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Monday, May 20, 2013

TaqMan Assay Vs SYBR Green Assay


Before going into the details of the Assays let's have some basics on real time PCR,

Real Time PCR differs from the convention PCR/ End point PCR is that in real time PCR both amplification and quantification of the target DNA molecule is possible.

In real time PCR the amplified DNA is detected in real time, where as in convention PCR detection is possible only at the end of PCR by performing agarose gel electrophoresis for the PCR product.

TaqMan Assay


Taqman real time pcr

TaqMan assay includes an oligonucleotide probe called the TaqMan Probe, containing a fluorescent reporter dye at the 5' end and quencher at the 3' end. This oligonucleotide is developed in such a way that it will bind to downstream of the primer binding site in the target DNA molecule.

TaqMan assay utilizes the nuclease activity of taq DNA polymerase, during the elongation step in the PCR, taq DNA polymerase displaces the bound reporter dye.

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.

when the reporter and quencher are inn close proximity, the flourescence emitted by the reporter dye will be readily quenched by the quencher, when the reporter is cleaved by the taq DNA polymerase, reporter moves away from the quencher thereby no quenching occurs, resulting in the emission of flourescent signal.

When the PCR cycles proceeds, resulting in more amplification of the target molecules proportionally fluorescent signal will also increase.

Advantages of TaqMan Assay
  • 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 Assay

The main disadvantage of TaqMan assay is that synthesis of different probes is required for different sequences sometimes makes it difficult.

SYBR Green Assay

SYBR Green dye, a dye which binds to the double stranded DNA, helps in detection of the amplified product. SYBR Green dye shows increased flourescence when binds to a double stranded DNA

During the PCR, Taq DNA polymerase amplifies the target DNA molecules, when SYBR green is present it binds to the double stranded DNA and gives flourescence siganl, as the PCR proceeds more and more amplification occurs and there by more binding of SYBR Green to the double stranded DNA resulting in the increase in flourescent signal.



Advantages of SYBR Green Assay
  • 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 Green Dye

The primary disadvantage is that it may generate false positive signals; i.e., because the SYBR Green 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.

Taqman Vs SYBR Green - Lab Rap Battle - Video by Life Technologies Corp


References
Life Technology - Technical Resources
TaqMan & SYBR Green are registered Trade Marks of Invirogen (Life Technologies Corporation)