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TaqMan® and SYBR® Green Chemistries

Applied Biosystems has developed two types of chemistries used to detect PCR products using real-time PCR instruments:

  • TaqMan® chemistry (also known as “fluorogenic 5' nuclease chemistry”)
  • SYBR® Green I dye chemistry
Overview of TaqMan®- and SYBR®-Green Based Detection
 
TaqMan®-Based Detection
SYBR®-Green Based Detection
Chemistry Overview Uses a fluorogenic probe to enable the detection of a specific PCR product as it accumulates during PCR cycles. Uses SYBR Green I dye, a highly specific, double-stranded DNA binding dye, to detect PCR product as it accumulates during PCR cycles.
Specificity Detects specific amplification products only. Detects all amplified double-stranded DNA, including non-specific reaction products.
Applications
  • One-step RT-PCR for RNA quantitation
  • Two-step RT-PCR for RNA quantitation
  • DNA/cDNA quantitation
    - Allelic Discrimination
    - Plus/Minus assays using an internal positive control (IPC)
  • One-step RT-PCR for RNA quantitation
  • Two-step RT-PCR for RNA quantitation
  • DNA/cDNA quantitation
Advantages
  • Specific hybridization between probe and target is required to generate fluorescent signal, significantly reducing background and false positives.
  • You can label probes with different, distinguishable reporter dyes, which allows you to amplify two distinct sequences in one reaction tube.
  • Post-PCR processing is eliminated, which reduces assay labor and material costs.
  • Enables you to monitor the amplification of any double-stranded DNA sequence.
  • No probes are required, which reduces your assay setup and running costs.
  • Multiple dyes can bind to a single amplified molecule, increasing sensitivity for detecting amplification products.
Disadvantages A different probe has to be synthesized for each unique target sequence. Because SYBR Green I dye binds to any double-stranded DNA—including nonspecific double-stranded DNA sequences—it may generate false positive signals.
TaqMan vs. SYBR Green Chemistry
Figure 1:
Comparison of TaqMan®- 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. The development of fluorogenic labeled probes also made it possible to eliminate post-PCR processing for the analysis of probe degradation.

How TaqMan Sequence Detection Chemistry Works

The TaqMan chemistry uses a fluorogenic probe to enable the detection of a specific PCR product as it accumulates during PCR.

TaqMan vs. SYBR Green Chemistry
Figure 2:
Overview of TaqMan®- Probe-Based Assay Chemistry

Step-by-Step Process

  1. An oligonucleotide probe is constructed containing a reporter fluorescent 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) through space.
  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.
Two Types of TaqMan® Probes

Applied Biosystems offers 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

Applied Biosystems recommends the general use of TaqMan MGB probes for allelic discrimination assays, especially when conventional TaqMan probes exceed 30 nucleotides. The TaqMan MGB probes contain:

  • A nonfluorescent quencher at the 3' end - The SDS 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

The advantages of the TaqMan chemistry are as follows:

  • Specific hybridization between probe and target is required to generate fluorescent signal
  • Probes can be labeled with different, distinguishable reporter dyes, which allows amplification 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 of the TaqMan chemistry is that the synthesis of different probes is required for different sequences

SYBR Green I Dye Chemistry


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

Applied Biosystems has developed conditions that permit the use of the SYBR Green I dye in PCR without PCR inhibition and increased sensitivity of detection compared to ethidium bromide.

How the SYBR Green I Dye Chemistry Works

The SYBR Green I dye chemistry uses the SYBR Green I dye to detect 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 Green I dye is added to a sample, it immediately binds to all double-stranded DNA present in the sample.
  2. During the PCR, AmpliTaq Gold® DNA Polymerase amplifies the target sequence, which creates the PCR products, or "amplicons."
  3. The SYBR Green I dye then binds to each new copy of double-stranded DNA.
  4. As the PCR progresses, more amplicons are created. Since the SYBR Green I dye binds to all double-stranded DNA, the result is an increase in fluorescence intensity proportionate to the amount of PCR product produced.
Advantages of SYBR Green I Dye
  • It can be used to monitor the amplification of any double-stranded DNA sequence
  • No probe is required, which reduces assay setup and running costs
Disadvantage of SYBR Green I Dye

The primary disadvantage of the SYBR Green I dye chemistry is that it may generate false positive signals; i.e., because the SYBR Green I dye binds to any double-stranded DNA, it can also bind to nonspecific double-stranded DNA sequences.

Additional Consideration

Another aspect of using DNA binding dyes is that multiple dyes bind to a single amplified molecule. This increases the sensitivity for detecting amplification products. 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.