Diese Arbeit wurde vom Office of Science (BER), US Department of Energy, Cooperative Agreement nicht unterstützt. De-FC02-02ER63453, die National Science Foundation Mikrobielle Genome Sequencing Program (Auszeichnung Zahlen 0626826 und 0731916). Wir danken John Glass für seine technische Kompetenz und Beratung und K. Eric Wommack für seine Unterstützung bei der Umwelt-Probenentnahme.

1. Herstellung von Lösungen Vor der Durchführung Hydroxyapatit-Chromatographie, muss Phosphatpuffer hergestellt werden und die Hydroxyapatit muss richtig hydratisiert. <ol><li> 1M Phosphat-Lösung, pH 6,8: In einem 1L Flasche auflösen 119.98g Natriumphosphat einbasigen in 1L steril, DEPC-behandeltem H 2 O. Bereiten Sie eine 1M Natriumdihydrogenphosphat-Lösung in einem 1L Flasche durch Auflösen von 141.96g von Natriumdihydrogenphosphat in 1L steril, DEPC-behandeltem H 2 O. Kombinieren Sie die Mono-und Di-Lösungen mit einem Verhältnis von 1:1. Küvette auf einer Rührplatte und mischen mit einem magnetischen Rührstab. Der pH-Wert auf 6,8 durch Zugabe von Natronlauge (zur pH-Erhöhung) oder Phosphorsäure (zur pH-Abnahme). </li><li> 0,12 M Phosphatpuffer: In einem 500 ml-Kolben, kombinieren 30ml 1M Phosphat-Lösung, pH 6,8, 2,5 ml 10% Natriumdodecylsulfat (SDS) und 5 ml 0,5 M EDTA. Add sterile, DEPC-behandeltem H2O auf ein Volumen von 225ml. Der pH-Wert auf 6,8. Add sterile, DEPC-behandeltem H2O auf 250ml. Autoclave. </li><li> 0,18 M Phosphatpuffer: In einem 500 ml-Kolben, kombinieren 45ml 1M Phosphat-Lösung, pH 6,8, 2,5 ml 10% Natriumdodecylsulfat (SDS) und 5 ml 0,5 M EDTA. Add sterile, DEPC-behandeltem H 2 O auf ein Volumen von 225ml. Der pH-Wert auf 6,8. Add sterile, DEPC-behandeltem H 2 O auf 250ml. Autoclave. </li><li> 0,20 M Phosphatpuffer: In einem 500 ml-Kolben, kombinieren 50ml 1M Phosphat-Lösung, pH 6,8, 2,5 ml 10% Natriumdodecylsulfat (SDS) und 5 ml 0,5 M EDTA. Add sterile, DEPC-behandeltem H 2 O auf ein Volumen von 225ml. Der pH-Wert auf 6,8. Add sterile, DEPC-behandeltem H 2 O auf 250ml. Autoclave. </li><li> 0.40 M Phosphatpuffer: in einer 500ml Flasche, kombinieren 100ml 1M Phosphat-Lösung, pH 6,8, 2,5 ml 10% Natriumdodecylsulfat (SDS) und 5 ml 0,5 M EDTA. Add sterile, DEPC-behandeltem H 2 O auf ein Volumen von 225ml. Der pH-Wert auf 6,8. Add sterile, DEPC-behandeltem H 2 O auf 250ml. Autoclave. </li><li> &quot;1,00 M Phosphat-Puffer&quot; (tatsächliche Konzentration von Phosphat in dieser Lösung ist 0,91 M): In einem 500 ml-Kolben, kombinieren 30ml 1M Phosphat-Lösung, pH 6,8, 2,5 ml 10% Natriumdodecylsulfat (SDS) und 5 ml 0,5 M EDTA. Add sterile, DEPC-behandeltem H2O auf ein Volumen von 225ml. Der pH-Wert auf 6,8. Add sterile, DEPC-behandeltem H 2 O auf 250ml. Autoclave. </li><li> Hydroxyapatit Hydratation: Gewicht aus 1g aus Hydroxylapatit und hinzufügen, um eine 50ml Tube. Add 6ml von 0,12 M Phosphat-Puffer auf die Hydroxyapatit und umkehren zu mischen. Vor dem Einsatz bringen Temperatur auf 60 ° C und gründlich mischen. Shop für längere Zeit bei 4 ° C. </li><li> Vor dem Gebrauch Gleichgewicht aller Phosphat-Puffer und hydratisiert Hydroxyapatit bei 60 ° C. </li></ol> 2. Vorbereitung von Econo-Column <ol><li> Absperrhahn schließen. Spülen Spalte mehrmals mit deionisiertem H 2 O Durch wiederholtes Umdrehen der Säule und Dekantieren. </li><li> Entfernen Sie den Hahn aus der Econo-Säule und der Säule Autoklaven zu sterilisieren. </li><li> Ersetzen und schließen Hahn. 1ml von Sigmacote die sterile Econo-Säule und Mantel aller Glasflächen. Öffnen Sie den Wasserhahn und dekantieren. </li><li> Befestigen Sie die Spalte, um einen zirkulierenden Wasserbad und eingestellte Temperatur auf 60 ° C. Um einsure den geringsten Wärmeverlust über die Säule das Einwickeln der Spalte in einer isolierenden Mittel wie Aluminiumfolie oder Schaum Rohrisolierung wird empfohlen. </li><li> Spülen Sie die Econo-Säule zweimal mit sterilem, RNase-freien H 2 O dann zweimal mit sterilem, RNase-frei 0,12 M Phosphatpuffer. </li></ol> 3. Hydroxypaptite Chromatographie <ol><li> Sicherstellen, dass der Hahn geschlossen ist, langsam zugeben 2ml vorbereiteten, resuspendiert Hydroxyapatit an der Unterseite des Econo-Säule mit einer sterilen 2 ml serologischen Pipette. </li><li> Lassen Sie die Hydroxylapatit unter der Schwerkraft für 30 Minuten absetzen. </li><li> Abfluss-Puffer aus der Säule und in der Nähe Hahn. Kombinieren Nukleinsäuren mit 0,12 M Phosphat-Puffer in einem Endvolumen von 500 ul und Inkubieren bei 60 ° C für 10 Minuten in einem Heizblock oder Wasserbad. </li><li> Schnell gelten Nukleinsäuren in 0,12 M Phosphat-Puffer auf die Hydroxyapatit mit einem 2ml serologischen Pipette darf dabei nicht um die Spalte zu stören vorbereitet. </li><li> Lassen Sie die Nukleinsäuren an das Hydroxyapatit für 30 Minuten zu binden. </li><li> Legen Sie eine 15ml Tube unter der Spalte, öffnen Sie den Hahn und sammelt die ersten Probe mit ssDNA. Absperrhahn schließen. </li><li> Add 6ml von 0,12 M Phosphat-Puffer auf die Hydroxyapatit darf dabei nicht um die Spalte zu stören, öffnen Sie den Hahn, und sammeln Einzelstrang-DNA in der 15ml Tube aus dem vorherigen Schritt. Absperrhahn schließen. </li><li> Add 1 Volumen Phenol: Chloroform: Isoamylalkohol (25:25:1 v / v / v) Lösung für das Rohr, kräftig mischen und zentrifugieren bei 3500 xg für 15 Minuten. Überstand mit ssDNA zu einem neuen 15ml Tube. </li><li> Wiederholen 3,76 und 3,87 mit 6ml von 0,20 M Phosphat-Puffer, um eLaute und reinigen RNA aus der Säule achten Sie darauf, ein neues 15ml Tube für die Sammlung verwenden. </li><li> Wiederholen 3,76 und 3,87 mit 6ml von 0,40 M Phosphat-Puffer zu eluieren und zu reinigen dsDNA aus der Spalte achten Sie darauf, ein neues 15ml Tube für die Sammlung verwenden. </li><li> Wiederholen 3,76 und 3,87 mit 6ml von 1,00 M Phosphat-Puffer auf die Säule der verbleibenden dsDNA achten Sie darauf, ein neues 15ml Tube für die Sammlung verwenden Streifen. </li></ol> 4. Entsalzung Nukleinsäure-Proben <ol><li> Transfer-4-5 ml Probe an einen Amicon Ultra-4 Zentrifugalfilter Gerät mit einer 30.000 Molekulargewicht off Ultracel Membran geschnitten ausgestattet. </li><li> Konzentrieren Probe &lt;500μl durch Spinnen bei 6.000 xg, 30 ° C, 5 Minuten (oder bis konzentrierten Volumen erreicht ist) in einem Festwinkelrotor. Entsorgen Sie durchströmen. </li><li> Fügen Sie den Rest der Probe (n) der Amicon Ultra-4 Zentrifugalfilter Gerät und bringen die Lautstärke von bis zu 4-5ml mit RNase frei 1X TE-Puffer. </li><li> Konzentrieren Probe &lt;500μl durch Spinnen bei 6.000 xg, 30 ° C, 5 Minuten (oder bis konzentrierten Volumen erreicht ist) in einem Festwinkelrotor. Entsorgen Sie durchströmen. </li><li> Add 4-5 ml RNase frei 1X TE-Puffer auf die Amicon Ultra-4 Zentrifugalfilter Gerät. Konzentrieren Probe &lt;500μl durch Zentrifugation bei 6.000 xg, 30 ° C, 5 Minuten (oder bis konzentrierten Volumen erreicht ist) in einem Festwinkelrotor. Entsorgen Sie durchströmen. </li><li> Wiederholen Sie Schritt 4.5 eine zusätzliche 5-mal vollständig entsalzt Nukleinsäureprobe (s). </li><li> Restlichen Probe (n) in ein steriles 1,7 ml Eppendorf-Röhrchen. Add 1:10 Volumen 3M Natriumacetat, pH 7,0, zwei Bände mit 100% Ethanol und 1μl der Glycoblue. Gut mischen durch Vortexen. </li><li> Zentrifuge bei 28.000 xg, 4 ° C, 60 Minuten. Dekantieren darf dabei nicht das Pellet stören. Fügen Sie 300μl 70% Ethanol. Spin bei 28.000 xg, Raumtemperatur für 10 Minuten. Dekantieren, trocken und resuspendieren jeder Fraktion in einem geeigneten Volumen RNase-freiem TE-Puffer. </li></ol> 5. Repräsentative Ergebnisse: Abbildung 1 fasst die Methoden für den Einsatz Hydroxyapatit-Chromatographie zur Fraktionierung ssDNA, dsDNA und RNA Nukleinsäuren aus einer gemischten virale Assemblage vorgestellt. Diese Methode nutzt die Ladungs-Wechselwirkung zwischen den negativ geladenen Phosphat-Rückgrat der Nukleinsäuren und die positiv geladenen Ca 2 +-Ionen in der Hydroxylapatit und ermöglicht die effiziente Fraktionierung von Nukleinsäure-Subtypen (ssDNA, dsDNA und RNA) mit steigender Phosphat-Puffer-Konzentrationen 12. Die Hydroxyapatit Fraktionierung von einem in vitro-&quot;Community&quot; bekannt ssDNA (M13mp18), dsDNA (Lambda) und RNA (MS2 und phi6) viralen Genomen ist in Abbildung 2 dargestellt. Die Nukleinsäuren wurden in gleichen Konzentrationen kombiniert, angewandt auf die Hydroxyapatit-Säule und eluiert mit einer zunehmenden Konzentration von Phosphat-Puffer. Jeder Nukleinsäure-Subtyp eluiert unabhängig von den anderen mit weniger als 9% von einem Bruchteil-over zum nächsten zu befördern. Die Anwendung dieser Technik, um Nukleinsäuren aus einer viralen Gemeinde aus der Chesapeake Bay gesammelt isoliert ist in Abbildung 3 dargestellt. Die gesamte Nukleinsäure-Ausbeute von gereinigten Viren isoliert, um die Hydroxyapatit-Säule aufgetragen wurde. Genomic Material, bestehend aus ssDNA, RNA und dsDNA wurde unabhängig mit 0,12 M, 0,20 M und 0.40M/1.00M Konzentrationen von Phosphat-Puffer bzw. eluiert. Doppelsträngige DNA ist bei Phosphat-Konzentrationen von 0,40 M und 1,00 M eluiert und in diesem Fall dominiert diese virale Community im Vergleich zu ssDNA und RNA-Genotypen. Diese Beobachtung steht im Einklang mit der erwarteten virale Community Zusammensetzung der Meeres-und Mündungs-Umgebungen, in denen die Mehrheit der erzielbare Nukleinsäuren dsDNA 13. <img src="/files/ftp_upload/3146/3146fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Flussdiagramm des Hydroxyapatit-Chromatographie-Verfahren zur Fraktionierung von Nukleinsäuren. Eine Mischung aus ssDNA, dsDNA und RNA in 0,12 M Phosphat-Puffer bereit ist, 60 ° C erhitzt und auf eine Hydroxyapatit-Säule bei konstant 60 ° C gehalten mit einem zirkulierenden Wasserbad. Nukleinsäuren (ssDNA, RNA und dsDNA) sind vom Hydroxyapatit mit steigenden Konzentrationen von Phosphat-Puffer eluiert. Diese Zahl ist mit freundlicher Genehmigung von American Society of Microbiology reproduziert worden und war ursprünglich in Andrews-Pfannkoch et al featured. 2010 12. <img src="/files/ftp_upload/3146/3146fig2.jpg" alt="Abbildung 2"/> Abbildung 2. Trennung von bekannten viralen Nukleinsäuren mittels Hydroxyapatit-Chromatographie. Equal Konzentrationen von M13mp18 ssDNA, MS2 ssRNA, phi6 dsRNA und Lambda-dsDNA (Spuren 2-5, jeweils) wurden vereinigt (Spur 6) und auf eine Hydroxyapatit-Säule. Single-DNA (M13mp18), RNA (MS2/phi6) und dsDNA (Lambda) waren unabhängig von der Hydroxyapatit-Säule mit 0,12 M (Spur 8), 0,18 M (Spur 9) und 0.40M/1.00M (Spur 10) eluiert . Ein 1kb ladder (Bahnen 1, 7) wurde verwendet, um Genomgrößen bestätigen. Diese Zahl ist mit freundlicher Genehmigung von American Society of Microbiology reproduziert worden und war ursprünglich in Andrews-Pfannkoch et al featured. 2010 12. <img src="/files/ftp_upload/3146/3146fig3.jpg" alt="Abbildung 3"/> Abbildung 3. Separation von viralen Nukleinsäuren aus einer Gemeinde innerhalb der Chesapeake Bay getrennt. Nukleinsäuren wurden isoliert und auf eine Hydroxyapatit-Säule. ssDNA (Spur 0,12 M), RNA (Spur 0,20 M) und dsDNA (Spuren 0.40M/1.0M) wurden unabhängig voneinander eluiert mit Phosphatpuffer Konzentrationen von 0,12 M, 0,20 M und 0.40M/1.00M jeweils. Hallo Messe DNA Ladder (Spur L1) und 1kb Ladder (Spur L2) wurden verwendet, um visuell bestätigen ungefähre Molekulargewicht und die Masse der Nukleinsäuren. Diese Zahl ist mit freundlicher Genehmigung von American Society of Microbiology reproduziert worden und war ursprünglich in Andrews-Pfannkoch et al featured. 2010 12.

<ol>
	<li>Whitman, W. B., Coleman, D. C., Wiebe, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prokaryotes:+The+Unseen+Majority.">Prokaryotes: The Unseen Majority.</a> Proceedings of the National Academy of Sciences of the United States of America. 95, 6578-65 (1998).</li><li>Hendrix, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriophages:+evolution+of+the+majority.">Bacteriophages: evolution of the majority.</a> Theor Popul Biol. 61, 471-471 (2002).</li><li>Weinbauer, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriophages:+Evolution+of+the+Majority.">Bacteriophages: Evolution of the Majority.</a> FEMS Microbiol Rev. 28, 127-127 (2004).</li><li>Dinsdale, E. A., Edwards, R. A., Hall, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Metabolic+Profiling+of+Nine+Biomes.">Functional Metabolic Profiling of Nine Biomes.</a> Nature. 455, 830-830 (2008).</li><li>McDaniel, L., Breitbart, M., Mobberley, 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+Analysis+of+Lysogeny+in+Tampa+Bay:+Implications+for+Prophage+Gene+Expression.">Metagenomic Analysis of Lysogeny in Tampa Bay: Implications for Prophage Gene Expression.</a> PLoS ONE. 3, e3263-e3263 (2008).</li><li>Williamson, S. J., Rusch, D. B., Yooseph, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Sorcerer+II+Global+Ocean+Sampling+Expedition:+Metagenomic+Characterization+of+Viruses+within+Aquatic+Microbial+Samples.">The Sorcerer II Global Ocean Sampling Expedition: Metagenomic Characterization of Viruses within Aquatic Microbial Samples.</a> PLoS ONE. 3, e1456-e1456 (2008).</li><li>Lang, A. S., Rise, M. L., Culley, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+Viruses+in+the+Sea.">RNA Viruses in the Sea.</a> FEMS Microbiol Rev. 33, 295-295 (2009).</li><li>Ng, T. F., Manire, C., Borrowman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+a+Novel+Single-Stranded+DNA+Virus+from+a+Sea+Turtle+Fibropapilloma+by+using+Viral+Metagenomics.">Discovery of a Novel Single-Stranded DNA Virus from a Sea Turtle Fibropapilloma by using Viral Metagenomics.</a> J. Virol. 83, 2500-2500 (2009).</li><li>Rosario, K., Duffy, S., Breitbart, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+Circovirus-like+Genome+Architectures+Revealed+by+Environmental+Metagenomics.">Diverse Circovirus-like Genome Architectures Revealed by Environmental Metagenomics.</a> J Gen Virol. 90, 2418-2418 (2009).</li><li>Culley, A. I., Lang, A. S., Suttle, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenomic+Analysis+of+Coastal+RNA+Virus+Communities.">Metagenomic Analysis of Coastal RNA Virus Communities.</a> Science. 312, 1795-1795 (2006).</li><li>Bernardi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromotography+of+Nucleic+Acids+on+Hydroxyapatite.">Chromotography of Nucleic Acids on Hydroxyapatite.</a> Nature. 209, 779-779 (1965).</li><li>Andrews-Pfannkoch, C., Fadrosh, D. W., Thorpe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxyapatite-Mediated+Separation+of+Double-Stranded+DNA,+Single-Stranded+DNA+and+RNA+Genomes+from+Natural+Viral+Assemblages.">Hydroxyapatite-Mediated Separation of Double-Stranded DNA, Single-Stranded DNA and RNA Genomes from Natural Viral Assemblages.</a> Applied and environmental microbiology. 76, 5039-5039 (2010).</li><li>Wommack, K. E., Colwell, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virioplankton:+Viruses+in+Aquatic+Ecosystems.">Virioplankton: Viruses in Aquatic Ecosystems.</a> Microbiol Mol Biol Rev. 64, 69-69 (2000).</li></ol>

Keine Interessenskonflikte erklärt.

Die Hydroxyapatit-Chromatographie-Methodik präsentiert hier ist eine hocheffiziente und robustes Werkzeug für die Fraktionierung von Nukleinsäuren aus gemischten virale Assemblagen, wenn das Ziel, die gesamte Nukleinsäure Zusammensetzung der Gemeinde-Studie ist. Im Allgemeinen wird ssDNA, RNA und dsDNA aus der Spalte pho-Phosphat-Puffer-Konzentrationen von mehr als etwa ~ 0und 0,40 M bzw. eluieren. Allerdings kann jeder Zubereitung von Hydroxyapatit haben eine etwas andere Zusammensetzung und Elutionsprofil...

<table border="1"><tbody><tr><th> Material Name </th><th> Firma </th><th> Katalog-Nummer </th><th> Katalog-Nummer </th></tr><tr><td> Econo-Column </td><td> Bio-Rad </td><td> 737-0717 </td><td> 0.7cm ID-Paket / 2 </td></tr><tr><td> Hydroxyapatit </td><td> Bio-Rad </td><td> 130-0520 </td><td> DNA Grade Bio-Gel HTP Gel, 100g/td&gt; </tr><tr><td> Sodium Phosphate, Einbasisches </td><td> VWR </td><td> VW1497-01 </td><td> Monohydrate, Crystal 500g </td></tr><tr><td> Sodium Phosphate, Dibasic </td><td> VWR </td><td> VW1496-01 </td><td> Wasserfrei, Pulver 500g </td></tr><tr><td> DEPC-behandeltem Wasser </td><td> Invitrogen </td><td> AM9922 </td><td> 1L </td></tr><tr><td> 10% SDS-Lösung </td><td> Invitrogen </td><td> 24730-020 </td><td> UltraPure, 1L </td></tr><tr><td> 0,5 M EDTA </td><td> Invitrogen </td><td> AM9262 </td><td> pH 8,0, 1L </td></tr><tr><td> Sigmacote </td><td> Sigma-Aldrich </td><td> SL2-25ML </td><td></td></tr><tr><td> 2ml serologische pippette </td><td> VWR </td><td> 89130-884 </td><td> Polystyrol, steril </td></tr><tr><td> BD Falcon Zentrifugenröhrchen </td><td> VWR </td><td> 21008-936 </td><td> 15ml, steril </td></tr><tr><td> Phenol: Chloroform: Isoamylalkohol (25:24:1 v / v / v) </td><td> Invitrogen </td><td> 15593-031 </td><td> UltraPure, 100ml </td></tr><tr><td> Amicon Ultra-4 Kreiselpumpen Geräte </td><td> Millipore </td><td> UFC803024 </td><td> Ultracel-30-Membran </td></tr><tr><td> 20X TE-Puffer, RNase-frei </td><td> Invitrogen </td><td> T11493 </td><td> 100ml </td></tr><tr><td> Glycoblue </td><td> Invitrogen </td><td> AM9516 </td><td> 15mg/ml </td></tr></tbody></table>

separation of single-stranded dna, double-stranded dna and rna from an environmental viral community using hydroxyapatite chromatography

Viren, vor allem Bakteriophagen (Phagen), sind die zahlreichen biologischen Einheiten auf der Erde 1,2. Viren modulieren Wirtszelle Fülle und Vielfalt, um den Kreislauf der Nährstoffe beitragen, zu verändern Wirtszelle Phänotyp, und Einfluss auf die Entwicklung der beiden Wirtszelle und Virus-Communities durch die seitlichen Transfer von Genen 3. Zahlreiche Studien haben die erstaunliche genetische Vielfalt der Viren und ihre funktionelle Potenzial in einer Vielzahl von natürlichen Umgebungen hervorgehoben. Metagenom Techniken wurden verwendet, um die taxonomische Diversität und funktionale Potenzial von komplexen viralen Assemblagen, deren Mitglieder enthalten Einzelstrang-DNA (ssDNA), doppelsträngige DNA (dsDNA) und RNA-Genotypen 4-9 studieren. Aktuelle Bibliothek Bau-Protokolle verwendet werden, um Umwelt-DNA-haltigen oder RNA-Viren enthalten Studie erfordern eine erste Nuklease Behandlung zur Entfernung nontargeted Templates 10. Allerdings erfordert ein umfassendes Verständnis des kollektiven Gen Ergänzung des Virus-Community und Viren Vielfalt Wissen aller Mitglieder, unabhängig von Genom Zusammensetzung. Die Fraktionierung des gereinigten Nukleinsäure-Subtypen bietet einen wirksamen Mechanismus, durch die virale Assemblagen, ohne dabei eine Teilmenge der Gemeinde genetische Signatur zu studieren. Hydroxyapatit, eine kristalline Form von Calciumphosphat, hat in der Trennung von Nukleinsäuren, sowie Proteine ​​und Mikroben eingesetzt worden, seit den 1960er Jahren 11. Durch die Ausnutzung der Ladungs-Wechselwirkung zwischen den positiv geladenen Ca 2 +-Ionen des Hydroxylapatit und der negativ geladenen Phosphat-Rückgrat der Nukleinsäure-Subtypen, ist es möglich, bevorzugt eluieren jede Nukleinsäure-Subtyp unabhängig von den anderen. Vor kurzem haben wir diese Strategie, unabhängig fraktionieren die Genome von ssDNA, dsDNA und RNA-Viren enthalten in der Vorbereitung der DNA-Sequenzierung 12 verwendet. Hier stellen wir ein Verfahren zur Fraktionierung und Rückgewinnung von ssDNA, dsDNA und RNA viralen Nukleinsäuren aus gemischten virale Assemblagen mit Hydroxyapatit-Chromatographie.

Watch this Scientific Journal Video about Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography at JoVE.com

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Wir beschreiben eine effiziente Methode, um Einzelstrang-DNA, doppelsträngige DNA-und RNA-Molekülen aus Umwelt-virale Gemeinden zu trennen. Nukleinsäuren sind fraktionierte mit Hydroxyapatit-Chromatographie mit steigenden Konzentrationen von Phosphat-haltigen Puffer. Diese Methode erlaubt die Isolierung aller viralen Nukleinsäure-Typen aus Umweltproben.

Trennung von Einzelstrang-DNA, doppelsträngige DNA und RNA aus ökologischer Viral Gemeinschaft mit Hydroxyapatit-Chromatographie

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

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JoVE Journal

Immunology and Infection

31.5K Aufrufe.  The J. Craig Venter Institute. Wir beschreiben eine effiziente Methode, um Einzelstrang-DNA, doppelsträngige DNA-und RNA-Molekülen aus Umwelt-virale Gemeinden zu trennen. Nukleinsäuren sind fraktionierte mit Hydroxyapatit-Chromatographie mit steigenden Konzentrationen von Phosphat-haltigen Puffer. Diese Methode erlaubt die Isolierung aller viralen Nukleinsäure-Typen aus Umweltproben.

Serie: Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Erzeugung von rekombinanten Influenza Virus von Plasmid-DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

Forschung

Immunologie und Infektion

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

Isolierung von Fidelity Varianten von RNA-Viren und Charakterisierung von Virus-Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

Entwicklung von Zell-spezifischen Anti-HIV-gp120-Aptamere für siRNA

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

Herstellung viraler DNA aus Nukleokapside

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

A Simple Chelex Protokoll zur DNA-Extraktion aus<em&gt; Anopheles</em&gt; Spp.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

Einrichtung einer<em&gt; In-vitro-</em&gt; System zur intrazelluläre Verhalten der Studie<em&gt; Candida glabrata</em&gt; Menschen THP-1-Makrophagen

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Mit Klick-Chemie, um die Wirkung von Viral Infection auf Host-Zell-RNA Synthese Messen

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Mittelung der Virushülle Glykoprotein-Spikes von Electron Cryotomography Rekonstruktionen mit Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

Development of an Economical DNA Delivery System by &quot;Acufection&quot; and its Application to Skin Research

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

Entwicklung eines wirtschaftlich DNA Delivery System von „Acufection“ und seine Anwendung auf die Hautforschung

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.