Thermostable DNA polymerase from a viral metagenome is a potent RT-PCR enzyme.
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.
Lucigen Corporation, Middleton, Wisconsin, United States of America.
Michael J
MJ
Moser
Robert A
RA
DiFrancesco
Krishne
K
Gowda
Audrey J
AJ
Klingele
Darby R
DR
Sugar
Stacy
S
Stocki
David A
DA
Mead
Thomas W
TW
Schoenfeld
D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004795:Enzyme Stability; D015723:Gene Library; D045482:Hot Springs / Q000821:virology; D006801:Humans; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification; D017909:Levivirus / Q000235:genetics / Q000302:isolation & purification; D054892:Metagenome / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature; D014780:Viruses / Q000235:genetics*
D012333:RNA, Messenger; C068508:Tth polymerase; D004259:DNA-Directed DNA Polymerase