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Real-time PCR of nonspecific DNA-binding dyes

Nonspecific DNA binding dyes only allow the determination of the presence or absence of an amplicon. No information on the precise nature of the product can be determined. The dye STBR Green I emits fluorescence when bound to double-stranded DNA. The amount of intercalated dye will increase as the number of DNA molecules increases. The emitted fluorescence is directly proportional to the copy number. Nonspecific DNA binding dyes allow measuring gene expression levels and the detection of pathogens. Unfortunately, intercalation dyes detect accumulation of both specific and nonspecific PCR products. 

The solution structure of the fluorescent bis-intercalator dye which contains a benzothiazole moiety similar as present in SYBR Green I illustrates the nature of the binding to the DNA helix.

                       TOTO               Models of the TOTO-dsDNA complex (PDB ID 108D)
In the structure of the DNA complex with the fluorescent bis-intercalator,  bis-intercalating dye 1,1'-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene) bis[4-(3-methyl -2,3- dihydro benzo-1,3-thiazolyl-2-methylidene) quinolinium] tetraiodide (TOTO) in the benzothiazole ring system is twisted relative to the quinoline in the uncomplexed TOTO molecule. TOTO is selective for the CTAG-CTAG  double-helix which is explained by its ability to adapt to the base pair propeller twist of dsDNA which optimizes stacking and the hydrophobic interaction between the thymidine methyl group and the benzothiazole ring. A 3000-fold fluorescence enhancement occurs upon binding of TOTO to dsDNA. In the complex, the nucleobase forms a clamp around the benzothiazole ring and the quinolinium ring of TOTO such that it can no longer rotate around the cyanine methane bonds. The clamping prevents rotation of the dye, and the chromophore loses excitation energy by fluorescence instead.


PDP ID 108d : Spielmann HP, Wemmer DE, Jacobsen JP.; Solution structure of a DNA complex with the fluorescent bis-intercalator TOTO determined by NMR spectroscopy. Biochemistry. 1995 Jul 11;34(27):8542-53.]