Why or why not? In One-step RT kit, it has both enzymes to reverse transcription and PCR. Is it possible to use only one enzyme to finish it?
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....
for more info. you see this link below...
https://www.promega.com/products/pcr/rt-pcr/tth-dna-polymerase/
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.
The method you choose will affect your results and how you conclude the findings an experimental design depends on the objectives of the experiment.
Is this for analytical or for some preparative cDNA applications?
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