ABSTRACT
Asymmetric cyanine dyes bind to the minor groove of double stranded DNA (dsDNA) owing to their crescent configuration; therefore, these dyes are widely used as a dsDNA probes. BOXTO-MEE is derived from BOXTO by adding the polar methoxyethoxyethyl tail in order to increase solubility, dissociation rate kinetics, and stability. As a result, BOXTO-MEE showed significant reduction in nonspecific amplification (primer dimers) without significant effect on target sequence amplification, PCR efficiency, and standard curve correlation coefficient. BETIBO is another example of an asymmetric cyanine dye that can binds to dsDNA but is less efficient than BOXTO-MEE for use in real-time PCR. Statistical analysis of reproducibility results shows that BETIBO is not strong enough to be used for quantifying low nucleic acid quantities. Statistical analysis for BOXTO-MEE results shows that there is no significant difference between the efficiency and correlation coefficient achieved by BOXTO-MEE and SYBR Green I, but a significant difference in the dynamic range is observed because BOXTO-MEE has a wider dynamic range. BOXTO-MEE stock solution was stable at -20 degrees C for more than 1 year and 40 microM solution was stable for 45 days (at least) at 4 degrees C.
Subject(s)
Carbocyanines/chemistry , DNA/chemistry , Fluorescent Dyes/chemistry , Organic Chemicals/chemistry , Benzothiazoles , Binding Sites , Carbocyanines/metabolism , DNA/metabolism , DNA Primers , DNA Probes , Diamines , Fluorescent Dyes/metabolism , Kinetics , Nucleic Acid Amplification Techniques/methods , Organic Chemicals/metabolism , Polymerase Chain Reaction/methods , Quinolines , Reproducibility of Results , Solubility , TemperatureABSTRACT
The unsymmetrical cyanine dyes BOXTO (4-[6-(benzoxazole-2-yl-(3-methyl-)-2,3-dihydro-(benzo-1,3-thiazole)-2- methylidene)]-1-methyl-quinolinium chloride)and its positive divalent derivative BOXTO-PRO (4-[(3-methyl-6-(benzoxazole-2-yl)-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)]-1-(3-trimethylammonium-propyl)-quinolinium dibromide) were studied as real-time PCR reporting fluorescent dyes and compared to SYBR GREEN I (SG)(2-[N- (3-dimethylaminopropyl)-N-propylamino] -4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl- quinolinium).Unmodified BOXTO showed no inhibitory effects on real-time PCR,while BOXTO-PRO showed complete inhibition. Sufficient fluorescent signal was acquired when 0.5-1.0 meu M BOXTO was used with RotorGene and iCycler platforms.Statistical analysis showed that there is no significant difference between the efficiency and dynamic range of BOXTO and SG.BOXTO stock solution (1.5 mM) was stable at -20 degree C for more than one year and 40 meu M BOXTO solution was more stable than 5x SG when both were stored at 4 degree C for 45 days.
Subject(s)
Fluorescent Dyes/chemistry , Polymerase Chain Reaction/methods , Quinolinium Compounds/chemistry , Thiazoles/chemistry , DNA Primers/genetics , Molecular Structure , TemperatureABSTRACT
FRET primer real-time PCR chemistry depends on internally labeled primers with FRET dyes linked to their 3' end. The best distance between the FRET dyes for obtaining the largest signal and the lowest background is six nucleotides. In this study the forward primer was labeled with FAM and the reverse primer with Texas red; the labeled primers meet in cycle two of PCR. At the end of the elongation step FAM is excited to emit fluorescence which will excite Texas red to emit new fluorescence that correlates directly with the quantity of PCR product accumulated. FRET primer techniques amplify short amplicons with unique thermal cycling steps, 0 s at 85 degrees C for denaturation, 7 s for annealing, and 2 s for elongation. The FRET primer technique was very efficient (92.6, 97.2, and 100%), correlation coefficients were high (1.0, 0.999, and 0.999), and total run time was very short (20, 45, and 40 min per 40 cycles with LightCycler, iCycler, and RotorGene 3000, respectively). When FRET-labeled primers were compared with similar but unlabeled primers it was observed that the FRET primer technique had a lower Ct value and was more efficient than use of unlabeled primers detected by use of SYBR Green I.
