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. 2004 Jul 12;32(12):e103.
doi: 10.1093/nar/gnh101.

Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications

Affiliations

Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications

Hubert Zipper et al. Nucleic Acids Res. .

Abstract

The detection of double-stranded (ds) DNA by SYBR Green I (SG) is important in many molecular biology methods including gel electrophoresis, dsDNA quantification in solution and real-time PCR. Biophysical studies at defined dye/base pair ratios (dbprs) were used to determine the structure-property relationships that affect methods applying SG. These studies revealed the occurrence of intercalation, followed by surface binding at dbprs above approximately 0.15. Only the latter led to a significant increase in fluorescence. Studies with poly(dA)* poly(dT) and poly(dG)* poly(dC) homopolymers showed sequence-specific binding of SG. Also, salts had a marked impact on SG fluorescence. We also noted binding of SG to single-stranded (ss) DNA, although SG/ssDNA fluorescence was at least approximately 11-fold lower than with dsDNA. To perform these studies, we determined the structure of SG by mass spectrometry and NMR analysis to be [2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]. For comparison, the structure of PicoGreen (PG) was also determined and is [2-[N-bis-(3-dimethylaminopropyl)-amino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]+. These structure-property relationships help in the design of methods that use SG, in particular dsDNA quantification in solution and real-time PCR.

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Figures

Figure 1
Figure 1
Dependence of the relative fluorescence intensity on the dbpr of the SG/dsDNA complex. Samples containing 0.15 or 0.015 μM base pairs of λDNA were incubated with increasing dye concentrations ranging from 0 to 2.1 μM or from 0.15 to 1.52 μM, respectively.
Figure 2
Figure 2
Dependences of differential absorption values (A) and fluorescence intensities (B) of SG bound to λDNA (triangle), dA · dT (circle) and dG · dC (square). TES buffer pH 7.5 containing 15 μM base pairs of a nucleic acid type was used for differential absorption measurements. Fluorescence intensity studies were performed in TE buffer pH 7.5 containing 0.15 μM base pairs of a nucleic acids polymer.
Figure 3
Figure 3
The influence of NaCl on the dbpr dependence of SG/dsDNA differential absorption values (A) and fluorescence (B). (A) Differential absorption values at 494 nm of SG/λDNA complexes at increasing dbprs in TE buffer pH 7.5 that did not contain NaCl (circle) and in TES buffer pH 7.5 that contained 100 mM NaCl (solid square). (B) Samples containing 0.15 μM bp of λDNA were titrated with increasing amounts of SG ranging from 0 to 1.5 μM in the presence of 0 mM (solid squares), 25 mM (solid circles), 50 mM (solid triangles, up) and 300 mM (solid triangles, down) NaCl.
Figure 4
Figure 4
Comparison of fluorescence of SG/ssDNA complexes and SG/dsDNA complexes. (A) Dependence of fluorescence intensities of SG/ssDNA complexes on dbr in comparison with the corresponding SG/dsDNA complexes. Samples containing 0.15 μM (in bp) EcoRI-digested pACYC184 plasmid (solid squares) or 0.3 μM (in bases) heat-denatured, EcoRI-digested pACYC184 plasmid (solid triangles) were incubated with increasing concentrations of SG (0–1.5 or 0–3 μM, respectively). For reasons of comparison, the fluorescence intensities of the SG/dsDNA complex and of the SG/ssDNA complex were set to 100% at a dbpr of 10 and 0.25, respectively. (B) Fluorescence spectra of SG in complex with dsDNA (EcoRI-digested pACYC184) at dbprs of 1 (dashed line) and 10 (solid line) and with ssDNA (heat-denatured EcoRI-digested pACYC184) at dbr of 1 (dash dot dot line) and 10 (dash dot line).

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