Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme

Michael J Moser,Thomas W Schoenfeld,David A Mead,Darby R Sugar,Robert A Difrancesco,Stacy Stocki,Audrey J Klingele,Krishne Gowda,John E Tavis

doi:10.1371/journal.pone.0038371

Abstract

Viral metagenomic libraries are a promising but previously untapped source of new reagent enzymes. Deep sequencing and functional screening of viral metagenomic DNA from a near-boiling thermal pool identified clones expressing thermostable DNA polymerase (Pol) activity. Among these, 3173 Pol demonstrated both high thermostability and innate reverse transcriptase (RT) activity. We describe the biochemistry of 3173 Pol and report its use in single-enzyme reverse transcription PCR (RT-PCR). Wild-type 3173 Pol contains a proofreading 3′-5′ exonuclease domain that confers high fidelity in PCR. An easier-to-use exonuclease-deficient derivative was incorporated into a PyroScript RT-PCR master mix and compared to one-enzyme (Tth) and two-enzyme (MMLV RT/Taq) RT-PCR systems for quantitative detection of MS2 RNA, influenza A RNA, and mRNA targets. Specificity and sensitivity of 3173 Pol-based RT-PCR were higher than Tth Pol and comparable to three common two-enzyme systems. The performance and simplified set-up make this enzyme a potential alternative for research and molecular diagnostics.

Highlights

Reverse transcription PCR (RT-PCR) is a powerful analytical and preparative method for detecting, quantifying and analyzing gene expression and RNA viruses
Most reverse transcription PCR (RT-PCR) protocols rely on two DNA polymerase (Pol) enzymes; a retroviral reverse transcriptase (RT) to copy RNA into cDNA and a thermostable DNA Pol to amplify the target sequence
We describe a unique single-enzyme alternative to the traditional format based on the innate reverse transcriptase activity of the thermostable 3173 Pol, which was recently isolated from a viral metagenomic library [1,2]

Summary

Introduction

Reverse transcription PCR (RT-PCR) is a powerful analytical and preparative method for detecting, quantifying and analyzing gene expression and RNA viruses. We believe this is the first report of a reagent enzyme produced from a viral metagenomic library, the first viral Pol shown to be fully thermostable in vitro and the first single-enzyme RT-PCR protocol with high sensitivity and specificity comparable to two-enzyme systems Despite their wide use and general reliability, existing twoenzyme RT-PCR systems have several documented performance problems attributed to deficiencies inherent in retroviral RTs: 1) poor reagent stability, 2) low fidelity, 3) frequent rearrangements during cDNA synthesis, 4) secondary enzymatic activities (i.e. RNase H and strand switching), 5) bias for specific primers and templates, and 6) inhibition of PCR Pol enzymes [3,4,5,6,7]. Alternative chemistries based on an improved RT-PCR enzyme are a means of addressing these shortcomings

Methods

Results

Conclusion