MMLV reverse transcriptase works on RNA as a template to make cDNA (reverse transcription). DNA Taq polymerase works best on it's natural target which is DNA. It has a poor reverse transcriptional activity when the Mg in the buffers is replaced with Mn. Because it is much less efficient than MMLV or AMV, it is not the preferred enzyme for reverse transcription, especially quantitative RT-PCR.
"One-step" RT-PCR is NOT one step. It is a 2 step process - reverse transcription followed by PCR amplification .A more accurate description is a "one--tube PCR" where both reactions occur separately and in succession in the same tube.
MMLV reverse transcriptase works on RNA as a template to make cDNA (reverse transcription). DNA Taq polymerase works best on it's natural target which is DNA. It has a poor reverse transcriptional activity when the Mg in the buffers is replaced with Mn. Because it is much less efficient than MMLV or AMV, it is not the preferred enzyme for reverse transcription, especially quantitative RT-PCR.
"One-step" RT-PCR is NOT one step. It is a 2 step process - reverse transcription followed by PCR amplification .A more accurate description is a "one--tube PCR" where both reactions occur separately and in succession in the same tube.
There is an enzyme called Tth polymerase. this enzyme can do reverse transcription as well as PCR amplification. But RT activity is only when Mn is present in buffer and amplification is only occur in the presence of Mg++. but this one enzyme does these both activity....
MMLV( Moloney Murine Leukemia Virus ) Reverse Transcriptase is an RNA-dependent DNA polymerase that can be used in cDNA synthesis( such as First - Strand cDNA synthesis when RT-PCR) with long mRNA templates ( >5 Kb). DNA Polymerase usually has a very low RT activity and is not an ideal Reverse Transcriptase ( except Tth DNA Polymerase in the presence of manganese).
People developed a range of hot start enzymes, like aptamer-based hot start Taq Polymerase, to minimize pre-cycling polymerase activity , which help increase the specificity and yield of PCR products, not for improving the RT activity. Therefore, non hot start DNA Polymerase can not replace MMLV RT when you do RT experiment.
MMLV is essential for reverse transcyptase, no doubt, but how do you ensure it has taken place or not, unless you have done the amplication and there you would need the DNA polymerase. That is why bothe enzymes have been provided to you.
of courese not, because there is a missed step in your procedure, how can the amplification reaction start if you haven't the suitable enzyme??? or how you amplification enzyme (annealing) can work if there was not the suitable template in the solution, so in conclusion every enzyme should be present to do it's work the first one to prepare the suitable template which is the cDNA ( from the RNA) and the second one to amplifying .