SyGreen vs SYBR Green: what are they and how to do they work?
What is SyGreen?
SyGreen is an asymmetrical cyanine DNA binding dye used in PCR Biosystems qPCR mixes and is registered in the UK under the trademark qPCRBIO SyGreen®. To find out more about our related products, please visit our dedicated product pages:
- qPCRBIO SyGreen Mix, for standard dye-based qPCR detection of DNA targets,
- qPCRBIO SyGreen Blue Mix, for standard dye-based qPCR detection of DNA targets,
- qPCRBIO SyGreen 1-Step Kits, for one step RT-qPCR from RNA targets.
What is SYBR Green?
SYBR Green is also an asymmetrical cyanine DNA binding dye, used under licence by multiple qPCR reagent manufacturers in dye-based qPCR mixes. The dye was developed by Molecular Probes Inc. and is now owned by Thermo Fisher Scientific. The IUPAC name for SYBR Green I is N’,N’-dimethyl-N-[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]-N-propylpropane-1,3-diamine with the CAS Number: 178918-96-2.
How do they work?
These intercalating dyes possess aromatic and planar groups that allow the intercalation of the molecule in the minor groove of double-stranded DNA spanning approximately 3.5-4 nucleotides. The positive groups present stabilise the dyes allowing for the coordination with the negatively charged DNA backbone.
Due to this mechanism of binding to DNA, they are not able to bind to single-strand nucleic acids, unless secondary structures are formed. On the other hand, fluorescence increases up to 1,000-fold upon intercalation with dsDNA.
When a dye intercalates between DNA base pairs, it emits fluorescence proportional to the amount of bound dye (and hence to the amount of DNA in a sample). This allows visualisation of nucleic acids on gels or fluorimeters in real time qPCR instruments. In the latter case, the number of intercalated dye molecules in a sample increases with the progress of a qPCR, leading to a corresponding increase in fluorescence with each cycle. At the onset of the reaction, the free dye emits a basal fluorescence level. As the reaction proceeds the fluorescence intensity increases exponentially, before it plateaus in later cycles, leading to the classic sigmoidal amplification plot.
Benefits and Limitations of Dye-Based qPCR
Benefits:
Versatile Target Detection: By combining a SyGreen or SYBR Green-type dye with primers, researchers can detect any target molecule of interest. This versatility allows for simple and cost-effective design of high-throughput singleplex qPCRs.
Reversible Fluorescent Signal Accumulation: The use of a DNA intercalating dye facilitates reversible accumulation of fluorescent signal. Because strong fluorescence arises only from intercalated dye, denaturation of DNA leads to a reduction in fluorescent signal. This characteristic enables specificity testing through melt curve analysis, which in turn allows for rapid evaluation of qPCR specificity (see more below).
Limitations:
Non-Specific Signal Generation: The intercalating dye can generate non-specific fluorescent signals when it binds to any dsDNA molecule present in the reaction tube. This can occur due to mis-priming, the presence of similar sequences in the sample, the formation of primer dimers, or potential primer hairpin structures. To validate results, additional analyses like melt curve analysis, gel electrophoresis, or Sanger sequencing should be performed.
Single Target Assay: Dye-based qPCR allows only one target to be assayed per reaction. For quantifying multiple targets in a sample, a corresponding number of reactions must be set up.
Melt Curve Analysis
Melt curve analysis serves to identify the melting point of a qPCR product, providing valuable insights into product specificity. This analysis involves an additional thermocycling step at the end of the qPCR cycling program. During this step, the reaction samples are gradually denatured by incrementally increasing the incubation temperature while collecting fluorescence data at each temperature interval (e.g., every 0.1-0.5 °C). Plotting the fluorescence intensity data against temperature generates the melt curve. In a reaction containing a specific product, fluorescence remains steady until the temperature approaches the target’s melting point, at which point it rapidly drops to baseline. The temperature at which fluorescence decreases to 50% of the maximum is considered the melting point or Tm of that molecule. Evaluating reaction specificity can be simplified by plotting the first-order derivative of fluorescence intensity (-dRFU) against temperature change (dT), known as dRFU/dT. For a reaction with a single specific product, this results in a parabolic peak centered around the product’s Tm, known as a melt peak.
For more in depth information on dye-based qPCR or qPCR in general please refer to our technical guide.
Disclaimer:
SYBR Green is a registered trademark of Molecular Probes Inc. and is owned by Thermo Fischer Scientific. PCR Biosystems Ltd. is in no way affiliated with any of these entities.
