Hello Md Fahmid Hossain Bhuiyan

No, you cannot perform TaqMan assay using average regular use PCR master mix.

If you wish to perform PCR using the regular PCR mix, the assay is no longer called TaqMan Assay because the defining feature of a TaqMan Assay is the probe. This small piece of DNA matched to the DNA template being measured has two special molecules attached, a fluorescent reporter dye (R) and a quencher (Q). While both molecules are attached to the probe, the fluorescence of the dye is suppressed by the quencher. These probes bind to the template DNA after it has been denatured into single strands but before it has begun duplicating, making sure that all duplications of the template interact with the probe.

During the PCR reaction, Taq DNA polymerase extends the primer through the polymerase activity, as it approaches the probe it displaces the probe and cleaves it through the 5′ to 3′ exonuclease activity. This separates the reporter dye and the quencher dye from the probe, which results in increased fluorescence of the reporter. Accumulation of PCR products is detected in “real-time” directly by monitoring the increase in fluorescence of the reporter dye with an automated PCR system.

The assay which you would wish to perform is called two-step reverse transcription-polymerase chain reaction. In this assay, two enzymes are used namely, reverse transcriptase to produce single-stranded cDNA copies, which are then used as templates in an amplification reaction catalyzed by a thermostable DNA polymerase. This assay is the traditional method of RT-PCR in which the two synthetic reactions are performed separately and sequentially.

The TaqMan Assay is a real-Time PCR assay which detects the accumulation of amplicon during the reaction. The data is then measured at the exponential phase of the PCR reaction. The assay which you may plan to perform using average regular use PCR master mix is a type of conventional PCR using agarose gel which is not as precise as qPCR. By using the regular use PCR master mix, you cannot perform qPCR because for qPCR one requires the fluorescent reporter molecule such as fluorescent dye, a labeled oligonucleotide primer or probe such as (TaqMan Probe) for fluorescent detection which is monitored by the automated PCR system. Real-Time PCR makes quantitation of DNA and RNA easier and more precise than conventional PCR.

So, if you wish to use the average regular use PCR master mix, you need to perform the two-step reverse transcription-polymerase chain reaction and not qPCR.

Best.

Malcolm Nobre I apologize for not stating my query in a more understandable manner. I am aware that TaqMqn assays require TaqMan probes. I wanted to know if I have the primers and the probes can I perform the TaqMan assay using conventional PCR mastermix, not using the TaqMan specific mastermix like TaqMan™ Universal PCR Master Mix or GoTaq® Probe qPCR and RT-qPCR Systems.

It is composed of recurring and invariable elements of PCR/qPCR reactions. For commercially available mixtures these are typically DNA or RNA dependent polymerase, dNTPS, MgCl2 and reaction buffer. But depending on the case, the master mix terminology can also include the DNA/RNA template or primers.

The use of a master mix type has the advantage of reducing reaction preparation time and reducing the number of pipetting steps.