We have developed two types of chemistries to detect PCR products using real-time PCR instruments:
Which real-time chemistry is right for you?
|SYBR Green–based detection||TaqMan-based detection|
Uses Applied Biosystems™ SYBR™ Green dye (a dsDNA binding dye) to detect PCR product as it accumulates during PCR.
Uses a fluorogenic probe specific to target gene to detect target as it accumulates during PCR.
|Sensitivity-low # of 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 Applied Biosystems™ 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 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 use in diagnostic procedures.