Engineering of a thermostable viral polymerase using metagenome-derived diversity for highly sensitive and specific RT-PCR.

ABSTRACT: Reverse transcription is an essential initial step in the analysis of RNA for most PCR-based amplification and detection methods. Despite advancements in these technologies, efficient conversion of RNAs that form stable secondary structures and double-stranded RNA targets remains challenging as retroviral-derived reverse transcriptases are often not sufficiently thermostable to catalyze synthesis at temperatures high enough to completely relax these structures. Here we describe the engineering and improvement of a thermostable viral family A polymerase with inherent reverse transcriptase activity for use in RT-PCR. Using the 3173 PyroPhage polymerase, previously identified from hot spring metagenomic sampling, and additional thermostable orthologs as a source of natural diversity, we used

SUBMITTER: Heller RC 

PROVIDER: S-EPMC6468311 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature