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Thermostable DNA polymerase from a viral metagenome is a potent RT-PCR enzyme.

Moser MJ, DiFrancesco RA, Gowda K, Klingele AJ, Sugar DR, Stocki S, Mead DA, Schoenfeld TW - PLoS ONE (2012)

Bottom Line: Wild-type 3173 Pol contains a proofreading 3'-5' exonuclease domain that confers high fidelity in PCR.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.

View Article: PubMed Central - PubMed

Affiliation: Lucigen Corporation, Middleton, Wisconsin, United States of America.

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.

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Comparison of 3173 Pol (PyroScript) RT-PCR mix with two enzyme RT-PCR systems in detection of MS2 and influenza A.Ten-fold serial dilutions of an MS2, an influenza A RNA preparation and a water only control (NTC) were amplified by one-step RT-PCR reagent mixes (PyroScript, qScript (Quanta), Transcriptor (Roche), and SuperScript (Invitrogen), as indicated. A. MS2 detection. Left panel: 2% agarose gel, each group of four wells are 10−4, 10−6, 10−7-fold target dilutions and NTC, MW is 100 bp DNA ladder (50 bp smallest band). Right Panel: RT-qPCR analysis of 10−3, 10−4, 10−5, and 10−6-fold target dilutions. B. Influenza A RNA detection. Left panel: 4–20% gradient polyacrylamide gel, each group of three wells are 10−6, 10−7-fold target dilutions and NTC, MW is 25 bp DNA ladder (50 bp smallest band). Right Panel: RT-qPCR analysis of 10−3, 10−4, 10−5, and 10−6-fold target dilutions.
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pone-0038371-g008: Comparison of 3173 Pol (PyroScript) RT-PCR mix with two enzyme RT-PCR systems in detection of MS2 and influenza A.Ten-fold serial dilutions of an MS2, an influenza A RNA preparation and a water only control (NTC) were amplified by one-step RT-PCR reagent mixes (PyroScript, qScript (Quanta), Transcriptor (Roche), and SuperScript (Invitrogen), as indicated. A. MS2 detection. Left panel: 2% agarose gel, each group of four wells are 10−4, 10−6, 10−7-fold target dilutions and NTC, MW is 100 bp DNA ladder (50 bp smallest band). Right Panel: RT-qPCR analysis of 10−3, 10−4, 10−5, and 10−6-fold target dilutions. B. Influenza A RNA detection. Left panel: 4–20% gradient polyacrylamide gel, each group of three wells are 10−6, 10−7-fold target dilutions and NTC, MW is 25 bp DNA ladder (50 bp smallest band). Right Panel: RT-qPCR analysis of 10−3, 10−4, 10−5, and 10−6-fold target dilutions.

Mentions: Since two-enzyme systems using MMLV RT derivatives and Taq Pol are far more commonly used than single-enzyme systems, we compared the performance of single-enzyme PyroScript mix to three widely used mixes that are based on the two enzyme MMLV RT plus Taq Pol combination, but are referred to as “one-step” systems. The comparators were: SuperScript® III One-Step RT-PCR System with Platinum® Taq DNA Polymerase (Life Technologies), the qScript™ One-Step SYBR® Green qRT-PCR Kit (Quanta), and the Transcriptor® One-Step RT-PCR Kit (Roche). MS2 RNA extract was amplified using primers targeting the 160 bp product from Figure 6. Three dilutions of MS2 RNA (the lower dilutions from Figure 6D) and a water-only control were amplified by 40 cycles of RT-qPCR using each of the respective reagents (Figure 8A). All of the reagents appeared to have similar limits of detection and amplified the expected product as seen by electrophoresis. All of the reagents produced a weak background amplification product of about 60 bp, from both the lowest RNA dilution and the water-only control. The Transcriptor kit reproducibly amplified more false product than did the other three. Similar slopes from plots of qPCR cycle threshold versus fold target dilution show that all four reagent mixes amplified the MS2 target with similar efficiency although the qScript reagent appeared to amplify the target a few cycles later than the other three mixes.


Thermostable DNA polymerase from a viral metagenome is a potent RT-PCR enzyme.

Moser MJ, DiFrancesco RA, Gowda K, Klingele AJ, Sugar DR, Stocki S, Mead DA, Schoenfeld TW - PLoS ONE (2012)

Comparison of 3173 Pol (PyroScript) RT-PCR mix with two enzyme RT-PCR systems in detection of MS2 and influenza A.Ten-fold serial dilutions of an MS2, an influenza A RNA preparation and a water only control (NTC) were amplified by one-step RT-PCR reagent mixes (PyroScript, qScript (Quanta), Transcriptor (Roche), and SuperScript (Invitrogen), as indicated. A. MS2 detection. Left panel: 2% agarose gel, each group of four wells are 10−4, 10−6, 10−7-fold target dilutions and NTC, MW is 100 bp DNA ladder (50 bp smallest band). Right Panel: RT-qPCR analysis of 10−3, 10−4, 10−5, and 10−6-fold target dilutions. B. Influenza A RNA detection. Left panel: 4–20% gradient polyacrylamide gel, each group of three wells are 10−6, 10−7-fold target dilutions and NTC, MW is 25 bp DNA ladder (50 bp smallest band). Right Panel: RT-qPCR analysis of 10−3, 10−4, 10−5, and 10−6-fold target dilutions.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366922&req=5

pone-0038371-g008: Comparison of 3173 Pol (PyroScript) RT-PCR mix with two enzyme RT-PCR systems in detection of MS2 and influenza A.Ten-fold serial dilutions of an MS2, an influenza A RNA preparation and a water only control (NTC) were amplified by one-step RT-PCR reagent mixes (PyroScript, qScript (Quanta), Transcriptor (Roche), and SuperScript (Invitrogen), as indicated. A. MS2 detection. Left panel: 2% agarose gel, each group of four wells are 10−4, 10−6, 10−7-fold target dilutions and NTC, MW is 100 bp DNA ladder (50 bp smallest band). Right Panel: RT-qPCR analysis of 10−3, 10−4, 10−5, and 10−6-fold target dilutions. B. Influenza A RNA detection. Left panel: 4–20% gradient polyacrylamide gel, each group of three wells are 10−6, 10−7-fold target dilutions and NTC, MW is 25 bp DNA ladder (50 bp smallest band). Right Panel: RT-qPCR analysis of 10−3, 10−4, 10−5, and 10−6-fold target dilutions.
Mentions: Since two-enzyme systems using MMLV RT derivatives and Taq Pol are far more commonly used than single-enzyme systems, we compared the performance of single-enzyme PyroScript mix to three widely used mixes that are based on the two enzyme MMLV RT plus Taq Pol combination, but are referred to as “one-step” systems. The comparators were: SuperScript® III One-Step RT-PCR System with Platinum® Taq DNA Polymerase (Life Technologies), the qScript™ One-Step SYBR® Green qRT-PCR Kit (Quanta), and the Transcriptor® One-Step RT-PCR Kit (Roche). MS2 RNA extract was amplified using primers targeting the 160 bp product from Figure 6. Three dilutions of MS2 RNA (the lower dilutions from Figure 6D) and a water-only control were amplified by 40 cycles of RT-qPCR using each of the respective reagents (Figure 8A). All of the reagents appeared to have similar limits of detection and amplified the expected product as seen by electrophoresis. All of the reagents produced a weak background amplification product of about 60 bp, from both the lowest RNA dilution and the water-only control. The Transcriptor kit reproducibly amplified more false product than did the other three. Similar slopes from plots of qPCR cycle threshold versus fold target dilution show that all four reagent mixes amplified the MS2 target with similar efficiency although the qScript reagent appeared to amplify the target a few cycles later than the other three mixes.

Bottom Line: Wild-type 3173 Pol contains a proofreading 3'-5' exonuclease domain that confers high fidelity in PCR.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.

View Article: PubMed Central - PubMed

Affiliation: Lucigen Corporation, Middleton, Wisconsin, United States of America.

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.

Show MeSH
Related in: MedlinePlus