Whether the same primer sequence (Used in the SYBRG-based qPCR) can be used to prepare TaqMan probe, if they are specifically amplifies the target? If not, what would be the exact difference or decision criteria to make a probe? I got the information that TaqMan probe sequence can be converted to use in SYBRG real time PCR as primer, but not exactly the vice-versa. Can anyone please help me to get some information on this aspect?
Well, the Taqmanprobe contains a fluorophore and a quencher. This quencher is degraded when this primer did bind to your DNA. What is left is the fluorophore which gets measured in the system. With SYBRgreen it is a completely different story: It is a normal primer without any tags and the fluorophore in your SYBRgreen solution intercalates beetween your amplified DNA double strands. As simply as this. You could use the SYBRgreen primers as Taqman probes but you need to tag them with a fluorophore on one side and a quencher on the other. I guess that might be too difficult.
Hi Shyam,
In Additional to @Teresa Otto, the Taqman probe based PCR is more accurate and less prone to the errors caused by the contaminants which can cause the fluorescence interference. The probe based method also gives the possibility of multiplexing which is not possible in the intercalation based PCR assays. Thus, this method is more dynamic and accurate but also expensive. Developing the probes is hard and expensive as compared to the normal primers used in the SYBR based assays.
Hello Shyam,
There're few imp. criteria to consider for Taqman probes. These could be informative in detail.
PREMIER Biosoft: http://www.premierbiosoft.com/tech_notes/TaqMan.html
Thermofisher manual:
https://tools.thermofisher.com/content/sfs/manuals/cms_042307.pdf
Good Luck!
There are some key differences between two in terms of Gene expression, Reproducibility, Specificity, application purposes etc. However, the following links could be informative.
@Surya Prasad Tiwari
Hi Sir,
Thanks for your valuable information. I will look into it.
Shyam
@Abhijeet Singh
@Teresa Otto
Hi,
Thank you very much for your kind support and informative answers.
Shyam
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