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

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.

Process control in cell culture technology using dielectric spectroscopy

“…The obvious drawback of these methods is the delay between sampling and the availability of results as well as the risk of contamination [Vojinovic et al, 2006]. Indirect methods, such as DNA concentration determination or fluorescent based measurements have been described [Myers, 1998, Rengarajan et al, 2002, Zipper et al, 2004. Furthermore indirect off-line methods, where the determination of the oxygen partial pressure, lactate or glucose concentration in the medium is used to monitor the process and to indirectly calculate the cell concentration have been described [Vojinovic et al, 2006].…”

Metal‐Induced Specific and Nonspecific Oligonucleotide Folding Studied by FRET and Related Biophysical and Bioanalytical Implications

“…At even higher Mg II concentrations, the overall fluorescence dropped due to quenching of SG fluorescence by Mg II . [46] The ionic strength problem stems from the lack of a rigid structural scaffold in this probe. As mentioned previously, this is less of a problem for naturally occurring functional nucleic acids.…”

An evolutionary Monte Carlo algorithm for predicting DNA hybridization

“…The SYBR Green I fluorescence intensity is proportional to the number of DNA bonds (Zipper et al, 2004). Therefore, its graph is the experimental confirmation of the simulation of the bond number.…”