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High-Fidelity Thermostable Reverse Transcriptase for RT PCR and Transcriptome Analysis High-fidelity Thermostable Reverse Transcriptase for RT PCR and Transcriptome analysis. www.lucigen.com Thomas Schoenfeld, Nicholas Hermersmann, Krishne Gowda, Darby Renneckar, David Mead, Michael J. Moser Lucigen Corporation, Middleton, WI 53562 USA 888-575-9695 email [email protected] ABSTRACT PyroScript™ RT-PCR Master Mix Kit Comparison Data • PyroScript RT-PCR 2X Master Mix Known limitations in reverse transcription enzymes (RTs) •Buffer compromise current RT PCR and RNA sequence analyses. Most PyroPhage RT Tth Polymerase •dNTPs common RT protocols use retroviral RTs that impair analysis due a. •PyroPhage RT Polymerase to low accuracy, strand switching and bias. Since none of these RT enzymes are truly thermostable, a second DNA polymerase is • RNA control needed to perform PCR amplification, which creates additional •Direct RT-PCR control handling steps and necessitate a compromise in optimal •Extractable Control conditions between the two enzymes. Recent screens of metagenomic libraries from hot spring viruses have uncovered • Control Primer Set novel DNA polymerases including PyroPhage™ RT. • 100 mM Magnesium Sulfate Thermostable PyroPhage RT is the first viral reverse transcriptase • Nuclease-free Water that is also capable of PCR. This allows single-enzyme, one-step RT PCR with sensitivity, specificity and accuracy equivalent to or Just add template, primers better than multienzyme RT PCR systems, with the added benefit and cycle of reduced hands-on time and downstream error. Extreme thermostability expedites sample preparation by allowing Compatible with end point (gel- complete denaturation to 94°C prior to reverse transcription and cDNA synthesis at 70°C, which improves analysis of highly based) or real-time analysis structured RNA templates. PyroPhage RT has extremely high PCR fidelity, equivalent to the most accurate PCR enzymes, raising the prospect of highly accurate transcriptome analysis with RT-PCR with PyroPhage RT PyroPhage RT vs. Tth Polymerase reduced amplification bias and strand switching as compared to Single-enzyme, one-step RT-PCR of a 160 bp amplicon using the alternatives. Additionally, PyroPhage RT was identified as part a 102 to 108-fold dilution series of MS2 RNA. of a viral replication complex that includes thermostable helicase a. a. Real-time and post-reaction melt data and primase subunits. These accessory proteins are now being b. Corresponding Agarose gel data. Tth polymerase used with Mn2+ as directed (Epicentre). expressed and studied as adjuncts to further improve PyroPhage Arrows show correct melt Tm (top) and amplicon (bottom). RT performance. Details on usage of the PyroPhage RT enzyme in both RT PCR and transcriptome analysis are provided. Thermophilic Phage Metagenomics b. T T T R R R er er d d e e e d d g g g a a a a a l l T T p V V V p oph R oph R L oph RT L L b r r r 0 b y y y No 10 No MM P No MM P MM P 100 Viral Metagenomics 144 bp 246 bp 298 bp β-actin β2-µglobulin Cyclophilin Thermophilic phage (electron micrographs, right) were isolated from the Hot Springs of Yellowstone National Park Linearity of RT-PCR PyroPhage RT vs. MMLV RT (left) and used to construct metagenomic libraries that were Triplicate single-enzyme RT-PCR amplifications of a 103 to screened to identify novel DNA polymerases. 108-fold dilution series of the RNA target. Amplification was 2-step RT-PCR human mRNA Applied Environmental Microbiology 74(13):4164-4174. 2008. detected by EvaGreen fluorescence. Total human Liver RNA (1 µg) was reverse transcribed by PyroScript RT-PCR 2X Master Mix, RNA Control and Control Moloney Murine Leukemia Virus or by PyroPhage RT, then Primer Set were used for the analysis. PCR amplified using Lucigen EconoTaq® PLUS Master Mix. a. Real-time and post-reaction melt data. Two-step cycling Shown are targets of 144, 246 and 298 bp. High Fidelity PCR with PyroPhage RT conditions were used. b. Linear regression of Cycle threshold (Ct) vs. log10 Dilution Factor. Polymerase Fidelity 9.0E+04 5'-3' exonuclease - CONCLUSIONS 8.0E+04 3'-5' exonuclease aStrong Strand displacement Strong 7.0E+04 Extension from nicks Strong 6.0E+04 Thermostability (T½ @95°C) 10 min. a. b. Km dNTPs 40 μM 5.0E+04 • Thermostable PyroPhage RT directly Km DNA 5.3 nM 4.0E+04 Processivity 47 nt Fidelity 7 X 104 3.0E+04 detects and RT-PCR amplifies viral RNA Template DNA / RNA 2.0E+04 and human transcripts. aThe 3’-5 exonuclease domain has been disabled in Pyrophage RT 1.0E+04 0.0E+00 • Effective for quantitative real-time and PhuPhussioionn PfuPfUlu tUralt r a EcoEncoTonoaqTaqPyroPhPyragoPe HhiaFgi ReT PyroPhPhaaggeeR RTT conventional RT-PCR analyses. er d • PyroPhage RT is effective in RT-LAMP. Accessory Proteins d a p p L p p p bp bp b bp bp b b b 3 4 4 7 9 0 bp 3 8 2 0b 0 9 • PyroScript RT-PCR 2X Master Mix is a 24 29 12 16 21 21 36 8 1 robust and convenient RT-PCR solution. Single Enzyme RT-PCR a.) Human 18S rRNA sequences were amplified from 100 pg total A549 cell line RNA. Seven primer sets targeting amplicons from 51 to 65% GC content and from 87 to 393 bp in length were tested. The PyroPhage RT gene is part of a 7.3 kb contig assembled from the Acknowledgements viral metagenome. This contig contains an apparent operon that b.) Viral RNA (Enterobacteriophage MS2, ATCC 15597-B1) This work was supported by NSF and NIH NIAID grants. includes the polA-like replicase (PyroPhage RT) and four apparent was amplified by 40 cycles of RT-PCR without background. accessory proteins that are being developed to improve RT PCR and Products from 89 to 362 bp in length were amplified. Sequencing of viral metagenomes was performed by JGI. transcriptome and RNA virome library preparation. • One-step single-enzyme RT-PCR cycling conditions: 15 sec @ 94˚C, (10s @ 94˚C, 30s @ 72˚C)*40.
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