Which Real-Time Chemistry Is Right for You?
|SYBR® Green-Based Detection||TaqMan®-Based Detections|
Uses SYBR® Green dye (a dsDNA binding dye) to detect PCR product as it accumulates during PCR.
Uses fluorogenic probe specific to target gene to detect target as it accumulates during PCR.
|Sensitivity-Low Copies||Variable*||1-10 copies|
|Typically Requires User Design, Experimental Optimization?|
|Gene Expression||Low level of quantitation||High level of quantitation|
DNA quantitation (pathogen detection)
Copy number variation
microRNA & small RNAs
|SYBR® Green Primers
SYBR® Green Master Mixes
TaqMan® Master Mixes
*Depends on template quality and primer/design optimization.
Initially, intercalator dyes were used to measure real-time PCR products. The primary disadvantage to these dyes is that they detect accumulation of both specific and nonspecific PCR products.
Development of TaqMan® Chemistry
Real-time systems for PCR were improved by the introduction of fluorogenic-labeled probes that use the 5' nuclease activity of Taq DNA polymerase. The availability of these fluorogenic probes enabled the development of a real-time method for detecting only specific amplification products.
Figure 2: Overview of TaqMan® Probe-Based Assay Chemistry.
Two Types of TaqMan® Probes
We offer two types of TaqMan® probes:
- TaqMan® probes (with TAMRA™ dye as the quencher dye)
- TaqMan® MGB probes
TaqMan® MGB Probes Recommended for Allelic Discrimination Assays
We recommend the general use of TaqMan® MGB probes for allelic discrimination assays, especially when conventional TaqMan® probes exceed 30 nucleotides. TaqMan® MGB probes contain:
- A nonfluorescent quencher at the 3' end—the real-time PCR instruments can measure the reporter dye contributions more precisely because the quencher does not fluoresce
- A minor groove binder at the 3' end—the minor groove binder increases the melting temperature (Tm) of probes, allowing the use of shorter probes
Consequently, the TaqMan® MGB probes exhibit greater differences in Tm values between matched and mismatched probes, which provide more accurate allelic discrimination.
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 primary disadvantage is that the synthesis of different probes is required for different sequences.
SYBR® Chemistry or other double-stranded DNA binding dyes
Small molecules that bind to double-stranded DNA can be divided into two classes:
- Minor-groove binders
Regardless of the binding method, there are two requirements for a DNA binding dye for real-time detection of PCR:
- Increased fluorescence when bound to double-stranded DNA
- No inhibition of PCR
We have developed conditions that permit the use of the SYBR® Green I dye in PCR with little PCR inhibition and increased sensitivity of detection compared to ethidium bromide. Additionally, we have newer SYBR® Green dyes that fluoresce more brightly and inhibit PCR less than the original SYBR® Green I.
How SYBR® Dye Chemistry Works
SYBR® dye detects polymerase chain reaction (PCR) products by binding to double-stranded DNA formed during PCR. Here’s how it works:
- When SYBR® dye is added to a sample, it immediately binds to all double-stranded DNA present in the sample.
- During the PCR, DNA polymerase amplifies the target sequence, which creates the PCR products.
- SYBR® dye then binds to each new copy of double-stranded DNA.
- As the PCR progresses, more PCR product is created. SYBR® dye binds to all double-stranded DNA, so the result is an increase in fluorescence intensity proportioned to the amount of PCR product produced.
Advantages of SYBR® Dye
- It can be used to monitor the amplification of any double-stranded DNA sequence.
- No probe is required, which can reduce assay setup and running costs, assuming that your PCR primers are well designed and your reaction is well characterized.
Disadvantage of SYBR® Dye
The primary disadvantage is that it may generate false positive signals; i.e., because the SYBR® dye binds to any double-stranded DNA, it can also bind to nonspecific double-stranded DNA sequences. Therefore, it is extremely important to have well-designed primers that do not amplify non-target sequences, and that melt curve analysis be performed.
Another aspect of using DNA binding dyes is that multiple dye molecules may bind to a single amplified DNA molecule. A consequence of multiple dye binding is that the amount of signal is dependent on the mass of double-stranded DNA produced in the reaction. Thus, if the amplification efficiencies are the same, amplification of a longer product will generate more signal than a shorter one. This is in contrast to the use of a fluorogenic probe, in which a single fluorophore is released from quenching for each amplified molecule synthesized, regardless of its length.
For Research Use Only. Not for human or animal therapeutic or diagnostic use.