Esta investigación fue apoyada por la Oficina de Ciencia (BER), EE.UU. Departamento de Energía, del Acuerdo de Cooperación no. De-FC02-02ER63453 Microbiana de la Fundación Nacional de Ciencias del Programa de la secuenciación del genoma (los números 0626826 y 0731916 premio). Agradecemos a John Glass por su experiencia y asesoramiento técnico y K. Eric Wommack por su ayuda con la recogida de muestras ambientales.

1. Preparación de las soluciones Antes de realizar la cromatografía de hidroxiapatita, tampones de fosfato debe estar preparado y la hidroxiapatita debe estar correctamente hidratado. <ol><li> Solución de fosfato 1 M, pH 6,8: En un matraz de 1 litro se disuelven 119.98g de fosfato de sodio monobásico en 1 litro de agua estéril, tratada con DEPC H 2 O. Prepare un fosfato de sodio dibásico 1M solución en un frasco de 1 litro de disolución 141.96g de fosfato de sodio dibásico en 1 litro de agua estéril, tratada con DEPC H 2 O. Combine los mono-y di-solución en una proporción de 1:1. Lugar matraz en una placa de agitación y mezcla con una varilla de agitación magnética. Ajustar el pH a 6,8 mediante la adición de hidróxido de sodio (para aumentar el pH) o el ácido fosfórico (para disminuir el pH). </li><li> Tampón fosfato 0.12M: En un matraz de 500 ml, 30 ml de combinar la solución de fosfato 1 M, pH 6,8, 2,5 ml de sodio al 10% dodecilsulfato (SDS), y 5 ml de 0,5 M EDTA. Añadir estéril tratada con DEPC H2O hasta un volumen de 225 ml. Ajustar el pH a 6.8. Añadir estéril tratada con DEPC H2O a 250 ml. Autoclave. </li><li> Tampón fosfato 0.18M: En un matraz de 500 ml, 45 ml de combinar la solución de fosfato 1 M, pH 6,8, 2,5 ml de sodio al 10% dodecilsulfato (SDS), y 5 ml de 0,5 M EDTA. Añadir estéril tratada con DEPC H 2 O a un volumen de 225 ml. Ajustar el pH a 6.8. Añadir estéril tratada con DEPC H 2 O a 250 ml. Autoclave. </li><li> Tampón fosfato 0,20 M: En un matraz de 500 ml, 50 ml de combinar la solución de fosfato 1 M, pH 6,8, 2,5 ml de sodio al 10% dodecilsulfato (SDS), y 5 ml de 0,5 M EDTA. Añadir estéril tratada con DEPC H 2 O a un volumen de 225 ml. Ajustar el pH a 6.8. Añadir estéril tratada con DEPC H 2 O a 250 ml. Autoclave. </li><li> 0,40 M tampón fosfato: en un frasco de 500 ml, 100 ml de combinar la solución de fosfato 1 M, pH 6,8, 2,5 ml de sodio al 10% dodecilsulfato (SDS), y 5 ml de 0,5 M EDTA. Añadir estéril tratada con DEPC H 2 O a un volumen de 225 ml. Ajustar el pH a 6.8. Añadir estéril tratada con DEPC H 2 O a 250 ml. Autoclave. </li><li> &quot;Tampón fosfato 1.00M&quot; (concentración real de fosfato en esta solución es 0,91 M): En un matraz de 500 ml, 30 ml de combinar la solución de fosfato 1 M, pH6.8, 2,5 ml de sodio al 10% dodecilsulfato (SDS), y 5 ml de 0,5 M EDTA. Añadir estéril tratada con DEPC H2O hasta un volumen de 225 ml. Ajustar el pH a 6.8. Añadir estéril tratada con DEPC H 2 O a 250 ml. Autoclave. </li><li> Hidratación de hidroxiapatita: Peso de 1 g de hidroxiapatita y añadir a un tubo de 50 ml. Agregar 6 ml de tampón fosfato 0.12M a la hidroxiapatita e invierta para mezclar. Antes de su uso llevar la temperatura a 60 ° C y mezclar bien. Almacenar por largos períodos de tiempo a 4 ° C. </li><li> Antes de su uso, se equilibran todos los tampones de fosfato de hidroxiapatita y la hidratación a 60 ° C. </li></ol> 2. Preparación de la columna Econo- <ol><li> Cerrar llave de paso. Enjuague varias veces la columna H 2 O desionizada invirtiendo varias veces por la columna y decantación. </li><li> Retire la llave de paso de la Econo-columna y la columna de autoclave para esterilizar. </li><li> Vuelva a colocar y cerrar llave de paso. Añadir 1 ml de Sigmacote a la estéril Econo-columna y cubra todas las superficies de vidrio. Abrir la llave y se decanta. </li><li> Coloque la columna a un baño de agua circulante y ajustar la temperatura a 60 ° C. Para einsure la menor cantidad de pérdida de calor a través de la columna de envolver la columna en un agente aislante como papel de aluminio o aislamiento de tuberías de espuma se recomienda. </li><li> Enjuague la columna Econo-dos veces con agua estéril, libre de RNasa H 2 O y luego dos veces con agua estéril, libre de RNasa tampón fosfato 0.12M. </li></ol> 3. Hydroxypaptite Cromatografía <ol><li> Asegurarse de que la llave está cerrada, poco a poco añadir 2 ml de preparado de antemano, la hidroxiapatita se resuspendieron en la parte inferior de la columna Econo-ayuda de una pipeta serológica de 2 ml. </li><li> Deje que la hidroxiapatita de resolver por la fuerza de gravedad durante 30 minutos. </li><li> Drenaje de amortiguación de la columna y la llave de cierre. Combine los ácidos nucleicos con tampón fosfato 0.12M en un volumen final de 500 l, y se incuba a 60 ° C durante 10 minutos en un bloque de calor o baño de agua. </li><li> Aplicar rápidamente los ácidos nucleicos preparados en tampón fosfato 0.12M a la hidroxiapatita con una pipeta serológica de 2 ml, asegurándose de no molestar a la columna. </li><li> Permita que los ácidos nucleicos que se unen a la hidroxiapatita durante 30 minutos. </li><li> Colocar un tubo de 15 ml en la columna, abra la llave de paso y recoger la muestra inicial que contiene ADN de cadena simple. Cerrar llave de paso. </li><li> Agregar 6 ml de tampón fosfato 0.12M a la hidroxiapatita, asegurándose de no molestar a la columna, abra la llave de paso, y cobrar de una sola cadena de ADN en el tubo de 15 ml de la etapa anterior. Cerrar llave de paso. </li><li> Añadir un volumen de un fenol: cloroformo: alcohol isoamílico (25:25:1 v / v / v) en el tubo, mezclar vigorosamente y se centrifuga a 3500 xg durante 15 minutos. La transferencia de ADN de cadena simple que contiene sobrenadante a un tubo de 15 ml nuevos. </li><li> Repita 3,76 y 3,87 con 6 ml de solución amortiguadora de fosfato 0,20 M de elaúd y purificar el ARN de la columna y asegúrese de usar un tubo de 15 ml nueva colección. </li><li> Repita 3,76 y 3,87 con 6 ml de solución amortiguadora de fosfato 0,40 m para eluir y purificar DNA de doble cadena de la columna y asegúrese de usar un tubo de 15 ml nueva colección. </li><li> Repita 3,76 y 3,87 con 6 ml de tampón fosfato 1.00M para despojar a la columna de cualquier dsDNA residual asegurándose de usar un tubo de 15 ml nueva colección. </li></ol> 4. Desalinización de muestras de ácidos nucleicos <ol><li> Transferencia de 4 de 5 ml de muestra a un Amicon Ultra-4 dispositivo centrífugo de filtro equipado con un peso molecular 30.000 cortar la membrana Ultracel. </li><li> Concentrar la muestra a &lt;500μl al girar a 6.000 xg, 30 ° C, a 5 minutos (o hasta que el volumen de concentrado se logra) en un rotor de ángulo fijo. Deseche el flujo a través. </li><li> Agregue el resto de la muestra (s) a la Amicon Ultra-4 dispositivo de filtro centrífugo y llevar el volumen hasta 4 5 ml con RNasa libre Buffer 1X TE. </li><li> Concentrar la muestra a &lt;500μl al girar a 6.000 xg, 30 ° C, a 5 minutos (o hasta que el volumen de concentrado se logra) en un rotor de ángulo fijo. Deseche el flujo a través. </li><li> Agregar 4 5 ml de RNasa libre Buffer 1X TE a la Amicon Ultra-4 dispositivo de filtro centrífugo. Concentrar la muestra a &lt;500μl por centrifugación a 6.000 xg, 30 ° C, a 5 minutos (o hasta que el volumen de concentrado se logra) en un rotor de ángulo fijo. Deseche el flujo a través. </li><li> Repita el paso 4.5 un adicional de 5 veces para desalar por completo muestra de ácido nucleico (s). </li><li> La transferencia de la muestra restante (s) a un tubo estéril eppendorf 1.7ml. Añadir un volumen de acetato de sodio 3M 1/10th, pH 7,0, dos volúmenes de etanol al 100%, y 1μl de Glycoblue. Mezclar bien por agitación. </li><li> Centrifugar a 28.000 xg, 4 º C, 60 minutos. Decantar con cuidado de no perturbar el sedimento. Agregar 300μl de etanol al 70%. Giran a 28.000 xg temperatura ambiente, durante 10 minutos. Se decanta, seco y resuspender cada fracción en un volumen adecuado de RNasa libre de buffer TE. </li></ol> 5. Los resultados representativos: La Figura 1 resume los métodos presentados para el uso de cromatografía de hidroxiapatita para fraccionar ssDNA, dsDNA y los ácidos nucleicos ARN viral a partir de un conjunto mixto. Este método explota la interacción de cargas entre el esqueleto de fosfato con carga negativa de los ácidos nucleicos y la carga positiva iones Ca 2 + presentes en la hidroxiapatita y permite el fraccionamiento eficiente de los subtipos de ácido nucleico (ssDNA, dsDNA y ARN) con el aumento de las concentraciones de tampón fosfato 12. El fraccionamiento de una hidroxiapatita in vitro &quot;comunidad&quot; de los más conocidos ssDNA (M13mp18), ADN de doble cadena (lambda) y ARN (MS2 y phi6) genomas virales se ilustra en la Figura 2. Los ácidos nucleicos se combinaron en concentraciones iguales, aplicada a la columna de hidroxiapatita y se eluyeron con un aumento de la concentración de tampón fosfato. Cada subtipo de ácido nucleico eluye independiente de los demás con menos del 9% acumulado de excedentes de una fracción a otra. La aplicación de esta técnica a los ácidos nucleicos aislados de una comunidad viral recogidos en la bahía de Chesapeake se muestra en la Figura 3. El rendimiento total de ácidos nucleicos aislados de virus purificado se aplicó a la columna de hidroxiapatita. Material genómico que consta de ssDNA, el ARN y el ADN de doble cadena se eluyó de forma independiente con 0.12M, y las concentraciones de 0,20 M de tampón fosfato 0.40M/1.00M respectivamente. Doble hebra de ADN se eluye en las concentraciones de fosfato de 0,40 m y 1.00M y en este caso, domina esta comunidad viral en comparación con los genotipos ssDNA y el ARN. Esta observación es consistente con la composición de la comunidad espera viral de ambientes marinos y estuarinos donde la mayoría de los ácidos nucleicos son recuperables dsDNA 13. <img src="/files/ftp_upload/3146/3146fig1.jpg" alt="Figura 1"/> Figura 1. Diagrama de flujo del método de cromatografía de hidroxiapatita para el fraccionamiento de los ácidos nucleicos. Una mezcla de ssDNA, dsDNA y ARN preparado en tampón fosfato 0.12M se calienta a 60 ° C y se aplica a una columna de hidroxiapatita mantenido a una temperatura constante de 60 ° C con un baño de agua circulante. Los ácidos nucleicos (ADN de cadena simple, ARN y ADN de doble cadena) se eluyen de la hidroxiapatita con concentraciones crecientes de un tampón fosfato que contienen. Esta cifra ha sido reproducido con el permiso de la Sociedad Americana de Microbiología y fue presentado originalmente en Andrews-Pfannkoch et al. 2010 12. <img src="/files/ftp_upload/3146/3146fig2.jpg" alt="Figura 2"/> Figura 2. Separación de los más conocidos ácidos nucleicos virales usando la cromatografía de hidroxiapatita. Concentraciones iguales de M13mp18 ssDNA, ssRNA MS2, dsRNA phi6 y lambda ADN de doble cadena (carriles 2-5, respectivamente) se combinaron (calle 6) y se aplicó a una columna de hidroxiapatita. Single-ADN de cadena (M13mp18), el ARN (MS2/phi6) y ADN de doble cadena (lambda) se eluyeron con independencia de la columna de hidroxiapatita con 0.12M (carril 8), 0.18M (línea 9) y 0.40M/1.00M (calle 10) . Una escalera de 1kb (líneas 1, 7) se utilizó para confirmar tamaño del genoma. Esta cifra ha sido reproducido con el permiso de la Sociedad Americana de Microbiología y fue presentado originalmente en Andrews-Pfannkoch et al. 2010 12. <img src="/files/ftp_upload/3146/3146fig3.jpg" alt="Figura 3"/> Figura 3. Separación de ácidos nucleicos virales aisladas de una comunidad dentro de la bahía de Chesapeake. Los ácidos nucleicos fueron aislados y se aplicó a una columna de hidroxiapatita. ssDNA (carril 0.12M), el ARN (carril de 0,20 M) y ADN de doble cadena (carriles 0.40M/1.0M) se eluye de forma independiente mediante la concentración de tampón fosfato de 0.12M, 0,20 M y 0.40M/1.00M, respectivamente. Misa de alta escalera del ADN (carril L1) y Ladder 1kb (carril L2) fueron utilizados para confirmar visualmente peso molecular aproximado y la masa de los ácidos nucleicos. Esta cifra ha sido reproducido con el permiso de la Sociedad Americana de Microbiología y fue presentado originalmente en Andrews-Pfannkoch et al. 2010 12.

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No hay conflictos de interés declarado.

La metodología de la cromatografía de hidroxiapatita que aquí se presenta es una herramienta muy eficiente y robusto para el fraccionamiento de los ácidos nucleicos de los conjuntos mixtos viral, cuando el objetivo es el estudio de la composición total de ácidos nucleicos de la comunidad. Por lo general, ssDNA, el ARN y ADN de doble cadena se eluyen de la columna de las concentraciones de pho tampón fosfato mayor que 0 y 0,40 m aproximadamente ~ respectivamente. Sin embargo, cada preparación de hidroxia...

<table border="1"><tbody><tr><th> Nombre del Material </th><th> Empresa </th><th> Número de catálogo </th><th> Número de catálogo </th></tr><tr><td> Econo-Columna </td><td> Bio-Rad </td><td> 737-0717 </td><td> 0.7cm paquete de ID / 2 </td></tr><tr><td> Hidroxiapatita </td><td> Bio-Rad </td><td> 130-0520 </td><td> Grado de ADN de Bio-Gel Gel HTP, 100g/td&gt; </tr><tr><td> El fosfato de sodio, monobásico </td><td> VWR </td><td> VW1497-01 </td><td> Monohidrato, cristal 500g </td></tr><tr><td> Fosfato sódico, dibásico </td><td> VWR </td><td> VW1496-01 </td><td> Anhidro, en polvo de 500 g </td></tr><tr><td> El agua tratada con DEPC </td><td> Invitrogen </td><td> AM9922 </td><td> 1L </td></tr><tr><td> 10% SDS solución </td><td> Invitrogen </td><td> 24730-020 </td><td> UltraPure, 1L </td></tr><tr><td> 0,5 M EDTA </td><td> Invitrogen </td><td> AM9262 </td><td> pH 8,0, 1 litro </td></tr><tr><td> Sigmacote </td><td> Sigma-Aldrich </td><td> SL2-25ML </td><td></td></tr><tr><td> 2 ml serológicas pippette </td><td> VWR </td><td> 89130-884 </td><td> Poliestireno, estéril </td></tr><tr><td> BD Falcon Tubos de centrífuga </td><td> VWR </td><td> 21008-936 </td><td> 15 ml, estéril </td></tr><tr><td> Fenol: cloroformo: alcohol isoamílico (25:24:1 v / v / v) </td><td> Invitrogen </td><td> 15593-031 </td><td> UltraPure, 100ml </td></tr><tr><td> Amicon Ultra-4 dispositivos centrífugos </td><td> Millipore </td><td> UFC803024 </td><td> Ultracel-30 de membrana </td></tr><tr><td> 20X buffer TE, RNasa libre </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

Los virus, particularmente los bacteriófagos (fagos), son las entidades biológicas más numerosas en 1,2 la Tierra. Los virus modulan la abundancia de la célula huésped y la diversidad, contribuir a los ciclos de los nutrientes, alterar anfitrión fenotipo de las células, y la influencia de la evolución de la célula huésped y de las comunidades viral a través de la transferencia lateral de genes 3. Numerosos estudios han puesto de relieve la asombrosa diversidad genética de los virus y su potencial funcional en una variedad de ambientes naturales. Técnicas de metagenómica se han utilizado para estudiar la diversidad taxonómica y el potencial funcional de los complejos conjuntos viral cuyos miembros contienen ADN de cadena sencilla (ssDNA), el ADN de doble cadena (dsDNA) y los genotipos de ARN 4-9. Los protocolos actuales de construcción de la biblioteca para estudiar el medio ambiente que contienen ADN o ARN que contienen los virus requieren un tratamiento inicial de la nucleasa con el fin de quitar las plantillas no focalizados a 10. Sin embargo, una comprensión global de la dotación genética colectiva de la comunidad de virus y la diversidad del virus requiere el conocimiento de todos los miembros, independientemente de la composición del genoma. Fraccionamiento de ácidos nucleicos purificados subtipos proporciona un mecanismo eficaz para que el estudio conjuntos viral sin tener que sacrificar una parte de la firma genética de la comunidad. Hidroxiapatita, una forma cristalina de fosfato de calcio, se ha empleado en la separación de los ácidos nucleicos, así como las proteínas y los microbios, desde 1960 11. Mediante la explotación de la interacción de cargas entre la carga positiva iones Ca 2 + de la hidroxiapatita y el esqueleto de fosfato con carga negativa de los subtipos de ácidos nucleicos, es posible eluir preferentemente cada subtipo de ácidos nucleicos independiente de los demás. Recientemente hemos empleado esta estrategia de forma independiente a fraccionar los genomas de ssDNA, dsDNA y los virus que contienen ARN en la preparación de la secuenciación del ADN 12. A continuación, presentamos un método para el fraccionamiento y la recuperación de ssDNA, dsDNA y el ARN viral ácidos nucleicos de los conjuntos mixtos viral mediante cromatografía 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

Se describe un método eficiente para separar ADN de cadena sencilla, doble cadena de ADN y las moléculas de ARN viral de las comunidades del medio ambiente. Los ácidos nucleicos son fraccionados utilizando cromatografía de hidroxiapatita con una creciente concentración de fosfato que contienen buffers. Este método permite el aislamiento de todos los tipos de ácido nucleico viral de las muestras ambientales.

La separación de una sola hebra de ADN, el ADN de doble cadena y el ARN de una comunidad ambiental viral mediante cromatografía de hidroxiapatita

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 vistas.  The J. Craig Venter Institute. Se describe un método eficiente para separar ADN de cadena sencilla, doble cadena de ADN y las moléculas de ARN viral de las comunidades del medio ambiente. Los ácidos nucleicos son fraccionados utilizando cromatografía de hidroxiapatita con una creciente concentración de fosfato que contienen buffers. Este método permite el aislamiento de todos los tipos de ácido nucleico viral de las muestras ambientales.

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.

Generación de recombinantes del virus de influenza a partir del ADN plásmido

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

Investigación

Inmunología e Infección

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.

El aislamiento de las variantes de los virus de ARN de fidelidad y caracterización de la frecuencia de mutación de virus

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.

Desarrollo de programas específicos de células de tipo anti-gp120 del VIH aptámeros para la entrega de 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.

Preparación de ADN viral de nucleocápside

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 protocolo Chelex simple para la extracción de ADN a partir 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.

Establecimiento de un<em&gt; In vitro</em&gt; Sistema para estudiar el comportamiento intracelular de<em&gt; Candida glabrata</em&gt; En 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 Química para medir el efecto de la infección viral en el Host-Cell ARN Síntesis

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.

Un promedio de glicoproteína de la envoltura viral Espigas de Electron cryotomography reconstrucciones utilizando 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.

Desarrollo de un sistema de entrega de ADN económico por &quot;Acufection&quot; y su aplicación a la investigación de la piel

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.