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.
SpecificityMedium* High
Sensitivity-low # of copiesVariable* 1-10 copies
ReproducibilityMedium* High
Multiplexing  yes
Predesigned assays  yes
Typically requires user design, experimental optimizationyes  
Gene expressionLow level of quantitation High level of quantitation
ApplicationsGene expression
DNA quantitation (pathogen detection)
CHiP		 Gene expression
DNA quantitation
CHiP
SNP genotyping
Copy number variation
Pathway analysis
microRNA & small RNAs
Mutation detection
Protein analysis
Multiplexing
 SYBR Green primers

SYBR Green master mixes

 TaqMan Assays

TaqMan master mixes

*Depends on template quality and primer/design optimization.

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Figure 1: Comparison of TaqMan-based and SYBR Green–based detection workflows.

TaqMan chemistry

Background

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.

Step-by-step process

 

  1. An oligonucleotide probe is constructed containing a fluorescent reporter dye on the 5' end and a quencher dye on the 3' end. While the probe is intact, the proximity of the quencher dye greatly reduces the fluorescence emitted by the reporter dye by fluorescence resonance energy transfer (FRET).
  2. If the target sequence is present, the probe anneals downstream from one of the primer sites and is cleaved by the 5' nuclease activity of Taq DNA polymerase as this primer is extended.
  3. This cleavage of the probe:
    -- Separates the reporter dye from the quencher dye, increasing the reporter dye signal.
    -- Removes the probe from the target strand, allowing primer extension to continue to the end of the template strand. Thus, inclusion of the probe does not inhibit the overall PCR process.
  4. Additional reporter dye molecules are cleaved from their respective probes with each cycle, resulting in an increase in fluorescence intensity proportional to the amount of amplicon produced.
 


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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

Background

Small molecules that bind to double-stranded DNA can be divided into two classes:

  • Intercalators
  • 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:

Step-by-step process

  1. When SYBR dye is added to a sample, it immediately binds to all double-stranded DNA present in the sample.
  2. During PCR, DNA polymerase amplifies the target sequence which creates the PCR products.
  3. SYBR dye then binds to each new copy of double-stranded DNA.
  4. 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.

Additional consideration

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.