Determination of Micro-Quantities of DNA Using DNAse and Fluorescence of Hoechst 33258 and Light-Scattering

Journal of Fluorescence
N L VekshinA M Lvov

Abstract

The DNA hydrolysis by deoxyribonuclease (DNAse I) in aqueous solution was studied, using fluorescence spectroscopy and high-sensitive light-scattering detection. Specific hydrolysis of high-polymer DNA or fragmented DNA by the enzyme led to a strong decrease in the fluorescence of the Hoechst dye. The hydrolysis of mitochondrial DNA was accompanied by a decrease in the fluorescence of the dye only in 1.6 times. Hydrolysis within minutes and even hours led to appearance of large polynucleotide fragments, but not to short oligonucleotides, that was confirmed using polarized fluorescence and highly sensitive measurement of light-scattering. At the moment of the time of formation of a complex between DNA and DNAse I, a strong light-scattering occurred, which then dropped sharply during hydrolysis of high-molecular DNA, and slowly decreased during hydrolysis of fragmented DNA. The proposed methods can be applied for selective detection of trace amounts of various types of DNA, as well as for studying their physic-chemical properties.

References

Nov 18, 1985·FEBS Letters·T K Palva, E T Palva
Mar 1, 1980·Analytical Biochemistry·C Labarca, K Paigen
Mar 21, 1998·Proceedings of the National Academy of Sciences of the United States of America·U KettlingM Eigen
Apr 30, 2009·Annual Review of Biomedical Engineering·Gang BaoAndrew Tsourkas

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Citations

May 5, 2020·ACS Sensors·Giraso Keza Monia KabandanaChengpeng Chen

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Methods Mentioned

BETA
light-scattering
Fluorescence
light scattering

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