Name: pyrosequencing for microbial identification and characterization
Uploaded: 2013-08-22T12:00:00
Duration: 12 min 37 s
Description: Watch this Scientific Journal Video about Pyrosequencing for Microbial Identification and Characterization at JoVE.com

This project was support in part by Johns Hopkins University, Office of the Provost through the Gateway Science Initiative and Qiagen Inc.

Introduction: A single bacterial colony should be used to inoculate an appropriate culture broth that is incubated overnight. A bacterial pellet is then processed to purify genomic DNA using a commercially available genomic isolation kit. The purified genomic DNA should have a 260/280 (nm) ratio &gt;1.8 to assure that the sample is free of protein contamination. The amount of genomic DNA required for generation of the pyrosequencing template is 10-20 ng.
A. Amplification of the Template
(PyroMark PCR Handbook; <a href="http://www.qiagen.com/handbooks" target="_blank">www.qiagen.com/handbooks</a>1)
Setting up the PCR reaction
Note: One primer must be biotinylated at its 5' end, and it's recommended that it be HPLC purified for template preparation. We recommend the PyroMark Assay Design Software 2.0 for PCR and sequence primer design that are optimized for short-read sequencing, however, Primer-BLAST (<a href="http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome" target="_blank">http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome</a>) from NCBI can also be used for primer design.
<ol>
 <li>Thaw all required solutions (listed in Table 1) and mix each thoroughly. Set up the reaction according to Table 1. All work can be done at room temperature.</li>
</ol>
Note: The PCR Master Mix contains MgCl2 for a final concentration of 1.5 mM, which provides satisfactory results in most cases. If a higher Mg2+ concentration is required, up to 3.5 &mu;l of 25 mM MgCl2 can be added to a reaction.
<ol start="2">
 <li>Thoroughly mix the reaction mix by gently pipetting up and down, then dispense appropriate volumes into PCR tubes.</li>
 <li>Add template DNA to each PCR tube. We recommend 10 ng of genomic DNA.</li>
 <li>Program the thermal cycler according to Table 2. A final hold at 4 &deg;C is recommended for convenience. If using a thermal cycler without a heated lid, overlay reactions with 100 &mu;l mineral oil.</li>
 <li>Place the PCR tubes in the cycler and start the cycling program.</li>
</ol>
B. Vacuum Workstation Protocol
Immobilizing the Biotinylated PCR products
<ol>
 <li>Make a master mix according to Figure 4.</li>
</ol>
Note: Before pipetting, gently shake the bottle of streptavidin-coated Sepharose beads to ensure a homogenous suspension.
<ol start="2">
 <li>Depending on the volume of PCR product used, dispense 60-75 &mu;l master mix into each well of a PCR plate to give a total volume of 80 &mu;l per well.</li>
 <li>Add 5-20 &mu;l biotinylated PCR product to each well.</li>
 <li>Seal the wells with strip caps and agitate the PCR plate at 1,400 rpm for 5-10 min at room temperature (15-25 &deg;C) using an orbital shaker.</li>
</ol>
Denaturation of DNA and addition of sequencing primer
<ol>
 <li>Dilute the sequencing primers to 0.3 &mu;M with annealing buffer and dispense 25 &mu;l into each well of a reaction plate. Position the plate on the workstation.</li>
 <li>Fill the workstation troughs according to the Figure 5 diagram.</li>
 <li>Turn on the vacuum pump, and make sure the switch on the vacuum tool is set to on (Figure 6). Flush the filter probes with high-purity water (Milli-Q 18.2 MW x cm or equivalent) in trough 5 (Figure 5). Refill the trough with fresh high-purity water for use in step 12.</li>
 <li>Working quickly, position the PCR plate with beads on the workstation. Ensure that both plates are in the same orientation as when the samples were loaded.</li>
 <li>With the vacuum switch ON, lower the vacuum tool into the wells of the PCR plate for 20 sec or until all volume has been aspirated. The beads will be captured by the vacuum tool (Figure 7).</li>
 <li>With the vacuum still ON, flush the tool with 70 % ethanol (trough 1) for 20 sec or until all volume has been aspirated.</li>
 <li>With vacuum ON, flush the tool with denaturation solution (trough 2) for 20 sec or until all volume has been aspirated.</li>
</ol>
Note: The Denaturation Solution contains sodium hydroxide, which is an eye and skin irritant. Always wear a lab coat, gloves, and protective goggles.
<ol start="8">
 <li>With vacuum ON, flush the tool with wash buffer (trough 3) for 20 sec or until all volume has been aspirated.</li>
 <li>With vacuum ON, raise the vacuum tool to beyond 90&deg; vertical for 5 sec, to allow all liquid to drain out of the vacuum tool. Turn the pump OFF, and align the vacuum tool with the reaction plate. Lower the vacuum tool into the wells and gently shake from side to side to release the beads into the wells containing sequencing primer.</li>
 <li>In order to clean the vacuum tool, agitate the filter probes in high-purity water (trough 4) for 10 sec.</li>
 <li>Switch the vacuum ON and flush the tool with high-purity water (trough 5) for 20 sec or until all volume has been aspirated.</li>
 <li>Raise the vacuum tool to beyond 90&deg;C vertical for 5 sec, then switch the vacuum OFF and store in the "Parking" position.</li>
</ol>
Annealing sequencing primer to DNA strands
<ol>
 <li>Place the reaction plate in a pre-warmed plate holder.</li>
 <li>Heat the plate on a heating block at 80 &deg;C for 2 min.</li>
 <li>Remove the plate from the holder and place on the counter to cool at room temperature for 5 min.</li>
</ol>
Pyromark Q24 Operation
Note: Switch on the instrument using the power switch located above the power cord. Startup can take up to 1 min.
<ol>
 <li>Fill a cartridge according to the instructions provided in the PyroMark Gold Q24 Reagents Handbook 2. To determine volumes needed, set up a run file in the instrument software, and under the Tools menu select Pre Run Information</li>
 <li>If the instrument was already on, ensure that it is not performing a run and then open the lid. An audible warning signal will be emitted if the lid is opened when it is unsafe to do so.</li>
 <li>Open the cartridge gate and insert the cartridge with the label facing forward (Figure 8). Ensure that the cartridge is properly inserted (a line should be visible at the front) and close the gate.</li>
 <li>Open the plate-holding frame and position the plate with samples inside the instrument.</li>
 <li>Close the plate-holding frame and the instrument lid (Figure 9).</li>
</ol>
Starting a run
<ol>
 <li>Insert the USB stick with the run files created with the instrument software into the USB port at the front of the instrument.</li>
 <li>Select "Run" in the main menu and press "OK".</li>
 <li>Use the scroll arrows to select the desired run file and press "Select".</li>
</ol>
Note: The instrument will begin dispensing reagents when all preset pressure and temperature levels are reached (this may take several minutes). During a run, the instrument screen displays the pyrogram of the selected well in real-time. Use the scroll arrows to view the pyrogram of other wells.
Completing a run
<ol>
 <li>When the instrument confirms that the run is finished and the run file has been saved to the USB stick, press "Close".</li>
 <li>Remove the USB stick. If it was removed before the run finished, insert the USB stick. Select "Administration" and then "Copy Unsaved Runs".</li>
 <li>The run file can now be opened and analyzed using the instrument software and compared to a library of known microbes using the Identifire software.</li>
</ol>

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PyroMark PCR Handbook. , (2009).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PyroMark Q24 User Manual. , (2012).</li><li>Elahi, E., Ronaghi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrosequencing:+a+tool+for+DNA+sequencing+analysis.">Pyrosequencing: a tool for DNA sequencing analysis.</a> Methods Mol. Bio. 255, 211-219 (2004).</li><li>Fakhrai-Rad, H., Pourmand, N., Ronaghi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrosequencing:+and+accurate+detection+platform+for+single+nucleotide+polymorphisms.">Pyrosequencing: and accurate detection platform for single nucleotide polymorphisms.</a> Hum. Mutat. 19 (5), 479-485 (2002).</li><li>Langaee, T., Ronaghi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variation+analyses+by+pyrosequencing.">Genetic variation analyses by pyrosequencing.</a> Mutat. Res. 573 (1-2), 96-102 (2005).</li><li>Liu, Z., Lozupone, C., Hamady, M., Bushman, F. D., Knight, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+pyrosequencing+reads+suffice+for+accurate+microbial+community+analysis.">Short pyrosequencing reads suffice for accurate microbial community analysis.</a> Nucleic Acids Res. 35 (18), e120 (2007).</li><li>Novais, R. C., Thorstenson, Y. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+Pyrosequencing+for+microbiology:+From+genes+to+genomes.">The evolution of Pyrosequencing for microbiology: From genes to genomes.</a> J. Microbiol. Methods. 86 (1), 1-7 (2011).</li><li>J, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenomic+pyrosequencing+and+microbial+identification.">Metagenomic pyrosequencing and microbial identification.</a> Clin. Chem. 55 (5), 856-866 (2009).</li><li>Quince, C., Lanzen, A., Curtis, T. P., Davenport, R. J., Hall, N., Head, I. M., Read, L. F., Sloan, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+determination+of+microbial+diversity+from+454+pyrosequencing+data.">Accurate determination of microbial diversity from 454 pyrosequencing data.</a> Nat. Methods. (9), 639-6341 (2009).</li><li>Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M., Nyren, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+DNA+sequencing+using+detection+of+pyrophosphate+release.">Real-time DNA sequencing using detection of pyrophosphate release.</a> Anal. Biochem. 242, 84-89 (1996).</li><li>King, C., Scott-Horton, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrosequencing:+A+simple+method+for+accurate+genotyping.">Pyrosequencing: A simple method for accurate genotyping.</a> J. Vis. Exp. (11), e630 (2008).</li><li>Rothberg, J. W., Heinz, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+semi-conductor+device+that+enabling+non-optical+genome+sequencing.">An integrated semi-conductor device that enabling non-optical genome sequencing.</a> Nature. 475, 348-352 (2011).</li><li>Levene, M. J., Korlach, J., Turner, S. W., Foquet, M., Craighead, H. G., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zero-Mode+Waveguides+for+Single-Molecule+Analysis+at+high+concentrations.">Zero-Mode Waveguides for Single-Molecule Analysis at high concentrations.</a> Science. 299, 682-686 (2003).</li><li>Eid, J., Fehr, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-Time+DNA+Sequencing+from+Single+Polymerase+Molecules.">Real-Time DNA Sequencing from Single Polymerase Molecules.</a> Science. 323, 133-138 (2009).</li></ol>

Production and Free Access to this article is sponsored by Qiagen. Authors Ahmed, Durocher, Jessen and Vardi are employees of Qiagen Inc. that produces reagents and instruments used in this article.

Several new next generation sequencing methods have been commercialized. Three of the most widely used methods are ion semiconductor sequencing, single molecule real time sequencing, and sequencing by synthesis (SBS).3-11 Each of these methods depends on sequencing by synthesis but employ novel platforms for detection of the incorporated nucleotides. In ion semiconductor sequencing, a single strand of DNA serves as a template for the sequencing strand. 12 Polymerase and nucleotides are added sequentially as in pyrosequencing. When a nucleotide is added to the growing DNA strand, a hydrogen ion is released. The hydrogen ion is detected by a field effect transistor based sensor.
Single molecule real time sequencing depends on the zero mode wave guide (ZMW).13 The ZMW is a small structure where a single polymerase molecule is attached to the bottom of the well. It is illuminated in such a way that a florescent molecule can be detected. Each nucleotide is tagged with a fluorescent molecule. As a fluorescently tagged nucleotide is incorporated, a detector identifies the nucleotide. When the next nucleotide is added, the florescent molecule is cleaved.14 
Sequencing by synthesis (SBS) uses a unique method to amplify the target DNA such that clusters of unique sequences are generated.15 Single stranded templates are generated and then the complementary strand is synthesized. Each nucleotide is labeled with a fluorescent molecule and after each base addition the fluorescence of the added base is recorded.

Pyrosequencing is a rapid and accurate method to sequence nucleic acids that is based on the principle of "sequencing by synthesis". "Sequencing by synthesis" involves using a single strand DNA template to synthesize the complementary strand one base at a time, and detecting the incorporated base (A, T, G, or C) at each step by detection of a chemiluminescent signal. The pyrosequencing reaction includes template DNA, dNTPs (dATP, dGTP, dTTP, dCTP), DNA polymerase, ATP sulfurylase, luciferin, luciferase, and apyrase. The synthesis reaction is initiated by the addition of one of the four dNTPs and DNA polymerase to the single stranded biotinylated template DNA. DNA polymerase incorporates the complementary dNTP onto the template, with the subsequent release of pyrophosphate (Figure 1). ATP sulfurylase proportionately converts the pyrophosphate to ATP. ATP acts as a catalyst for the luciferase-mediated conversion of luciferin to oxyluciferin, which generates light (Figure 2). The intensity of light is proportional to the number of nucleotides incorporated and determines if one or more specific dNTP's (dATP, dTTP, dGTP, or dCTP) is present on the template strand sequentially (Figure 3). Any unincorporated dNTP is degraded by apyrase prior to addition of the next dNTP for continuation of the synthesis reaction. This "sequencing by synthesis" reaction is repeated with addition of each of the four dNTPs until the DNA sequence of the single stranded DNA template is determined.
To view an animation of the pyrosequencing reaction <a href="http://www.jove.com/files/ftp_upload/50405/50405movie1.mov" target="_blank">Click here to view movie</a>.

<table border=1>
<tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>PyroMark PCR Master Mix, 2x</td>
 <td>Qiagen</td>
 <td>978703</td>
 <td></td>
 </tr>
 <tr>
 <td>CoralLoad Concentrate, 10x</td>
 <td>Qiagen</td>
 <td>978703, 978705</td>
 <td></td>
 </tr>
 <tr>
 <td>Q-Solution, 5x</td>
 <td>Qiagen</td>
 <td>978703, 978705</td>
 <td></td>
 </tr>
 <tr>
 <td>MgCl2, 25 mM</td>
 <td>Qiagen</td>
 <td>978703, 978705</td>
 <td></td>
 </tr>
 <tr>
 <td>RNase-Free Water</td>
 <td>Qiagen</td>
 <td>129112</td>
 <td></td>
 </tr>
 <tr>
 <td>Primers</td>
 <td>Qiagen</td>
 <td>978776 or 978777</td>
 <td>Primers should be purchased from an established oligonucleotide manufacturer (i.e. QIAGEN Pyromark Custom Assays, IDT, etc). Lyophilized primers should be dissolved in TE to provide a stock solution of 100 &mu;M.</td>
 </tr>
 <tr>
 <td>Pyromark Gold Q24 Reagents</td>
 <td>Qiagen</td>
 <td>970802</td>
 <td></td>
 </tr>
 <tr>
 <td>High-purity water (Milli-Q 18.2 M&Omega; x cm or equivalent)</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Streptavidin Sepharose High Performance Beads</td>
 <td>GE Healthcare</td>
 <td>17-5113-07</td>
 <td></td>
 </tr>
 <tr>
 <td>PyroMark Annealing Buffer</td>
 <td>Qiagen</td>
 <td>979009</td>
 <td></td>
 </tr>
 <tr>
 <td>PyroMark Denaturation Solution</td>
 <td>Qiagen</td>
 <td>979007</td>
 <td></td>
 </tr>
 <tr>
 <td>PyroMark Wash Buffer</td>
 <td>Qiagen</td>
 <td>979008</td>
 <td></td>
 </tr>
 <tr>
 <td>PyroMark Q24</td>
 <td>Qiagen</td>
 <td>9001514</td>
 <td></td>
 </tr>
 <tr>
 <td>PyroMark Q24 Cartridge</td>
 <td>Qiagen</td>
 <td>979202</td>
 <td></td>
 </tr>
 <tr>
 <td>PyroMark Q96 HS Tip Holder Box</td>
 <td>Qiagen</td>
 <td>979105</td>
 <td></td>
 </tr>
 <tr>
 <td>PyroMark Q24 Vacuum Workstation</td>
 <td>Qiagen</td>
 <td>9001516</td>
 <td></td>
 </tr>
 <tr>
 <td>PyroMark Q24 Plate Holder</td>
 <td>Qiagen</td>
 <td>9022273</td>
 <td></td>
 </tr>
 <tr>
 <td>Orbital shaker</td>
 <td>Fisher</td>
 <td>11-675-297</td>
 <td></td>
 </tr>
 <tr>
 <td>Heat block</td>
 <td>Fisher</td>
 <td>11-718Q</td>
 <td></td>
 </tr>
 </tbody>
</table>

pyrosequencing for microbial identification and characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

The results of a typical pyrosequencing run using the software shows the location of the forward and reverse primers used for amplicon generation, and the sequencing primer used for the pyrosequencing reaction within the conserved 16S ribosomal sequence (Figure 10).The hypervariable sequence between the primer sites allows for bacterial identification of a large number of bacterial species using the conserved sequencing primer (5'-TACATGCAAGTCGA).As an internal quality control, the DNA sequence bracketing the variable region can be checked to assure that the correct DNA region has been analyzed. The pyrosequencing software allows for comparison and alignment of the generated sequence to an internal database of bacterial ribosomal sequences for bacterial identification. In addition, a sequence can be analyzed to determine mutations that confer antibiotic drug resistance. For example, analysis of mutations in the 23S ribosomal genes of Helicobacter pylori demonstrates two mutation patterns (GAA or AGA) that confer antibiotic resistance (Figure 11). Analysis of multiple isolates from the same patient can be concurrently assayed to track the emergence of drug resistance over time to aid in epidemiological investigations of microbial drug resistance outbreaks.
<img alt="Figure 1" src="/files/ftp_upload/50405/50405fig1.jpg" /> 
Figure 1. Biotinylated PCR product is used as a template to incorporate dNTPs by DNA polymerase, leading to the generation of pyrophosphate (PPi).
<img alt="Figure 2" src="/files/ftp_upload/50405/50405fig2.jpg" /> 
Figure 2. ATP sulfurylase proportionately converts pyrophosphate to ATP. ATP acts as a catalyst for the luciferase-mediated conversion of luciferin to oxyluciferin, which generates light that is proportional to the amount of ATP. The light is recorded as peak on the pyrogram trace and indicates nucleotide incorporation. The unincorporated dNTPs are degraded by apyrase before the next dNTP is added for continuation of the synthesis.
<img alt="Figure 3" src="/files/ftp_upload/50405/50405fig3.jpg" /> 
Figure 3. The intensity of light generated indicates if one or more specific dNTP's (dATP, dTTP, dGTP, or dCTP) was incorporated onto the template strand sequentially.
<img alt="Figure 4" src="/files/ftp_upload/50405/50405fig4.jpg" /> 
Figure 4. Flow chart for preparation of master mix to immobilize the biotinylated PCR product.
<img alt="Figure 5" src="/files/ftp_upload/50405/50405fig5.jpg" />
Figure 5. PyroMark workstation, with PCR plate, PyroMark plate, and trough locations.
<img alt="Figure 6" src="/files/ftp_upload/50405/50405fig6.jpg" /> 
Figure 6. Locations of the Vacuum switch ON and OFF positions.
<img alt="Figure 7" src="/files/ftp_upload/50405/50405fig7.jpg" /> 
Figure 7. Vacuum tool. Proper handling of the vacuum tool.
<img alt="Figure 8" src="/files/ftp_upload/50405/50405fig8.jpg" /> 
Figure 8. Cartridge gate opened with cartridge in place.
<img alt="Figure 9" src="/files/ftp_upload/50405/50405fig9.jpg" /> 
Figure 9. Cartridge properly inserted with gate closed.
<img alt="Figure 10" src="/files/ftp_upload/50405/50405fig10.jpg" /> 
Figure 10. Pyrosequencing based bacterial identification results. PCR primers are designed for conserved regions of the DNA template, and the sequencing primer is positioned immediately upstream of a well-characterized identifying hypervariable DNA sequence within the amplicon (shown in blue).
<img alt="Figure 11" src="/files/ftp_upload/50405/50405fig11.jpg"/> 
Figure 11. Detection of antibiotic drug resistance in Helicobacter pylori using pyrosequencing. Analysis of mutations in the 23S genes that confer antibacterial resistance in Helicobacter pylori containing GAA or AGA sequences. <a href="https://www.jove.com/files/ftp_upload/50405/50405fig11large.jpg" target="_blank">Click here to view larger figure</a>.
<table border="1" fo:keep-together.within-page="always">
 <tbody>
 <tr>
 <td>Component</td>
 <td>Volume per reaction</td>
 <td>Final concentration</td>
 </tr>
 <tr>
 <td>Pyro PCR Master Mix, 2x</td>
 <td>12.5 &mu;l</td>
 <td>1x</td>
 </tr>
 <tr>
 <td>CoralLoad Concentrate, 10x</td>
 <td>2.5 &mu;l</td>
 <td>1x</td>
 </tr>
 <tr>
 <td>25 mM MgCl2 (optional)</td>
 <td>Variable</td>
 <td>&ge;1.5 mM</td>
 </tr>
 <tr>
 <td>Q-Solution, 5x (optional)</td>
 <td>5 &mu;l</td>
 <td>1x</td>
 </tr>
 <tr>
 <td>Primer A/Primer B</td>
 <td>Variable/Variable</td>
 <td>0.2 &mu;M/0.2 &mu;M</td>
 </tr>
 <tr>
 <td>RNase-free water</td>
 <td>Variable</td>
 <td>-</td>
 </tr>
 <tr>
 <td>Total Volume (after adding template DNA)</td>
 <td>25 &mu;l</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
Table 1. PCR Reaction Mixture.
<table border="1" fo:keep-together.within-page="always"> 
 <tbody>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Additional comments</td>
 </tr>
 <tr>
 <td>Initial PCR activation step</td>
 <td>15 min</td>
 <td>95 &deg;C</td>
 <td>HotStartTaq DNA Polymerase is activated</td>
 </tr>
 <tr>
 <td>3 step cycling: Denaturation</td>
 <td>30 sec</td>
 <td>94 &deg;C</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Annealing</td>
 <td>30 sec</td>
 <td>60 &deg;C 
 56 &deg;C</td>
 <td>For genomic DNA 
 For bisulfite converted DNA</td>
 </tr>
 <tr>
 <td>Extension</td>
 <td>30 sec</td>
 <td>72 &deg;C</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Number of cycles</td>
 <td>45</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Final extension</td>
 <td>10 min</td>
 <td>72 &deg;C</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
Table 2. PCR Cycle Specifications.

Watch this Scientific Journal Video about Pyrosequencing for Microbial Identification and Characterization at JoVE.com

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

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Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in understanding the gene regulation and spatiotemporal expression patterns of target genes. On the other hand, it is also critical to establish promoter evaluation tools and genetic transformation techniques that are fast, efficient, and reproducible. In this study, we investigated the spatiotemporal expression pattern of the rhizobial symbiosis-specific nodule inception (NIN) promoter of Phaseolus vulgaris in the transgenic hairy roots. Using plant genome databases and analysis tools we identified, isolated, and cloned the P. vulgaris NIN promoter in a transcriptional fusion to the chimeric reporter &#946;-glucuronidase (GUS) GUS-enhanced::GFP. Further, this protocol describes a rapid and versatile system of genetic transformation in the P. vulgaris using Agrobacterium rhizogenes induced hairy roots. This system generates &#8805;2 cm hairy roots at 10 to 12 days after transformation. Next, we assessed the spatiotemporal expression of NIN promoter in Rhizobium inoculated hairy roots at periodic intervals of post-inoculation. Our results depicted by GUS activity show that the NIN promoter was active during the process of nodulation. Together, the present protocol demonstrates how to identify, isolate, clone, and characterize a plant promoter in the common bean hairy roots. Moreover, this protocol is easy to use in non-specialized laboratories.

Promoter expression analyses are crucial to improving the understanding of gene regulation and the spatiotemporal expression of target genes. Herein we present a protocol to identify, isolate, and clone a plant promoter. Further, we describe the characterization of the nodule-specific promoter in the common bean hairy roots.

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in ...

Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. Deviations from what is considered a healthy microbiota composition (dysbiosis) may impair vital functions leading to pathologic conditions. Recent and ongoing research efforts have been directed toward the characterization of associations between microbial composition and human health and disease.
Advances in high-throughput sequencing technologies enable characterization of the gut microbial composition. These methods include 16S rRNA-amplicon sequencing and shotgun sequencing. 16S rRNA-amplicon sequencing is used to profile taxonomical composition, while shotgun sequencing provides additional information about gene predictions and functional annotation. An advantage in using a targeted sequencing method of the 16S rRNA gene variable region is its substantially lower cost compared to shotgun sequencing. Sequence differences in the 16S rRNA gene are used as a microbial fingerprint to identify and quantify different taxa within an individual sample.
Major international efforts have enlisted standards for 16S rRNA-amplicon sequencing. However, several studies report a common source of variation caused by batch effect. To minimize this effect, uniformed protocols for sample collection, processing, and sequencing must be implemented. This protocol proposes the integration of broadly used protocols starting from fecal sample collection to data analyses. This protocol includes a column-free, direct-PCR approach that enables simultaneous handling and DNA extraction of large numbers of fecal samples, along with PCR amplification of the V4 region. In addition, the protocol describes the analysis pipeline and provides a script using the latest version of QIIME (QIIME 2 version 2017.7.0 and DADA2). This step-by-step protocol is aimed to guide those interested in initiating the use of 16S rRNA-amplicon sequencing in a robust, reproductive, easy to use, detailed way.

This manuscript describes a detailed standardized protocol of high-throughput 16S rRNA-amplicon sequencing. The protocol introduces an integrated, uniformed, feasible, and inexpensive protocol starting from fecal sample collection through data analyses. This protocol enables analysis of large numbers of samples with rigorous standards and several controls.

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. ...

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors FAPs and Myogenic Progenitors MPs in Skeletal Muscle in the Rat

This protocol outlines a method to isolate Fibro-adipogenic progenitors (FAPs) and myogenic progenitors (MPs) from rat skeletal muscle. Utilization of the rat in muscle injury models provides increased tissue availability from atrophic muscle for the analysis and a larger repertoire of validated methods to assess muscle strength and gait in free-moving animals.

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in ...

In Silico Identification and Characterization of circRNAs During Host-Pathogen Interactions

Circular RNAs (circRNAs) are a class of non-coding RNAs that are formed via back-splicing. These circRNAs are predominantly studied for their roles as regulators of various biological processes. Notably, emerging evidence demonstrates that host circRNAs can be differentially expressed (DE) upon infection with&#160;pathogens (e.g., influenza and coronaviruses), suggesting a role for circRNAs in regulating host innate immune responses. However, investigations on the role of circRNAs during pathogenic infections are limited by the knowledge and skills required to carry out the necessary bioinformatic analysis to identify DE circRNAs from RNA sequencing (RNA-seq) data. Bioinformatics prediction and identification of circRNAs is crucial before any verification, and functional studies using costly and time-consuming wet-lab techniques. To solve this issue, a step-by-step protocol of in silico prediction and characterization of circRNAs using RNA-seq data is provided in this manuscript. The protocol can be divided into four steps: 1) Prediction and quantification of DE circRNAs via the CIRIquant pipeline; 2) Annotation via circBase and characterization of DE circRNAs; 3) CircRNA-miRNA interaction prediction through Circr pipeline; 4) functional enrichment analysis of circRNA parental genes using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). This pipeline will be useful in driving future in vitro and in vivo research to further unravel the role of circRNAs in host-pathogen interactions.

In Silico Identification and Characterization of circRNAs During Host-Pathogen Interactions

The protocol submitted here explains the complete in silico pipeline needed to predict and functionally characterize circRNAs from RNA-sequencing transcriptome data studying host-pathogen interactions.

Circular RNAs (circRNAs) are a class of non-coding RNAs that are formed via back-splicing. These circRNAs are predominantly studied for their roles as ...