Before you do qPCR, if you plan to use the probe method, you must first determine the fluorescence and quencher of the probe you are doing.
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Of course, you may think that SYBR Green is more convenient, but in fact, the non-specific amplification of SYBR Green is its most fatal weakness. The specificity of the probe method is higher than that of SYBR GREEN. However, the probe method still has its problem, which is that not all instruments are compatible.
This is obvious that the ABI instrument in multiplex PCR cannot use ROX as a fluorescent probe, because ROX must be used as a substrate control fluorescence in ABI instruments. Of course, there are also some ABI instrument models that do not support a particularly large number of channels, which will have a relatively large impact.
: In addition to this, there is another point to note is that the fluorescence of VIC, due to patent issues, so many people will use HEX instead, but in fact, there is still a certain difference between HEX and VICIn particular, you should choose carefully if you plan to use dual fluorescence for SNP site detection）。As a result, the fluorescence intensity measured on ABI's instrument will be slightly different when using the VIC probe and the HEX probe (Other brands of models don't seem to be able to be distinguished）。Of course, there is also an alternative called Yakima Yellow, which can replace VIC, but this requires careful selection of quenching groups.
Of course, in addition to the screening of fluorophores, it is more important to screen the quencher of TaqMan probes. For example, try not to choose tamra as the quencher.
Why?Because tamra itself is a fluorophore, although it also has a quenching effect, it will enhance the background fluorescence intensity. Of course, you may not be able to tell the difference between BHQ1 and BHQ2. The main reason is that among the commonly used fluorophores, their quenching effect is basically the same, and both FAM and HEX can be killed, but when it comes to TaqMan probes for other fluorophores, remember to choose carefully:
The dual-labeled fluorescent probe is a powerful fluorescent probe, and I hope today's introduction will be helpful to you in your future scientific research!