4 replies to this topic

[genugene]

can anyone please elaborate why  DTT is used in Reverse Transcription reaction? Thanks in advance.

Edited by bioforum, 14 January 2005 - 09:38 AM.

[seqgirl]

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.

[genugene]

seqgirl, on Jan 12 2005, 01:55 PM, said:

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.........

[badcell]

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!

[gtran89]

you're right. dtt is required to protect RT and Taq polymerase in that it reduces disulfide bonds. It is also required for RNase inhibitor activity, where most require a minimum of 1mM.