Why is DTT used in reverse transcription (RT) reaction? - (Jan/11/2005 )
can anyone please elaborate why DTT is used in Reverse Transcription reaction? Thanks in advance.
Dithiothreitol is a reducing agent, so it helps to break bonds (like disulfide bonds) which will loosen the secondard structure of the RNA and facilitate RT enzyme initiation of transcription and processivity.
Thank you for your reply. But normally there is no disulfide bond in the mRNAs, so i wonder.........
In low concentrations, DTT is used to stabilize proteins which possess free sulfhydryl groups. Thus, its addition to the RT mix would serve the purpose of protecting the enzymatic activity of the reverse transcriptase. I'm not sure if this is the reason by which DTT is added to RT, but is the only answer I can come up with. I've done thousands of RTs and never wonder why was I adding the DTT!
Hi
(sorry for my english) I think that you use DTT to be in a reductor state like in the nucleus, the enzyme functions so in an optimal manner.
have anyone ever used RT from NEB before. Do i need to add DDT to RT or it is already in the RT buffer?
DTT is used to reduce disulfide bonds in RNases needed by RNases for stability, thereby inhibiting RNase activity and preserving mRNA for the RT reaction. Please see:
Chen Z, Ling J, Gallie DR. RNase activity requires formation of disulfide bonds and is regulated by the redox state. Plant Mol Biol. 2004 May;55(1):83-96.
Mercaptoethanol should be functioning the same right? To destroy the disulphide bond of Rnase?
correct.
if DTT is used as an RNase inhibitor, how about RNaseOUT (Invitrogen protocolof RT-PCR), an RNase inhibitor also? Why do we use 2 RNase ihibitors?