Two major types of chemistries may be utilized to detect the products of your qPCR cycles, each with particular advantages. Understanding the differences between the two will ensure you choose the most effective detection chemistry for the particular assay you are designing.
Detection of qPCR products may be achieved through the use of DNA binding fluorescent dyes. The dyes bind to any double-stranded DNA, which causes an increase in the intensity of their fluorescent emissions, as compared to their fluorescence in the unbound form.
As more PCR cycles are completed, copies of the original double-stranded DNA accumulate, allowing the dye to bind to additional double-stranded DNA. The fluorescence intensity continues to increase in proportion to the increase in the amount of PCR amplicons present.
DNA-binding dyes may be used for either singleplex or multiplex assays.
Using a DNA-binding dye for your assay has several advantages. Overall assay design is much simpler, as there is no need to synthesize a probe for each target. This often means lower costs, increased convenience and decreased preparation time. They are easier to use, and appropriate for a wide range of applications.
The major disadvantage to using a dye for your assay is the non-specific binding of the dye to any double-stranded products. Since the dye will bind to any double-stranded DNA sequence, false positives are a concern, along with increased levels of background noise relative to your signal detection. In addition, some dyes cannot be used at high concentrations, as the dye will redistribute during the melt curve analysis.
Types of Dyes
SYBR Green is the most commonly used fluorescent qPCR dye. It is a cyanine dye, and its high binding affinity for double-stranded DNA is due to the two positive charges it contains under typical PCR reaction conditions. After it is added to the sample, it binds by intercalating between the bases of the double-stranded DNA.
When the dye is bound to double-stranded DNA, blue light is absorbed by the complex and green light is emitted.
- Low fluorescence
- PCR inhibition at high concentrations
- Favored binding to GC-rich regions
Next Generation Dyes [Chai Green, EvaGreen, LC Green]
Although SYBR Green continues to be the most commonly used fluorescent dye for qPCR, there are several other fluorescent dye options available. These alternate dyes can present many of the same advantages as SYBR Green, but with fewer of the corresponding limitations associated with SYBR Green.
Optimal dye concentrations for these dyes are higher than for SYBR Green, maximizing the PCR signal. Saturation of the double-stranded DNA binding sites eliminates the potential for dye “jumping” during your melt curve acquisition, which makes these dyes highly suitable for applications involving high resolution melt curve (HRM) analysis, such as mutation or polymorphism detection.
WHY CHOOSE AN ALTERNATIVE TO SYBR GREEN?
- Lower PCR inhibition
- Provide better results for melt curve analysis
- Can be used at saturating concentrations
- Greatly increased fluorescent intensity