Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications.
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.
Zipper
Hubert
H
Laboratory of Biochemistry, Institute for Interfacial Engineering, University of Stuttgart, 70569 Stuttgart, Germany.
Brunner
Herwig
H
Bernhagen
Jürgen
J
Vitzthum
Frank
F
D052160:Benzothiazoles; D002412:Cations / Q000494:pharmacology; D004247:DNA / Q000032:analysis / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D003959:Diamines; D004587:Electrophoresis, Agar Gel; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D007364:Intercalating Agents / Q000737:chemistry* / Q000378:metabolism; D046911:Macromolecular Substances; D009930:Organic Chemicals / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011804:Quinolines; D013053:Spectrophotometry
D052160:Benzothiazoles; D002412:Cations; D004277:DNA, Single-Stranded; D003959:Diamines; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D046911:Macromolecular Substances; D009930:Organic Chemicals; C099476:PicoGreen; D011804:Quinolines; C098022:SYBR Green I; D004247:DNA