Abstract

Two methods have been developed for the detection of DNA immobilized on the surface of microtiter wells. An intercalating dye, 3,6-diaminoacridine, is used in stain and rinse solutions, so that measured absorbance values (450 nm) reflect the sum of DNA-bound and free dye. With diaminoacridine, signal increases of 0.056 ± 0.010 were achieved on immobilizing double-stranded calf thymus DNA. An intercalant-enzyme conjugate, consisting of an average of four daunomycin moieties covalently bound to each glucose oxidase, was shown to provide a 10-fold signal enhancement (optimum 0.25 μM, with rinsing and peroxidase-o-dianisidine detection) compared to diaminoacridine, due to catalytic amplification; signals of 0.50 ± 0.05 were obtained. This conjugate possesses 56% of the activity of native glucose oxidase and was prepared using water-soluble carbodiimide and N-hydroxysuccinimide reagents. Single-stranded DNA was immobilized onto avidin-coated polystyrene plates and commercially available (Covalink) plates possessing secondary amine groups. Following hybridization with complementary DNA, detection was performed with the daunomycin-glucose oxidase conjugate. Both immobilization methods showed optimum DNA concentrations of 0.10 μg/mL, and maximum signal intensities were obtained when > 0.5 μg/mL complementary DNA was present in the hybridization solution. Some nonspecific binding of the intercalant-enzyme conjugate was suggested by results obtained with avidin-coated polystyrene plates, but not with Covalink plates.
Original languageEnglish
Pages (from-to)1197-1200
Number of pages4
JournalAnalytical Chemistry
Volume68
Issue number7
DOIs
Publication statusPublished - 1 Apr 1996
Externally publishedYes

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this