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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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Reverse transcriptase assays.A. Fluorogenic assay. RT activity was measured by detection of RNA:DNA heteroduplex by fluorescence of EvaGreen binding. Oligo dT primed poly A was incubated at 37°C and 65°C in the presence of indicated Pol enzymes in manufacturer recommended buffers and dTTP. Fluorescence measurements were obtained every 6 seconds for 10 minutes. The initial slopes from a plot of RFU vs. time in seconds were determined by linear least square regression from 30 to 150 seconds at 37°C and from 30 to 90 seconds at 65°C. Error bars are standard error of regression slope. B. RT primer extension assay. HEX-labeled dT20 primed poly A was incubated 10 minutes at 37°C and then 10 minutes at 65°C in the presence of indicated Pol enzymes and dTTP in manufacturer recommended buffers. Primer extension products were resolved by 10% denaturing PAGE and imaged on a Molecular Imager FX (Bio-Rad). Left facing triangle indicates migration of unextended dT20 primer and asterisk indicates bromophenol blue dye front. C. RT MS2-specific primer extension. 5′-labeled primer was annealed to MS2 RNA and incubated 10 minutes at 37°C and then 30 minutes at 65°C in the presence of indicated Pol enzymes with dNTPS (N = A,C,G,T) in manufacturer recommended buffers. Primer extension products were resolved by 5% denaturing PAGE. Lane 1 No RNA+MMLV RT; Lane 2: MS2 RNA No RT; Lane 3 MS2 RNA+MMLV RT, Lane 3 MS2 RNA+3173 Pol. Molecular weight in bases indicted. Red Arrow: ∼650 base MMLV extension product. Blue Arrow: ∼715 base PyroScript extension product. Green arrow: Non-templated MMLV reaction product.
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pone-0038371-g003: Reverse transcriptase assays.A. Fluorogenic assay. RT activity was measured by detection of RNA:DNA heteroduplex by fluorescence of EvaGreen binding. Oligo dT primed poly A was incubated at 37°C and 65°C in the presence of indicated Pol enzymes in manufacturer recommended buffers and dTTP. Fluorescence measurements were obtained every 6 seconds for 10 minutes. The initial slopes from a plot of RFU vs. time in seconds were determined by linear least square regression from 30 to 150 seconds at 37°C and from 30 to 90 seconds at 65°C. Error bars are standard error of regression slope. B. RT primer extension assay. HEX-labeled dT20 primed poly A was incubated 10 minutes at 37°C and then 10 minutes at 65°C in the presence of indicated Pol enzymes and dTTP in manufacturer recommended buffers. Primer extension products were resolved by 10% denaturing PAGE and imaged on a Molecular Imager FX (Bio-Rad). Left facing triangle indicates migration of unextended dT20 primer and asterisk indicates bromophenol blue dye front. C. RT MS2-specific primer extension. 5′-labeled primer was annealed to MS2 RNA and incubated 10 minutes at 37°C and then 30 minutes at 65°C in the presence of indicated Pol enzymes with dNTPS (N = A,C,G,T) in manufacturer recommended buffers. Primer extension products were resolved by 5% denaturing PAGE. Lane 1 No RNA+MMLV RT; Lane 2: MS2 RNA No RT; Lane 3 MS2 RNA+MMLV RT, Lane 3 MS2 RNA+3173 Pol. Molecular weight in bases indicted. Red Arrow: ∼650 base MMLV extension product. Blue Arrow: ∼715 base PyroScript extension product. Green arrow: Non-templated MMLV reaction product.

Mentions: Both radioactive and fluorogenic incorporation assays indicated strong RNA-dependent DNA synthesis (reverse transcription) activity for 3173 Pol in buffers containing either magnesium or manganese (not shown). We used two assays (Figure 3) to compare the RT activities of the wild-type and exonuclease deficient 3173 Pols to those of AMV and MMLV RTs at 37°C or 65°C on an oligo dT primed poly A substrate. The AMV and MMLV had higher RT activity at 37°C while the 3173 Pol RT was much more active at 65°C using the fluorogenic incorporation assay (Figure 3A). The Taq polymerase and no enzyme controls had no detectable RT activity at either temperature. Extension products from a 5′-fluorophore-labeled dT20 primer were resolved by denaturing polyacrylamide gel to further demonstrate RT activity and to assess the relative lengths of the extension products of the 3173 Pol and MMLV RT (Figure 3B). Both RTs were able to efficiently extend the primer when polyA RNA template was provided. The length distribution of the 3173 Pol cDNAs was visibly shorter than that produced by the MMLV RT, although a subset of the 3173 extension products appeared to be so large that they barely entered the gel. Incubation of the DNA primer:RNA template complex with the Taq Pol negative control resulted in a structure-dependent 5′-3′ exonuclease cleavage product that migrated at the dye front [39]. As an additional test to compare the RT activity of the 3173 Pol to that of MMLV-RT on a complex RNA substrate, a primer specific to bases 714 to 690 of the negative sense RNA MS2 genome [40] was 5′-fluorophore-labeled. The labeled cDNA primer was extended using extracted MS2 RNA as a template (Figure 3C). The 3173 Pol and MMLV RT were both able to extend the primer to produce faint, nearly full-length products although the 3173 Pol product was detectably longer than that of MMLV RT. The 3173 Pol also synthesized a larger amount of several shorter length extension products from 175 to 300 bases in length. The MMLV RT formed a visible template-independent product in the absence of added RNA template that was not resolved by the gel, while the 3173 Pol did not.


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)

Reverse transcriptase assays.A. Fluorogenic assay. RT activity was measured by detection of RNA:DNA heteroduplex by fluorescence of EvaGreen binding. Oligo dT primed poly A was incubated at 37°C and 65°C in the presence of indicated Pol enzymes in manufacturer recommended buffers and dTTP. Fluorescence measurements were obtained every 6 seconds for 10 minutes. The initial slopes from a plot of RFU vs. time in seconds were determined by linear least square regression from 30 to 150 seconds at 37°C and from 30 to 90 seconds at 65°C. Error bars are standard error of regression slope. B. RT primer extension assay. HEX-labeled dT20 primed poly A was incubated 10 minutes at 37°C and then 10 minutes at 65°C in the presence of indicated Pol enzymes and dTTP in manufacturer recommended buffers. Primer extension products were resolved by 10% denaturing PAGE and imaged on a Molecular Imager FX (Bio-Rad). Left facing triangle indicates migration of unextended dT20 primer and asterisk indicates bromophenol blue dye front. C. RT MS2-specific primer extension. 5′-labeled primer was annealed to MS2 RNA and incubated 10 minutes at 37°C and then 30 minutes at 65°C in the presence of indicated Pol enzymes with dNTPS (N = A,C,G,T) in manufacturer recommended buffers. Primer extension products were resolved by 5% denaturing PAGE. Lane 1 No RNA+MMLV RT; Lane 2: MS2 RNA No RT; Lane 3 MS2 RNA+MMLV RT, Lane 3 MS2 RNA+3173 Pol. Molecular weight in bases indicted. Red Arrow: ∼650 base MMLV extension product. Blue Arrow: ∼715 base PyroScript extension product. Green arrow: Non-templated MMLV reaction product.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366922&req=5

pone-0038371-g003: Reverse transcriptase assays.A. Fluorogenic assay. RT activity was measured by detection of RNA:DNA heteroduplex by fluorescence of EvaGreen binding. Oligo dT primed poly A was incubated at 37°C and 65°C in the presence of indicated Pol enzymes in manufacturer recommended buffers and dTTP. Fluorescence measurements were obtained every 6 seconds for 10 minutes. The initial slopes from a plot of RFU vs. time in seconds were determined by linear least square regression from 30 to 150 seconds at 37°C and from 30 to 90 seconds at 65°C. Error bars are standard error of regression slope. B. RT primer extension assay. HEX-labeled dT20 primed poly A was incubated 10 minutes at 37°C and then 10 minutes at 65°C in the presence of indicated Pol enzymes and dTTP in manufacturer recommended buffers. Primer extension products were resolved by 10% denaturing PAGE and imaged on a Molecular Imager FX (Bio-Rad). Left facing triangle indicates migration of unextended dT20 primer and asterisk indicates bromophenol blue dye front. C. RT MS2-specific primer extension. 5′-labeled primer was annealed to MS2 RNA and incubated 10 minutes at 37°C and then 30 minutes at 65°C in the presence of indicated Pol enzymes with dNTPS (N = A,C,G,T) in manufacturer recommended buffers. Primer extension products were resolved by 5% denaturing PAGE. Lane 1 No RNA+MMLV RT; Lane 2: MS2 RNA No RT; Lane 3 MS2 RNA+MMLV RT, Lane 3 MS2 RNA+3173 Pol. Molecular weight in bases indicted. Red Arrow: ∼650 base MMLV extension product. Blue Arrow: ∼715 base PyroScript extension product. Green arrow: Non-templated MMLV reaction product.
Mentions: Both radioactive and fluorogenic incorporation assays indicated strong RNA-dependent DNA synthesis (reverse transcription) activity for 3173 Pol in buffers containing either magnesium or manganese (not shown). We used two assays (Figure 3) to compare the RT activities of the wild-type and exonuclease deficient 3173 Pols to those of AMV and MMLV RTs at 37°C or 65°C on an oligo dT primed poly A substrate. The AMV and MMLV had higher RT activity at 37°C while the 3173 Pol RT was much more active at 65°C using the fluorogenic incorporation assay (Figure 3A). The Taq polymerase and no enzyme controls had no detectable RT activity at either temperature. Extension products from a 5′-fluorophore-labeled dT20 primer were resolved by denaturing polyacrylamide gel to further demonstrate RT activity and to assess the relative lengths of the extension products of the 3173 Pol and MMLV RT (Figure 3B). Both RTs were able to efficiently extend the primer when polyA RNA template was provided. The length distribution of the 3173 Pol cDNAs was visibly shorter than that produced by the MMLV RT, although a subset of the 3173 extension products appeared to be so large that they barely entered the gel. Incubation of the DNA primer:RNA template complex with the Taq Pol negative control resulted in a structure-dependent 5′-3′ exonuclease cleavage product that migrated at the dye front [39]. As an additional test to compare the RT activity of the 3173 Pol to that of MMLV-RT on a complex RNA substrate, a primer specific to bases 714 to 690 of the negative sense RNA MS2 genome [40] was 5′-fluorophore-labeled. The labeled cDNA primer was extended using extracted MS2 RNA as a template (Figure 3C). The 3173 Pol and MMLV RT were both able to extend the primer to produce faint, nearly full-length products although the 3173 Pol product was detectably longer than that of MMLV RT. The 3173 Pol also synthesized a larger amount of several shorter length extension products from 175 to 300 bases in length. The MMLV RT formed a visible template-independent product in the absence of added RNA template that was not resolved by the gel, while the 3173 Pol did not.

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