Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme
| Autor: | Darby R. Sugar, Stacy Stocki, Audrey J. Klingele, Thomas W. Schoenfeld, David A. Mead, Robert A. Difrancesco, Michael J. Moser, Krishne Gowda |
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| Rok vydání: | 2012 |
| Předmět: |
Viral Diseases
DNA polymerase viruses Applied Microbiology lcsh:Medicine Gene Expression DNA-Directed DNA Polymerase Biochemistry Hot Springs law.invention law Nucleic Acids Enzyme Stability Molecular Cell Biology Genomic library lcsh:Science Polymerase chain reaction 0303 health sciences Multidisciplinary biology Reverse Transcriptase Polymerase Chain Reaction Temperature Viral Replication Complex Genomics Enzymes Reverse transcription polymerase chain reaction Real-time polymerase chain reaction Infectious Diseases Influenza A virus Viruses Viral Enzymes Medicine Research Article Exonuclease Microbiology 03 medical and health sciences Diagnostic Medicine Virology Humans RNA Messenger Biology 030304 developmental biology Gene Library Levivirus 030306 microbiology lcsh:R Computational Biology Processivity Molecular biology Reverse transcriptase Viral Replication Influenza Viral Disease Diagnosis biology.protein Metagenome RNA lcsh:Q Metagenomics |
| Zdroj: | PLoS ONE PLoS ONE, Vol 7, Iss 6, p e38371 (2012) |
| ISSN: | 1932-6203 |
| DOI: | 10.1371/journal.pone.0038371 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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