I am new to In vitro transcription. Can anyone help me why do we add the following components in the IVT reaction? 1) MnCl2 2) DTT? 3) Magnesium chloride 4) pyrophosphatase
Hi Hardik,
your question is a bit generic, because the different component are needed at different stages of IVT and it would have been more thoughtful to ask the questions specifically for each reagent and the relative reaction.
Anyway, DTT is a reducing agent without a specific function that is used in virtually all reaction buffers to increase the life of enzymes in a reaction tube, limiting the natural oxidation that would inactivate them (check restriction enzymes, ligases, polymerases, etc...).
MgCl2 provides Mg2+ ions that are needed by many enzymes to catalyse their reactions: in this case all 3 classical phage RNA polymerases (T3, T7, SP6), the pyrophosphatase and the DNAse I that you should use at the end of the reaction to remove the DNA template require it, so it is a component that you need to provide throughout the whole reaction and has a specific function for the enzymes.
MnCl2, vice-versa, is only required by the DNAse I. Mn2+ triggers a different activity compared to Mg2+: it activates double strand break activity, while with Mg2+ the DNAse I cuts randomly only one strand of the dsDNA.
Finally, during IVT many triphosphate-nucleotides (NTPs) are incorporated in the nascent RNA releasing every time a pyrophosphate, which is an inhibitor of RNA polymerization. Consequently, pyrophosphatases are needed to break the pyrophosphates into single orthophosphate ions and increase the RNA yield in IVT.
Cheers.
Normally, you do not use MnCl2 in T7 RNA polymerase reaction mixtures. Only Mg(2+) ions are required by the enzyme to produce a regular RNA transcript. However, if you want to incorporate deoxynucleotides or 2'-methylated ribonucleotides into the transcript, you may add MnCl2, which promotes such a reaction. See, for example, the following paper: "Enzymatic synthesis of 2'-modified nucleic acids: identification of important phosphate and ribose moieties in RNase P substrates" (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC306953/).
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