Esta pesquisa foi suportada pelo Escritório de Ciência (BER), Departamento de Energia dos EUA, Acordo de Cooperação n º. De-FC02-02ER63453, o Programa Nacional Science Foundation Seqüenciamento do Genoma Microbiana (números 0626826 e 0731916 prêmio). Agradecemos a John de vidro para sua perícia técnica e aconselhamento e Eric K. Wommack por sua ajuda com a coleta de amostras ambientais.

1. Preparação de soluções Antes de realizar a cromatografia de hidroxiapatita, fosfato de buffers deve ser preparado e da hidroxiapatita deve ser adequadamente hidratado. <ol><li> Solução fosfato 1M, pH 6.8: Em um frasco 1L dissolver 119.98g de fosfato de sódio monobásico em 1L de estéril, DEPC tratados com H 2 O. Prepare uma solução de fosfato de sódio 1M dibásico em um frasco 1L, dissolvendo 141.96g de fosfato de sódio dibásico em 1L de estéril, DEPC tratados com H 2 O. Combine o mono-e di-soluções na proporção de 1:1. Frasco lugar em uma placa mexa e misture usando uma barra de agitação magnética. Ajustar o pH para 6,8 pela adição de hidróxido de sódio (para aumentar o pH) ou ácido fosfórico (para diminuir o pH). </li><li> Tampão fosfato 0.12M: Em um frasco de 500ml, combine 30ml de solução de fosfato 1M, pH 6,8, 2,5 ml de 10% de sódio dodecil sulfato (SDS), e 5 ml de EDTA 0.5M. Adicionar estéreis, tratados com DEPC H2O para um volume de 225ml. Ajustar o pH para 6,8. Adicionar estéreis, tratados com DEPC H2O a 250ml. Autoclave. </li><li> Tampão fosfato 0.18M: Em um frasco de 500ml, combine 45ml de solução de fosfato 1M, pH 6,8, 2,5 ml de 10% de sódio dodecil sulfato (SDS), e 5 ml de EDTA 0.5M. Adicionar estéril, DEPC tratados com H 2 O para um volume de 225ml. Ajustar o pH para 6,8. Adicionar estéril, DEPC tratados com H 2 O para 250ml. Autoclave. </li><li> Tampão fosfato 0,20 m: Em um frasco de 500ml, combine 50ml de solução de fosfato 1M, pH 6,8, 2,5 ml de 10% de sódio dodecil sulfato (SDS), e 5 ml de EDTA 0.5M. Adicionar estéril, DEPC tratados com H 2 O para um volume de 225ml. Ajustar o pH para 6,8. Adicionar estéril, DEPC tratados com H 2 O para 250ml. Autoclave. </li><li> Tampão fosfato 0,40 M: num frasco de 500ml, 100ml de combinar solução de fosfato 1M, pH 6,8, 2,5 ml de 10% de sódio dodecil sulfato (SDS), e 5 ml de EDTA 0.5M. Adicionar estéril, DEPC tratados com H 2 O para um volume de 225ml. Ajustar o pH para 6,8. Adicionar estéril, DEPC tratados com H 2 O para 250ml. Autoclave. </li><li> &quot;O fosfato 1.00M Buffer&quot; (concentração real de fosfato nesta solução é 0,91 M): Em um frasco de 500ml, combine 30ml de solução de fosfato 1M, pH6.8, 2,5 ml de 10% de sódio dodecil sulfato (SDS), e 5 ml de 0,5 M EDTA. Adicionar estéreis, tratados com DEPC H2O para um volume de 225ml. Ajustar o pH para 6,8. Adicionar estéril, DEPC tratados com H 2 O para 250ml. Autoclave. </li><li> Hidratação hidroxiapatita: Peso fora 1g de hidroxiapatita e adicionar a um tubo de 50ml. Adicionar 6ml de tampão fosfato 0.12M à hidroxiapatita e inverta para misturar. Antes do uso trazer temperatura para 60 ° C e misture bem. Armazenar por longos períodos de tempo a 4 ° C. </li><li> Antes do uso, equilibram todos os tampões fosfato e hidroxiapatita hidratado a 60 ° C. </li></ol> 2. Preparação de Econo-Column <ol><li> Fechar torneira. Enxaguar várias vezes a coluna com H 2 O deionizada por várias vezes invertendo a coluna e decantação. </li><li> Retire a torneira da coluna e Econo autoclave para esterilizar a coluna. </li><li> Substituir e fechar torneira. Adicionar 1 ml de Sigmacote ao estéril Econo-coluna e casaco todas as superfícies de vidro. Abra a torneira e decantar. </li><li> Anexar coluna para um banho de água circulante e ajustar a temperatura a 60 ° C. Para einsure o mínimo de perda de calor através da coluna envolvendo a coluna em um agente isolante, tais como folha de alumínio ou isolamento de tubos de espuma é recomendado. </li><li> Lavar a Econo-coluna duas vezes com estéril, livre de RNase H 2 O, em seguida, duas vezes com estéril, tampão fosfato 0.12M RNase. </li></ol> 3. Cromatografia Hydroxypaptite <ol><li> Certificando-se a torneira está fechada, lentamente, adicione 2 ml de pré-preparada de hidroxiapatita, em suspensão para o fundo da Econo-coluna usando uma pipeta estéril de 2ml sorológicos. </li><li> Permitir que a hidroxiapatita para resolver sob a força de gravidade por 30 minutos. </li><li> Dreno tampão de coluna e torneira perto. Combine ácidos nucléicos com tampão fosfato 0.12M em um volume final de 500 mL e incubar a 60 ° C por 10 minutos em um bloco de aquecimento ou banho-maria. </li><li> Aplicar rapidamente ácidos nucléicos preparadas em tampão fosfato 0.12M à hidroxiapatita, utilizando uma pipeta 2ml sorológicos tomando cuidado para não perturbar a coluna. </li><li> Permitir que os ácidos nucléicos de se ligar à hidroxiapatita por 30 minutos. </li><li> Coloque um tubo de 15ml na coluna, abra a torneira e coletar a amostra inicial contendo ssDNA. Fechar torneira. </li><li> Adicionar 6ml de tampão fosfato 0.12M à hidroxiapatita tomando cuidado para não perturbar a coluna, abrir a torneira, e recolher single-stranded DNA no tubo de 15ml da etapa anterior. Fechar torneira. </li><li> Adicionar um volume de um fenol: clorofórmio: álcool isoamílico (25:25:1 v / v / v) solução para o tubo, misturar vigorosamente e centrifugar a 3500 xg por 15 minutos. Transferência de ssDNA contendo sobrenadante para um novo tubo 15ml. </li><li> Repita 3,76 e 3,87 usando 6ml de tampão fosfato 0,20 m e paraalaúde e purificar RNA a partir da coluna certificando-se de usar um novo tubo para a coleta de 15ml. </li><li> Repita 3,76 e 3,87 usando 6ml de tampão fosfato 0,40 m para eluir e purificar dsDNA da coluna certificando-se de usar um novo tubo para a coleta de 15ml. </li><li> Repita 3,76 e 3,87 usando 6ml de tampão fosfato 1.00M para tirar a coluna de qualquer dsDNA residual certificando-se de usar um novo tubo para a coleta de 15ml. </li></ol> 4. Dessalinização Amostras de Ácido Nucleico <ol><li> Transferência de 4-5ml de amostra para um Amicon Ultra-4 dispositivo centrífugo filtro equipado com um peso molecular 30.000 cortado membrana Ultracel. </li><li> Concentre-se amostra para &lt;500μl girando a 6.000 xg, 30 ° C, a 5 minutos (ou até que seja alcançado volume concentrado) em um rotor de ângulo fixo. Descartar fluir. </li><li> Adicione o restante da amostra (s) para o Ultra-4 Amicon dispositivo centrífugo filtro e colocar o volume até 4-5ml com RNAse tampão TE 1X. </li><li> Concentre-se amostra para &lt;500μl girando a 6.000 xg, 30 ° C, a 5 minutos (ou até que seja alcançado volume concentrado) em um rotor de ângulo fixo. Descartar fluir. </li><li> Adicione 4-5ml de tampão TE Rnase livre 1X ao Ultra-4 Amicon dispositivo centrífugo filtro. Concentre-se amostra para &lt;500μl por centrifugação a 6.000 xg, 30 ° C, a 5 minutos (ou até que seja alcançado volume concentrado) em um rotor de ângulo fixo. Descartar fluir. </li><li> Repita o passo 4,5 um adicional de 5 vezes para dessalinizar amostra de ácido nucleico (s). </li><li> Transferência de amostra restante (s) para um tubo eppendorf estéril 1.7ml. Adicionar 1/10th volume de acetato de sódio 3M, pH 7.0, dois volumes de etanol 100%, e 1μl de Glycoblue. Misture bem em vórtice. </li><li> Centrifugar a 28.000 xg, 4 ° C, 60 minutos. Decantar tomando cuidado para não perturbar o sedimento. Adicionar 300 ul de etanol 70%. Giram a 28.000 xg temperatura ambiente, por 10 minutos. Decantar, seco e ressuspender cada fração em um volume adequado de RNase tampão TE. </li></ol> 5. Resultados representativos: A Figura 1 resume os métodos apresentados para a utilização de cromatografia de hidroxiapatita para fracionar ssDNA, dsDNA e ácidos nucléicos RNA a partir de um assemblage mista viral. Este método explora a interação carga entre a espinha dorsal de fosfato carregados negativamente dos ácidos nucléicos e Ca 2 + positivamente carregada íons presentes na hidroxiapatita e permite o fracionamento eficiente dos subtipos de ácido nucléico (ssDNA, dsDNA e RNA) com concentrações crescentes de fosfato tampão 12. O fracionamento de hidroxiapatita de uma in vitro &quot;comunidade&quot; de ssDNA conhecidos (M13mp18), dsDNA (lambda) e RNA (MS2 e phi6) genomas virais é ilustrada na Figura 2. Os ácidos nucléicos foram combinados em concentrações iguais, aplicada à coluna de hidroxiapatita e foram eluídos com uma crescente concentração de tampão fosfato. Cada subtipo de ácido nucléico elui independente das outras, com menos de 9% carry-over de uma fração para o próximo. A aplicação desta técnica para ácidos nucléicos isolados de uma comunidade viral coletado da Baía de Chesapeake é mostrado na Figura 3. A produção total de ácidos nucléicos isolados do vírus purificado foi aplicada à coluna de hidroxiapatita. Material genético consiste de ssDNA, RNA e dsDNA foi independentemente eluída com 0.12M, 0,20 m e concentrações 0.40M/1.00M de tampão fosfato, respectivamente. Double-stranded DNA é eluído em concentrações de fosfato de 0,40 m e 1.00M e, neste caso, domina esta comunidade viral em comparação com genótipos ssDNA e RNA. Esta observação é consistente com a composição da comunidade esperado viral de ambientes marinhos e estuarinos onde a maioria dos ácidos nucléicos são recuperáveis ​​dsDNA 13. <img src="/files/ftp_upload/3146/3146fig1.jpg" alt="Figura 1"/> Figura Diagrama 1. Do método de cromatografia de hidroxiapatita para o fracionamento de ácidos nucléicos. Uma mistura de ssDNA, dsDNA e RNA preparadas em tampão fosfato 0.12M é aquecida a 60 ° C e aplicada a uma coluna de hidroxiapatita mantida a 60 ° C constante através de um banho de água circulante. Ácidos nucléicos (ssDNA, RNA e dsDNA) são eluída da hidroxiapatita com concentrações crescentes de um tampão de fosfato contendo. Esta figura foi reproduzido com permissão da Sociedade Americana de Microbiologia e foi originalmente apresentado em al-Andrews et Pfannkoch. 2010 12. <img src="/files/ftp_upload/3146/3146fig2.jpg" alt="Figura 2"/> Figura 2. Separação de ácidos nucléicos virais conhecidos através de cromatografia de hidroxiapatita. Concentrações iguais de M13mp18 ssDNA, ssRNA MS2, dsRNA phi6 e dsDNA lambda (faixas 2-5, respectivamente) foram combinadas (faixa 6) e aplicado a uma coluna de hidroxiapatita. Single-stranded DNA (M13mp18), RNA (MS2/phi6) e dsDNA (lambda) foram independentemente eluída da coluna de hidroxiapatita utilizando 0.12M (faixa 8), 0.18M (faixa 9) e 0.40M/1.00M (faixa 10) . Uma escada 1kb (faixas 1, 7) foi utilizado para confirmar tamanhos genoma. Esta figura foi reproduzido com permissão da Sociedade Americana de Microbiologia e foi originalmente apresentado em al-Andrews et Pfannkoch. 2010 12. <img src="/files/ftp_upload/3146/3146fig3.jpg" alt="Figura 3"/> Figura 3. Separação de ácidos nucléicos virais isoladas de uma comunidade dentro da Baía de Chesapeake. Ácidos nucléicos foram isolados e aplicada a uma coluna de hidroxiapatita. ssDNA (lane 0.12M), RNA (pista 0,20 m) e dsDNA (pistas 0.40M/1.0M) foram independentemente eluída usando concentrações de tampão fosfato 0.12M, 0,20 m e 0.40M/1.00M, respectivamente. Oi Ladder DNA Mass (lane L1) e Ladder 1kb (lane L2) foram usados ​​para confirmar visualmente peso molecular aproximado e massa de ácidos nucléicos. Esta figura foi reproduzido com permissão da Sociedade Americana de Microbiologia e foi originalmente apresentado em al-Andrews et Pfannkoch. 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>

Não há conflitos de interesse declarados.

A metodologia de cromatografia de hidroxiapatita apresentado aqui é uma ferramenta altamente eficiente e robusto para o fracionamento de ácidos nucléicos a partir de conjuntos mistos viral, quando o objetivo é estudar a composição de ácidos nucléicos totais da comunidade. Geralmente, ssDNA, RNA e dsDNA vai saem da coluna pho concentrações de fosfato tampão maior que cerca de 0,40 m ~ 0and respectivamente. No entanto, cada preparação de hidroxiapatita pode ter uma composição ligeiramente diferente...

<table border="1"><tbody><tr><th> Nome do material </th><th> Companhia </th><th> Número Catálogo </th><th> Número Catálogo </th></tr><tr><td> Coluna Econo- </td><td> Bio-Rad </td><td> 737-0717 </td><td> 0,7 centímetros pacote de ID / 2 </td></tr><tr><td> Hidroxiapatita </td><td> Bio-Rad </td><td> 130-0520 </td><td> DNA Grade Bio-Gel Gel HTP, 100g/td&gt; </tr><tr><td> Fosfato de sódio monobásico </td><td> VWR </td><td> VW1497-01 </td><td> Monohidratada, Crystal 500g </td></tr><tr><td> Fosfato de sódio dibásico </td><td> VWR </td><td> VW1496-01 </td><td> Anidro, em pó 500g </td></tr><tr><td> DEPC tratados com água </td><td> Invitrogen </td><td> AM9922 </td><td> 1L </td></tr><tr><td> 10% da solução SDS </td><td> Invitrogen </td><td> 24730-020 </td><td> UltraPure, 1L </td></tr><tr><td> EDTA 0.5M </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 sorológicos pippette </td><td> VWR </td><td> 89130-884 </td><td> Poliestireno, estéril </td></tr><tr><td> Centrífuga BD Tubos Falcon </td><td> VWR </td><td> 21008-936 </td><td> 15ml, estéril </td></tr><tr><td> Fenol: clorofórmio: álcool isoamílico (25:24:1 v / v / v) </td><td> Invitrogen </td><td> 15593-031 </td><td> UltraPure, 100ml </td></tr><tr><td> Amicon Dispositivo Ultra-4 Centrífuga </td><td> Millipore </td><td> UFC803024 </td><td> Ultracel-30 membrana </td></tr><tr><td> 20X TE Buffer, Rnase livre </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

Vírus, especialmente os bacteriófagos (fagos), são as entidades mais numerosas biológica em 1,2 Terra. Vírus da célula hospedeira modular a abundância e diversidade, contribuem para a ciclagem de nutrientes, alterar anfitrião fenótipo celular, e influenciar a evolução de ambas as células do hospedeiro e as comunidades viral através da transferência lateral de genes 3. Numerosos estudos têm destacado a diversidade genética impressionante de vírus e seu potencial funcional em uma variedade de ambientes naturais. Metagenomic técnicas têm sido usadas para estudar a diversidade taxonômica e potencial funcional do complexo assemblages viral cujos membros contêm DNA de fita simples (ssDNA), double-stranded DNA (dsDNA) e genótipos RNA 4-9. Protocolos biblioteca atual de construção usado para estudo de DNA contendo ambientais ou RNA contendo vírus requerem um tratamento nuclease inicial, a fim de remover os modelos não-alvo 10. No entanto, uma compreensão abrangente do complemento gene coletiva da comunidade vírus e diversidade de vírus requer o conhecimento de todos os membros, independentemente da composição do genoma. Fracionamento de ácido nucléico purificadas subtipos fornece um mecanismo eficaz pelo qual o estudo assemblages viral sem sacrificar um subconjunto de assinatura genética da comunidade. Hidroxiapatita, uma forma cristalina de fosfato de cálcio, tem sido empregada na separação de ácidos nucléicos, bem como proteínas e micróbios, desde os anos 1960 11. Ao explorar a interação entre o Ca carga com carga positiva 2 + íons da hidroxiapatita e da espinha dorsal de fosfato carregados negativamente dos subtipos de ácido nucléico, é possível eluir preferencialmente cada subtipo de ácido nucléico independente dos outros. Recentemente, esta estratégia empregada de forma independente fracionar os genomas de ssDNA, dsDNA e RNA-vírus que contenham em preparação de seqüenciamento de DNA 12. Aqui, apresentamos um método para o fracionamento e recuperação de ácidos viral ssDNA, dsDNA e RNA nucleico de misturado assemblages viral utilizando cromatografia de hidroxiapatita.

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

Nós descrevemos um método eficiente para separar single-stranded DNA, dupla fita de DNA e RNA de moléculas ambientais comunidades viral. Os ácidos nucleicos são fracionados por cromatografia em fase de hidroxiapatita com concentrações crescentes de fosfato contendo buffers. Este método permite o isolamento de todos os tipos de ácido nucléico viral a partir de amostras ambientais.

Separação de Single-stranded DNA, Double-stranded DNA e RNA de uma Comunidade Ambiental Viral Usando Cromatografia hidroxiapatita

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

separation-single-stranded-dna-double-stranded-dna-rna-from-an

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JoVE Journal

Immunology and Infection

31.4K visualizações.  The J. Craig Venter Institute. Nós descrevemos um método eficiente para separar single-stranded DNA, dupla fita de DNA e RNA de moléculas ambientais comunidades viral. Os ácidos nucleicos são fracionados por cromatografia em fase de hidroxiapatita com concentrações crescentes de fosfato contendo buffers. Este método permite o isolamento de todos os tipos de ácido nucléico viral a partir de amostras ambientais.

Vídeo: 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.

Geração de recombinante do vírus Influenza de DNA plasmidial

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

Imunologia e Infecção

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 de variantes de vírus de RNA Fidelity e Caracterização de frequência de mutação de vírus

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.

Desenvolvimento de células de tipo específico anti-HIV gp120 aptâmeros para entrega 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.

Preparação de DNA viral a partir nucleocapsids

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.

Um Protocolo de Chelex simples para extração de DNA de<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.

Estabelecimento de um<em&gt; In vitro</em&gt; Sistema para estudar o comportamento intracelular de<em&gt; Candida glabrata</em&gt; Em humanos THP-1 macrófagos

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.

Usando Click Chemistry medir o efeito da infecção viral sobre a síntese de RNA do Host-Cell

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.

Calculando a média de Viral glicoproteína do envelope Spikes de Electron Cryotomography reconstruções com 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.

Desenvolvimento de um sistema de entrega de DNA Economical por &quot;Acufection&quot; e sua aplicação à 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 ...

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.