RTX (Reverse Transcriptase Xenopolymerase)

Reverse Transcriptase Xenopolymerase

RTX (Reverse Transcriptase Xenopolymerase) is a mutant variant of KOD DNA Polymerase (DNAP) with relaxed substrate specificity that permits DNA synthesis from either DNA or RNA template. KOD is a family B DNAP that derives from Thermococcus kodakarensis (KOD) and has 3’->5’ exonuclease activity, which is more commonly called “proof-reading activity.” [https://science.sciencemag.org/content/352/6293/1590.long]

Reagent Sharers

Ellington research group at the University of Texas at Austin

Summary

As a mutant derivative of KOD DNAP, RTX retains two important features. First, RTX performs reverse transcription with 3’->5’ activity thus making it a novel means of performing RT reactions with proof-reading. Additionally, as with KOD, RTX remains a thermostable polymerase with the capacity for thermocycling. These two features allow for high-fidelity, thermocycling-based amplification of an RNA template.

RTX has recently been used to develop a new single-enzyme RT-PCR detection method fo SARS-Cov-2 (https://www.biorxiv.org/content/10.1101/2020.03.29.013342v1).

Expression Information

RTX is currently available for T7-based expression in E. coli (i.e. BL21(DE3) strain) using a modified pET21 plasmid. This plasmid can be used for expression of polyhistidine-tagged RTX and the mutant version without exonuclease/proof-reading activity - called RTX(exo-). See the preprint for more information (more coming soon!).

Use in Protocols

See the preprint for more information (more coming soon!).

How to get the plasmids

Both plasmids are available on Addgene (https://www.addgene.org/102787/) (https://www.addgene.org/102786/) or can be requested by directly contacting the Ellington research group at the University of Texas at Austin (reagentellingtonlab@gmail.com).