Key Difference – SYBR Green vs Taqman
SYBR Green and Taqman are two methods employed to detect or watch amplification process of real-time PCR. SYBR Green is a method based on intercalating nucleic acid staining dye while Taqman is a method based on hydrolysis probe. Both technologies are designed to generate fluorescence during the PCR, which allows real-time PCR machine to monitor the reaction in “real time”. SYBR Green method is carried out using a fluorescent dye called SYBR green and detects the amplification by binding the dye to produced double stranded DNA. Taqman is carried out using dual-labeled probes and detects the amplification by degradation of the probe by Taq polymerase and releases of the fluorophore. This is the key difference between SYBR Green and Taqman.
CONTENTS
1. Overview and Key Difference
2. What is SYBR Green
3. What is Taqman
4. Side by Side Comparison – SYBR Green vs Taqman
5. Summary
What is SYBR Green?
SYBR Green is a fluorescent dye used to stain nucleic acids, especially double stranded DNA in Molecular Biology. SYBR Green method is used to quantify PCR products during the real-time PCR. Once it binds with DNA, the resulting DNA-dye complex absorbs blue light and emits intense green light. It happens due to the structural change that occurs in the dye molecule upon binding with double-stranded DNA. When PCR creates more and more DNA, more dye molecules bind with DNA, generating more fluorescence. Therefore, the fluorescence increases with the PCR product accumulation. Hence, the amount of PCR product can be quantitatively measured by the SYBR Green fluorescence detection.
SYBR green dye can also be used for DNA labelling in cytometry and fluorescent microscopy. Ethidium bromide has been successfully replaced by SYBR Green since Ethidium bromide is a carcinogenic dye with disposal problems during DNA visualization in the gel electrophoresis.
There are advantages and disadvantages of SYBR green method. This method is very sensitive, inexpensive and easy to use. However, due to its ability to bind to any double-stranded DNA, nonspecific binding can lead to over quantification of PCR product.
Figure 01: SYBR Green Technique
What is Taqman?
Taqman is an alternative method to SYBR Green to monitor real-time PCR process. This method depends on the 5’ – 3’ exonuclease activity of Taq polymerase enzyme to degrade the probes during the extension of the new strand and release of fluorophore. Dual-labeled probes are used in this method and it is based on the hydrolysis of probes. Probes are fluorescently labeled DNA oligonucleotides having a fluorescent reporter molecule (fluorophore) at the 5’ end and a quencher molecule at the 3’ end. They are designed to bind to the single stranded template at the opposite side of the primer anneals. Taq polymerase adds nucleotides to the primer and extends the new strand towards the dual-labeled probes. Once the Taq polymerase meets the probe, exonuclease action of the Taq polymerase activates and degrades the probe. Once it completes the synthesis of the new strand, the probe is subjected to complete degradation and release the fluorophore. The release of fluorophore generates fluorescence. Fluorescent Quencher molecule efficiently quenches the emitted light and creates the output for quantification of the PCR product. The release of fluorophores and quantity of the PCR products are proportional. Hence, quantification can be easily done by the Taqman method.
Figure 02: Taqman Method
Taqman method is used in real time PCR, quantification of gene expression, detection of genetic polymorphisms, quantification of chromosomal DNA deletions, bacterial identification, verification of microarray analysis, SNP genotyping etc.
What is the difference between SYBR green and Taqman?
SYBR Green vs Taqman
|
|
| SYBR Green is based on DNA binding dye. | Taqman depend on hybridization probes and 5’ to 3’ exonuclease activity of Taq polymerase. |
| Fluorescently Labeled Probes | |
| No fluorescently labeled probes are not required. | Dual-labeled probes are required. |
| Multiplex Gene Analysis | |
| It can not be used for multiplex gene targets. | It can be used for multiplex gene targets. |
| Cost | |
| This is less expensive. | This is more expensive. |
| Specificity | |
| This is less specific and binds with any double strand DNA | These are highly specific since probes detect the specific amplification products. |
| Effectiveness | |
| This is less effective. | This is highly effective. |
| Application | |
| This is used in real-time PCR, agarose gel visualization, DNA labeling etc. | This is used in real time PCR, quantification of gene expression, detection of genetic polymorphisms, etc. |
Summary – SYBR Green and Taqman
Taqman and SYBR green are two methods employed in real-time PCR (quantitative PCR). Both methods enable the quantification of the PCR product efficiently and rely on the emission of the fluorescence. Taqman method uses dual-labeled probes for detection of the accumulated DNA while SYBR Green method uses a fluorescent dye. Both these methods also have different applications in molecular biology.
Reference:
1. Tajadini, Mohamad Hasan, Mojtaba Panjehpour, and Shaghayegh Haghjooy Javanmard. “Comparison of SYBR Green and TaqMan methods in quantitative real-time polymerase chain reaction analysis of four adenosine receptor subtypes.” Advanced Biomedical Research. Medknow Publications & Media Pvt Ltd, 2014. Web. 13 Mar. 2017.
2. “Real-time PCR basic principles.” Real Time PCR, Quantitative (qPCR), Primers & Mastermix : Primerdesign Ltd. N.p., n.d. Web. 13 Mar. 2017.
3. “SYBR Green and Other Real-Time PCR Dyes.” Biocompare. N.p., 05 Apr. 2010. Web. 14 Mar. 2017
Image Courtesy:
1. “PCR with SYBR green” By –Ygonaar 23:09, 7 March 2006 (UTC) – It’s a graph create by Ygonaar with Power point, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=619528
2. “Taqman” By User:Braindamaged – Own work (Public Domain) via Commons Wikimedia