Questa ricerca è stata sostenuta dall&#39;Ufficio della Scienza (BER), US Department of Energy, Cooperativa accordo no. De-FC02-02ER63453, la National Science Foundation microbica Programma Genome Sequencing (numeri premio 0626826 e 0731916). Ringraziamo Giovanni in vetro per la sua competenza tecnica e di consulenza e K. Eric Wommack per la sua assistenza con la raccolta dei campioni ambientali.

1. Preparazione di soluzioni Prima di eseguire la cromatografia idrossiapatite, tamponi fosfato deve essere preparato e l&#39;idrossiapatite devono essere adeguatamente idratati. <ol><li> Soluzione fosfato 1M, pH 6,8: In un pallone da 1 litro sciogliere 119.98g di fosfato monobasico di sodio in 1 litro di sterile, DEPC trattati con H 2 O. Preparare una soluzione 1M fosfato di sodio bibasico in un pallone da 1 litro, sciogliendo 141.96g di fosfato bibasico di sodio in 1 litro di sterile, DEPC trattati con H 2 O. Combinano la mono-e di soluzioni con un rapporto di 1:1. Pallone posto su un piatto mescolare e miscelare con una barra magnetica mescolare. Regolare il pH a 6,8 con l&#39;aggiunta di idrossido di sodio (per aumentare il pH) o acido fosforico (per diminuire il pH). </li><li> Tampone fosfato 0.12M: in un pallone da 500 ml, unire 30 ml di soluzione 1M fosfato, pH 6,8, 2,5 ml di 10% di sodio dodecil solfato (SDS), e 5 ml di EDTA 0.5M. Aggiungi sterile, DEPC trattati con H2O ad un volume di 225ml. Regolare il pH a 6,8. Aggiungi sterile, DEPC trattati con H2O a 250ml. Autoclave. </li><li> Tampone fosfato 0.18M: in un pallone da 500 ml, unire 45ml di soluzione 1M fosfato, pH 6,8, 2,5 ml di 10% di sodio dodecil solfato (SDS), e 5 ml di EDTA 0.5M. Aggiungi sterile, DEPC trattati con H 2 O ad un volume di 225ml. Regolare il pH a 6,8. Aggiungi sterile, DEPC trattati con H 2 O a 250 ml. Autoclave. </li><li> Tampone fosfato 0.20m: in un pallone da 500 ml, unire 50 ml di soluzione 1M fosfato, pH 6,8, 2,5 ml di 10% di sodio dodecil solfato (SDS), e 5 ml di EDTA 0.5M. Aggiungi sterile, DEPC trattati con H 2 O ad un volume di 225ml. Regolare il pH a 6,8. Aggiungi sterile, DEPC trattati con H 2 O a 250 ml. Autoclave. </li><li> Tampone fosfato 0,40 M: in un pallone da 500 ml, 100 ml di soluzione di combinare fosfato 1M, pH 6,8, 2,5 ml di 10% di sodio dodecil solfato (SDS), e 5 ml di EDTA 0.5M. Aggiungi sterile, DEPC trattati con H 2 O ad un volume di 225ml. Regolare il pH a 6,8. Aggiungi sterile, DEPC trattati con H 2 O a 250 ml. Autoclave. </li><li> &quot;1.00m fosfato buffer&quot; (effettiva concentrazione di fosfato in questa soluzione è 0,91 M): in un pallone da 500 ml, unire 30 ml di soluzione di fosfato 1M, pH6.8, 2,5 ml di 10% di sodio dodecil solfato (SDS), e 5 ml di 0.5M EDTA. Aggiungi sterile, DEPC trattati con H2O ad un volume di 225ml. Regolare il pH a 6,8. Aggiungi sterile, DEPC trattati con H 2 O a 250 ml. Autoclave. </li><li> Idratazione idrossiapatite: Peso da 1g di idrossiapatite e aggiungere in una provetta da 50 ml. Aggiungere 6 ml di tampone fosfato 0.12M al idrossiapatite e miscelare. Prima dell&#39;uso portare la temperatura a 60 ° C e mescolare accuratamente. Conservare per lunghi periodi di tempo a 4 ° C. </li><li> Prima dell&#39;uso, equilibrare tutti i tamponi fosfato e idrossiapatite idratata a 60 ° C. </li></ol> 2. Preparazione di Econo-Colonna <ol><li> Chiudere rubinetto. Sciacquare colonna parecchie volte con acqua deionizzata H 2 O invertendo ripetutamente la colonna e decantazione. </li><li> Rimuovere il rubinetto dal Econo-colonna e la colonna autoclave per la sterilizzazione. </li><li> Sostituire il rubinetto e chiudi. Aggiungere 1ml di Sigmacote alla sterile Econo-colonna e cappotto tutte le superfici in vetro. Aprire il rubinetto e decantare. </li><li> Allegare colonna a un bagno d&#39;acqua circolante e impostare la temperatura a 60 ° C. Per einsure la minor quantità di perdita di calore attraverso la colonna avvolgendo la colonna in un agente isolanti come un foglio di alluminio o tubo di schiuma isolante è raccomandato. </li><li> Sciacquare la Econo-colonna due volte con sterili, RNasi-free H 2 O poi due volte con sterili, RNasi-free tampone fosfato 0.12M. </li></ol> 3. Hydroxypaptite cromatografia <ol><li> Assicurandosi che il rubinetto è chiuso, aggiungere lentamente 2 ml di pre-preparati, idrossiapatite risospese alla parte inferiore del Econo-colonna con una pipetta sterile 2ml sierologici. </li><li> Lasciare che il idrossiapatite di stabilirsi sotto la forza di gravità per 30 minuti. </li><li> Scolare buffer da colonna e rubinetto vicino. Combina acidi nucleici con tampone fosfato 0.12M in un volume finale di 500 microlitri e incubare a 60 ° C per 10 minuti in un blocco di calore o bagnomaria. </li><li> Applicare rapidamente gli acidi nucleici, preparato in tampone fosfato 0.12M alla idrossiapatite con una pipetta sierologica 2ml facendo attenzione a non disturbare la colonna. </li><li> Lasciare gli acidi nucleici di legarsi alla idrossiapatite per 30 minuti. </li><li> Posizionare un tubo 15ml sotto la colonna, aprire il rubinetto e raccogliere il campione iniziale, contenente ssDNA. Chiudere rubinetto. </li><li> Aggiungere 6 ml di tampone fosfato 0.12M alla idrossiapatite facendo attenzione a non disturbare la colonna, aprire il rubinetto e raccogliere DNA a singolo filamento nel tubo 15ml dal passaggio precedente. Chiudere rubinetto. </li><li> Aggiungere 1 volume di fenolo: cloroformio: alcool isoamilico (25:25:1 v / v / v) al tubo, mescolare vigorosamente e centrifugare a 3500 xg per 15 minuti. Trasferire il supernatante contenente ssDNA di un nuovo tubo 15ml. </li><li> Ripetere 3,76 e 3,87 con 6 ml di tampone fosfato 0.20m per postaliuto e RNA purificare dalla colonna facendo attenzione a utilizzare un nuovo tubo 15ml per la raccolta. </li><li> Ripetere 3,76 e 3,87 con 6 ml di tampone fosfato 0.40m per eluire e purificare dsDNA dalla colonna facendo attenzione a utilizzare un nuovo tubo 15ml per la raccolta. </li><li> Ripetere 3,76 e 3,87 con 6 ml di tampone fosfato 1,00 m di togliere la colonna di qualsiasi residuo dsDNA assicurandosi di utilizzare un nuovo tubo 15ml per la raccolta. </li></ol> 4. Dissalazione campioni acidi nucleici <ol><li> Trasferimento 4-5 ml di campione ad un Amicon Ultra-4 dispositivo centrifugo filtro dotato di un peso molecolare 30.000 tagliato membrana Ultracel. </li><li> Concentrato campione &lt;500μl di filatura a 6.000 xg, 30 ° C, 5 minuti (o fino a quando il volume concentrato si ottiene) in un rotore ad angolo fisso. Gettare flusso attraverso. </li><li> Aggiungere il resto del campione (s) per l&#39;Ultra-4 Amicon dispositivo centrifugo filtro e portare il volume fino a 4 da 5 ml con tampone TE 1X RNase libero. </li><li> Concentrato campione &lt;500μl di filatura a 6.000 xg, 30 ° C, 5 minuti (o fino a quando il volume concentrato si ottiene) in un rotore ad angolo fisso. Gettare flusso attraverso. </li><li> Aggiungere 4-5 ml di tampone TE 1X RNase libera al sistema Ultra-4 Amicon dispositivo centrifugo filtro. Concentrato campione &lt;500μl per centrifugazione a 6.000 xg, 30 ° C, 5 minuti (o fino a quando il volume concentrato si ottiene) in un rotore ad angolo fisso. Gettare flusso attraverso. </li><li> Ripetere il passo 4,5 un ulteriore 5 volte per desalt completamente campione di acido nucleico (s). </li><li> Trasferimento del campione rimanente (s) ad un tubo sterile 1.7ml eppendorf. Aggiungi 1/10th volume di acetato di sodio 3M, pH 7,0, due volumi di etanolo 100%, e 1ml di Glycoblue. Mescolare bene nel vortex. </li><li> Centrifugare a 28000 xg, a 4 ° C, 60 minuti. Decantare facendo attenzione a non disturbare il pellet. Aggiungere 300μl di etanolo 70%. Spin a 28.000 xg, temperatura ambiente per 10 minuti. Decantare, asciutto e risospendere ogni frazione in un volume adeguato di RNasi-free tampone TE. </li></ol> 5. Rappresentante dei risultati: La Figura 1 riassume i metodi presentati per l&#39;utilizzo di cromatografia idrossiapatite di frazionare ssDNA, dsDNA e gli acidi nucleici RNA da un assemblaggio misto virale. Questo metodo sfrutta l&#39;interazione di carica tra la dorsale fosfato con carica negativa degli acidi nucleici e la carica positiva di Ca 2 + ioni presenti nella idrossiapatite e consente il frazionamento efficiente dei sottotipi acido nucleico (ssDNA, dsDNA e RNA), con concentrazioni crescenti di buffer fosfato 12. Il frazionamento di un idrossiapatite in vitro &quot;comunità&quot; di noti ssDNA (M13mp18), dsDNA (lambda) e RNA (MS2 e phi6) genomi virali è illustrato in Figura 2. Gli acidi nucleici sono stati combinati in concentrazione pari, applicata alla colonna di idrossiapatite e sono stati eluiti con una crescente concentrazione del tampone fosfato. Ogni sottotipo acido nucleico eluisce indipendente dalle altre, con meno del 9% riporto da una frazione all&#39;altra. L&#39;applicazione di questa tecnica per gli acidi nucleici isolato da una comunità virale raccolti dalla Baia di Chesapeake è mostrata in Figura 3. La resa totale di acidi nucleici isolato dal virus purificato è stato applicato alla colonna di idrossiapatite. Materiale genomico composto da ssDNA, RNA e DNA a doppia elica era indipendentemente eluiti con 0.12M, 0.20m 0.40M/1.00M e le concentrazioni di tampone fosfato, rispettivamente. DNA a doppia elica viene eluita a concentrazioni di fosfato e di 1,00 m e 0,40 m in questo caso, domina questa comunità virale rispetto ai genotipi ssDNA e RNA. Questa osservazione è coerente con la composizione prevista comunità virale di ambienti marini e di estuario, dove la maggior parte degli acidi nucleici sono recuperabili dsDNA 13. <img src="/files/ftp_upload/3146/3146fig1.jpg" alt="Figura 1"/> Figura 1. Diagramma di flusso del metodo di cromatografia idrossiapatite per il frazionamento degli acidi nucleici. Una miscela di ssDNA, dsDNA e RNA, preparato in tampone fosfato 0.12M viene riscaldato a 60 ° C e applicata a una colonna di idrossiapatite mantenuto alla temperatura costante di 60 ° C con un bagno di acqua circolante. Acidi nucleici (ssDNA, RNA e dsDNA) sono eluiti dalla idrossiapatite con concentrazioni crescenti di un fosfato, contenente tampone. Questo dato è stato riprodotto con il permesso di Società Americana di Microbiologia ed è stato originariamente nel Andrews-Pfannkoch et al. 2010 12. <img src="/files/ftp_upload/3146/3146fig2.jpg" alt="Figura 2"/> Figura 2. Separazione delle note acidi nucleici virali mediante cromatografia idrossiapatite. Concentrazioni pari di M13mp18 ssDNA, MS2 ssRNA, phi6 dsRNA e lambda dsDNA (corsie 2-5, rispettivamente) sono stati combinati (corsia 6) e applicata a una colonna di idrossiapatite. Single-DNA (M13mp18), RNA (MS2/phi6) e dsDNA (lambda) erano indipendentemente eluito idrossiapatite con 0.12M (corsia 8), 0.18M (corsia 9) e 0.40M/1.00M (corsia 10) . Una scala 1kb (corsie 1, 7) è stato usato per confermare le dimensioni del genoma. Questo dato è stato riprodotto con il permesso di Società Americana di Microbiologia ed è stato originariamente nel Andrews-Pfannkoch et al. 2010 12. <img src="/files/ftp_upload/3146/3146fig3.jpg" alt="Figura 3"/> Figura 3. Separazione di acidi nucleici virale isolato da una comunità all&#39;interno della Baia di Chesapeake. Gli acidi nucleici sono stati isolati e applicato a una colonna di idrossiapatite. ssDNA (corsia 0.12M), RNA (corsia 0.20m) e dsDNA (corsie 0.40M/1.0M) erano indipendentemente eluiti utilizzando le concentrazioni di tampone fosfato di 0.12M, 0.20m e 0.40M/1.00M, rispettivamente. Ladder Messa hi DNA (corsia L1) e Ladder 1kb (corsia L2) sono stati utilizzati per confermare visivamente approssimativo peso molecolare e la massa degli acidi nucleici. Questo dato è stato riprodotto con il permesso di Società Americana di Microbiologia ed è stato originariamente nel Andrews-Pfannkoch et al. 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>

Nessun conflitto di interessi dichiarati.

La metodologia cromatografia idrossiapatite qui presentato è uno strumento altamente efficiente e robusto per il frazionamento degli acidi nucleici da assemblaggi misti virale, quando l&#39;obiettivo è quello di studiare la composizione totale di acidi nucleici della comunità. In generale, ssDNA, RNA e DNA a doppia elica si eluire dalla colonna pho concentrazioni di tampone fosfato maggiore di circa 0.40m 0e ~ rispettivamente. Tuttavia, ogni preparazione di idrossiapatite può avere una composizione leggerme...

<table border="1"><tbody><tr><th> Nome del materiale </th><th> Azienda </th><th> Numero di catalogo </th><th> Numero di catalogo </th></tr><tr><td> Econo-Colonna </td><td> Bio-Rad </td><td> 737-0717 </td><td> 0,7 centimetri pacchetto ID / 2 </td></tr><tr><td> Idrossiapatite </td><td> Bio-Rad </td><td> 130-0520 </td><td> DNA Grado Bio-Gel Gel HTP, 100g/td&gt; </tr><tr><td> Fosfato di sodio, monobasico </td><td> VWR </td><td> VW1497-01 </td><td> Monoidrato, Crystal 500g </td></tr><tr><td> Fosfato di sodio, bibasico </td><td> VWR </td><td> VW1496-01 </td><td> Anidro, Polvere 500g </td></tr><tr><td> DEPC trattati con acqua </td><td> Invitrogen </td><td> AM9922 </td><td> 1L </td></tr><tr><td> 10% SDS Soluzione </td><td> Invitrogen </td><td> 24730-020 </td><td> Ultrapura, 1L </td></tr><tr><td> 0.5M 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 sierologici pippette </td><td> VWR </td><td> 89130-884 </td><td> Polistirene, sterili </td></tr><tr><td> BD Falcon Centrifuga Tubi </td><td> VWR </td><td> 21008-936 </td><td> 15 ml, sterili </td></tr><tr><td> Fenolo: cloroformio: alcool isoamilico (25:24:1 v / v / v) </td><td> Invitrogen </td><td> 15593-031 </td><td> Ultrapura, 100ml </td></tr><tr><td> Amicon Ultra-4 dispositivo centrifugo </td><td> Millipore </td><td> UFC803024 </td><td> Ultracel-30 membrana </td></tr><tr><td> TE buffer 20X, RNasi libero </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

I virus, in particolare batteriofagi (fagi), sono le entità più numerosi biologica sulla Terra 1,2. I virus modulare cellula ospite abbondanza e la diversità, contribuiscono alla cicli dei nutrienti, alterare ospitare fenotipo delle cellule, e influenza l&#39;evoluzione di entrambi cellula ospite e le comunità virale attraverso il trasferimento laterale dei geni 3. Numerosi studi hanno messo in evidenza la diversità sconcertante genetica dei virus e delle loro potenzialità funzionali in una varietà di ambienti naturali. Tecniche di metagenomica sono state utilizzate per studiare la diversità tassonomica e le potenzialità funzionali di complessi assemblaggi virale i cui componenti contengono DNA a singolo filamento (ssDNA), DNA a doppia elica (dsDNA) e RNA genotipi 4-9. Attuali protocolli di costruzione della libreria usata per studiare l&#39;ambiente del DNA-RNA contenenti o contenenti virus richiedono un trattamento iniziale nucleasi al fine di rimuovere i modelli nontargeted 10. Tuttavia, una comprensione globale del complemento gene collettiva della comunità virus e la diversità del virus richiede la conoscenza di tutti i membri indipendentemente dalla composizione del genoma. Frazionamento di acido nucleico purificato sottotipi fornisce un meccanismo efficace per lo studio assemblaggi virale senza sacrificare un sottoinsieme della firma genetica della comunità. Idrossiapatite, una forma cristallina di fosfato di calcio, è stato impiegato nella separazione degli acidi nucleici, così come le proteine ​​e microbi, dal 1960 11. Sfruttando l&#39;interazione tra la carica a carica positiva Ca 2 + ioni di idrossiapatite e la spina dorsale fosfato con carica negativa dei sottotipi acidi nucleici, è possibile eluire preferenzialmente ogni sottotipo di acido nucleico indipendente dagli altri. Recentemente abbiamo utilizzato questa strategia per frazionare in maniera indipendente i genomi di ssDNA, dsDNA e RNA contenenti virus, in preparazione di sequenziamento del DNA 12. Qui, vi presentiamo un metodo per il frazionamento ed il recupero di ssDNA, dsDNA e RNA virale acidi nucleici da assemblaggi misti virale mediante cromatografia in idrossiapatite.

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

Descriviamo un metodo efficace per separare singolo filamento di DNA a doppio filamento di DNA e le molecole di RNA virale ambientale comunità. Gli acidi nucleici sono frazionate mediante cromatografia di idrossiapatite con concentrazioni crescenti di fosfati contenenti buffer. Questo metodo consente l&#39;isolamento di tutti i tipi virali acidi nucleici da campioni ambientali.

Separazione di DNA a singolo filamento, doppia elica del DNA e RNA da una Comunità Ambientale virale usando la cromatografia idrossiapatite

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

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Immunology and Infection

31.4K Views.  The J. Craig Venter Institute. Descriviamo un metodo efficace per separare singolo filamento di DNA a doppio filamento di DNA e le molecole di RNA virale ambientale comunità. Gli acidi nucleici sono frazionate mediante cromatografia di idrossiapatite con concentrazioni crescenti di fosfati contenenti buffer. Questo metodo consente l'isolamento di tutti i tipi virali acidi nucleici da campioni ambientali.

Video: 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.

Generazione di influenza da virus ricombinante del DNA plasmidi

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

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Immunologia e infezioni

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.

Isolamento di varianti di virus RNA Fidelity e caratterizzazione di frequenza Virus Mutation

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.

Sviluppo di Cell-tipo specifico anti-HIV gp120 aptameri per la consegna di 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.

Estrazione di DNA virale da nucleocapsidi

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.

Un protocollo Chelex semplice per l&#39;estrazione del DNA da<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.

Istituzione di un<em&gt; In vitro</em&gt; Sistema per studiare intracellulare Comportamento<em&gt; Candida glabrata</em&gt; In umani THP-1 macrofagi

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.

Utilizzando Clicca Chimica per misurare l&#39;effetto dell&#39;infezione virale sulla cellula ospite RNA Sintesi

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.

Della media di Viral Busta Glicoproteina Spikes da Electron Cryotomography Ricostruzioni con 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.

Sviluppo di un sistema di consegna economico DNA da &quot;Acufection&quot; e la sua applicazione alla ricerca della pelle

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.