Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme

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

Identification of genomic differences between Escherichia Colistrains pathogenic for poultry and E. Coli K-12 MG1655

Diseases of poultry caused by Escherichia coli result in significant economic losses every year. Specific virulence factors associated with E. coli strains pathogenic for poultry have been identified, but it is likely that others remain to be identified. The objective of this project was to identify novel virulence factors of avian E. coli. The signature-tagged mutagenesis technique was to be used for this purpose. Difficulties were encountered using that technique, and it was decided that suppression subtractive hybridization should be used to identify genomic differences between E. coli K-12 and avian E. coli pathogenic for poultry. A total of 62 fragments specific to two avian strains were identified. They were composed of sequences with homology to four types of fragments: plasmid sequences, phage sequences, sequences with known function and sequences without any currently known function. Two E. coli collections, a reference collection of diverse strains (ECOR) and a collection of 41 avian isolates, were screened for the presence of 25 of the 62 fragments. Eleven fragments were present in significantly more of the avian strains than of the ECOR strains. Some of the fragments were further characterized by sequencing the DNA flanking the fragments, and analyzing the sequences for the putative genes. One of the fragments was disrupted in the genome of an avian E. coli strain. The disruption mutant did not have reduced virulence in two animal models tested, which indicates that the disrupted gene may not be required for virulence. The role of the other fragment sequences in the virulence of avian E. coli has not yet been determined

Polymerase assay for detection of expression clones containing thermostable Pol activity from a boiling hot spring metagenomic library.

<p>Clones judged by sequence to encode complete <i>pol</i> genes were cultivated and thermostable proteins extracted as described in the Methods. Extension of a 37 nucleotide (nt) ROX-labeled primer <b>(peak 1)</b> on a 41 nt template oligonucleotide by a polymerase results in a shift from 37 to 41 nt <b>(peak 2)</b>. If a single nucleotide non-templated extension occurs as seen with Taq Pol, a peak at 42 nt results <b>(peak 3)</b>. Degradation of the ROX-labeled substrate by 3′ to 5′ exonuclease activity results in peaks of less than 37 nt <b>(peak 4)</b>. nt: size of standard markers in nucleotides.</p

Two step RT-PCR comparing 3173 Pol and MMLV RT.

<p><b>A.</b> MS2 viral RNA and <b>B., C.</b> total human liver RNA were reverse transcribed using either 3173 Pol or MMLV RT and then PCR amplified using Taq Polymerase. <b>Target amplicons</b>: <b>A.</b> MS2 RNA phage 77 bp amplicon, 2% gel, <b>B.</b> Human beta-actin 144 bp amplicon, 2% gel, <b>C.</b> Human beta-actin 821 bp amplicon, 1% gel. <b>Lanes</b>: <b>1</b>,<b>11</b>: oligo dT primer; <b>2–4</b>,<b>12–14</b>: Gene specific primers; <b>5</b>,<b>15</b>: random hexamers; <b>6</b>,<b>16</b>: random nonamers; <b>7</b>,<b>17</b>: No primer plus RT; <b>8</b>: No RT enzyme; <b>9</b>: PCR No Target Control; <b>10</b>: Molecular Weight Marker (MW), 100 bp (50 bp lowest) for Panels A, B and 1000 bp (300, 500, 700 lowest) for Panel C. Correct PCR product size indicated by black triangle.</p

RT-PCR conditions.

a<p>Not including thermal melt. NA is not applicable.</p

Biochemical characterization of 3173 Pol.

<p>The thermal profile of the 3173 Pol was determined by assay at the indicated temperatures. Activity relative to maximal (77°C) is shown.</p

RT-PCR detection of human transcript RNAs.

<p>Beta-actin, beta2-microglobulin and cyclophilin target sequences of the indicated sizes were amplified from human liver total RNA using the primers described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038371#pone-0038371-t001" target="_blank">Table 1</a>. 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.</p

Comparison of 3173 Pol (PyroScript) RT-PCR mix with two enzyme RT-PCR systems in detection of MS2 and influenza A.

<p>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. <b>A.</b> 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>B.</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.</p

Primers and Other Oligonucleotides.

*<p>ROX = Carboxy-X-rhodamine.</p>#<p>CF560 = CalFluor 560.</p

Single-enzyme, one step RT-PCR amplification of MS2 phage RNA using 3173 Pol.

<p>MS2 RNA was amplified by 40 cycles of RT-PCR using the primers shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038371#pone-0038371-t001" target="_blank">Table 1</a> and 3173 Pol. <b>A.</b> Products from 89 to 362 bp in length were amplified using one-step single-enzyme RT-PCR cycling conditions: 15 sec @ 94°C, (10 s @ 94°C, 30 s @ 72°C)*40. Products were resolved by 2% agarose gel electrophoresis. <b>B.</b> The MS2 RNA was diluted from 10¹ to 10⁷-fold and amplified using a primer pair corresponding to the 160 bp fragment in Panel A. Real-time PCR fluorescence in RFU (relative fluorescence units) vs. PCR cycles. <b>C.</b> Post-amplification thermal melt in -dRFU/dTemperature vs. Temperature (°C). Light blue region indicates melt curves for specific products. <b>D.</b> Standard curve PCR Cycle threshold vs. log₁₀ RNA copy number in triplicate with linear least squares best fit line.</p