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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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Related in: MedlinePlus

RT-PCR detection of human transcript RNAs.Beta-actin, beta2-microglobulin and cyclophilin target sequences of the indicated sizes were amplified from human liver total RNA using the primers described in Table 1. Shown are products of two step reactions where either MMLV RT or 3173 Pol were used for first strand cDNA synthesis, as indicated. Taq Pol was used for PCR. Products were resolved on a 1% agarose gel.
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pone-0038371-g005: RT-PCR detection of human transcript RNAs.Beta-actin, beta2-microglobulin and cyclophilin target sequences of the indicated sizes were amplified from human liver total RNA using the primers described in Table 1. Shown are products of two step reactions where either MMLV RT or 3173 Pol were used for first strand cDNA synthesis, as indicated. Taq Pol was used for PCR. Products were resolved on a 1% agarose gel.

Mentions: Under favorable conditions 3173 Pol did reverse transcribe mRNA transcripts (Figure 5). The 3173 Pol was compared to MMLV RT for the detection of three shorter target sequences in common high-abundance reference genes using the two-step RT-PCR protocol. Both enzymes appear to transcribe the targets with similar efficiency and specificity. The amount of PCR product for all three transcripts appeared visibly greater in the 3173 Pol reactions, although we cannot rule out the contribution of residual thermostable 3173 Pol to the PCR reaction yield.


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)

RT-PCR detection of human transcript RNAs.Beta-actin, beta2-microglobulin and cyclophilin target sequences of the indicated sizes were amplified from human liver total RNA using the primers described in Table 1. Shown are products of two step reactions where either MMLV RT or 3173 Pol were used for first strand cDNA synthesis, as indicated. Taq Pol was used for PCR. Products were resolved on a 1% agarose gel.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366922&req=5

pone-0038371-g005: RT-PCR detection of human transcript RNAs.Beta-actin, beta2-microglobulin and cyclophilin target sequences of the indicated sizes were amplified from human liver total RNA using the primers described in Table 1. Shown are products of two step reactions where either MMLV RT or 3173 Pol were used for first strand cDNA synthesis, as indicated. Taq Pol was used for PCR. Products were resolved on a 1% agarose gel.
Mentions: Under favorable conditions 3173 Pol did reverse transcribe mRNA transcripts (Figure 5). The 3173 Pol was compared to MMLV RT for the detection of three shorter target sequences in common high-abundance reference genes using the two-step RT-PCR protocol. Both enzymes appear to transcribe the targets with similar efficiency and specificity. The amount of PCR product for all three transcripts appeared visibly greater in the 3173 Pol reactions, although we cannot rule out the contribution of residual thermostable 3173 Pol to the PCR reaction yield.

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