Choosing the right chemistry and master mix is an important step when performing qPCR experiment. A master mix is a mixture of polymerase, salts, magnesium, dNTPs, and optimized reaction buffer. The template and primers are the only additions necessary to perform qPCR. Different formulations are good for different applications such as genotyping, gene expression, microRNA quantification, and virus detection. The most common detection methods when performing Real-Time PCR are SYBR and TaqMan.
1. SYBR® Green: A non-specific molecules that bind to double-stranded DNA resulting in a significant increase in its fluorescent intensity.
Based on two hybridization events, the forward and reverse primers.
Advantages of SYBR dye:
- It can be used to monitor the amplification of any double-stranded DNA
- No probe is required, which can reduce assay setup and running costs, if your PCR primers are well designed, and your reaction is well characterized.
Disadvantage of SYBR dye:
- May generate false positive signals and therefore it is extremely important to have well-designed primers that do not amplify non-target sequences, and that melt curve analysis be performed.
2. TaqMan: Fluorogenic-labeled probes that use the 5′ nuclease activity of Taq DNA polymerase. The master mix which bases its detection on 5’ nuclease activity requires three hybridization events: two PCR primers and one probe, with hydrolysis of the probe during extension of the primers.
Advantages of TaqMan chemistry:
- Specific hybridization between probe and target is required to generate fluorescent signal
- Probes can be labeled with different, distinguishable reporter dyes, which allows amplification and detection of two distinct sequences in one reaction tube
- Post-PCR processing is eliminated, which reduces assay labor and material costs
Disadvantages of TaqMan chemistry:
- The synthesis of different probes is required for different sequences.
If you need high specificity and a predesigned assay for gene expression experiment you better choose Taqman® chemistry. In order to choose the right master mix, select the options that make up your particular experiment, in the online selection tool and the recommended Master Mix will appear.