Hi All
Brief notes from the Reverse Transcriptase breakout session at the recent Reclone Symposium!
If anyone has interesting thoughts to share about expressing, assaying or improving RTs then please comment on this thread.
If you have any useful RT plasmids or reagents let us know and make them available through Reclone!
I’ve marked in the notes below what is already available.
Jenny
Notes from Reclone Symposium Breakout Session - Reverse Transcriptases
On going work
Adam shared that he is investigating improving 3 DNA polymerases with RT activity:
-
RTX and a more recent paper on the use of RTX. in a one-step RT-qPCR assay for COVID, which also details the more practical expression and purification steps of RTX. You can request RTX plasmids from the Ellington Lab! See this post on the forum for more details.
-
Tth and original 1991 paper here. Tth DNA Polymerase is available in the Open Enzyme Collection.
-
KlenTaq: the version in the linked paper is being taken forward due to it’s higher activity, but this paper is a great read, where they designed a smart mutant library to create an RT KlenTaq variant, but their goal was the design of the library rather than RT design so the final enzyme was somewhat unoptimised. KlenTaq variants has an amino acid responsible for binding to the RNA backbone and while most papers screen for highest RT activity, this paper went with binding. Klentaq1 DNA Polymerase (not variants in these papers) is available in the Open Enzyme Collection.
Why are there so few options for RTs relative to DNA polymerases? Range of factors including historically what the readily available retroviruses were for analysis. Lots of focus now on engineering DNA polymerases with RT activity because there are a lot more to play with and enhance rather than going out and trying to find them. Current options are also very good e.g. RTx from NEB performs well and comes in warm start version.
Do we have a good alignment of the possible options? Pipette Jockey made one for MashUp RT but there is a lot of info in literature and patents that could be overlaid on an alignment with reference - maybe a project for someone who is really into RTs?
Open question: how best to easily assay for RT activity?