Nos gustaría agradecer a la Fundación Canadiense para la Innovación, de la Columbia Británica Fondo para el Desarrollo del Conocimiento, Genoma Columbia Británica y el British Columbia Ministerio de Bosques y de rango para el apoyo a los estudios en curso de la productividad del suelo forestal. SL fue apoyado por una beca de la Fundación Centro TULA financiado por la diversidad microbiana y la evolución.

Parte I: La extracción de ADN Procedimiento 1. 8 horas son necesarias, para el procesamiento de seis muestras de exclusión de la cuantificación de ADN. Antes de iniciar la extracción, tendrá que pre-chill mortero, mano de mortero y una espátula en un congelador a -20 ° C o el uso de nitrógeno líquido. Además, pre-frío tubos de 50 ml que contiene 20 ml de cloroformo alcohol isoamílico (24:1) en el hielo. Ajuste el horno de hibridación a 65 ° C para precalentar. Compruebe solución CTAB, si se cristaliza caliente a 60 ° C para fundir los cristales. Completar la solución de desnaturalización y tampón de extracción mediante la adición de los últimos ingredientes justo antes de iniciar la extracción (ver receta a continuación). Llevar a cabo todos los procedimientos de extracción en una campana extractora. 2. Prepare el tampón de desnaturalización y tampón de extracción. Nota: Para maximizar el rendimiento de ADN, es fundamental para hacer precisamente buffers siguiendo las instrucciones. 3. Poner 2 g de suelo en un mortero previamente enfriado. Nota: Suelo recolectado en el campo debe ser congelado en hielo seco para el transporte y se almacenan a -80 ° C. Deshielo y el suelo tamizado con un tamiz de 2 mm antes de la extracción de ADN. Puede aumentar el importe de partida de suelo de hasta 10 g. 4. Añadir 1 ml de solución de desnaturalización de la tierra. 5. Congelar y triturar el suelo en N 2 líquido utilizando un mortero hasta que las partículas del suelo mirar en polvo y homogénea. Repetir dos veces por congelación y la trituración. Nota: Mantenga la muestra de suelo helado durante la molienda, utilizando nitrógeno líquido suficiente para cubrir la totalidad de la muestra de suelo. Fusión leve durante la molienda es aceptable. 6. Transferir el suelo de tierra a un tubo cónico de 50 ml con una espátula de pre-enfriado. Nota: La muestra de suelo de tierra pueden ser almacenadas a -80 ° C en este punto, si es necesario. 7. Añadir 9 ml de tampón de extracción. Para mezclar la solución y el suelo, agitar brevemente a baja velocidad. Nota: Para garantizar el buffer es homogénea, que se caliente a 60 ° C durante 10-15 minutos antes de su uso. Mantenga el resto de amortiguamiento a 60 ° C a lo largo de la extracción. Vortex a velocidad 8 (en una escala donde 10 es el máximo). 8. Incubar durante 40 min a 65 ° C en un horno de hibridación. Durante la incubación, girar continuamente los tubos a la velocidad más baja, o invierta suavemente los tubos cada 10 min. Nota: Coloque el tubo en horizontal en un horno de hibridación para permitir buffer ponerse en contacto con el suelo sobre una superficie más grande. La solución puede parecer un poco viscosa durante la incubación. 9. Centrifugar a 1800 x g, durante 10 minutos a 10-14 ° C. Transferir el sobrenadante en el pre-enfriado tubo cónico de 50 ml que contienen 20 ml de cloroformo-alcohol isoamílico (24:1), mantener en hielo. Nota: Cuando se transfiere el sobrenadante, tenga cuidado de no alterar la capa orgánica (capa inferior). Agregar 1-2 ml de sobrenadante, si es necesario con el fin de recoger sólo sobrenadante limpio. A menudo se puede ver una capa de color beige (aproximadamente 1-3 mm de espesor) en la parte superior del sobrenadante. No moleste a esta capa al recoger el sobrenadante. Pruebe a deslizar la punta a lo largo de la pared del tubo para evitar la rotura de esta capa de color beige. Si el sobrenadante es demasiado turbia, repetir la extracción de alcohol de cloroformo-isoamílico una vez más en el sobrenadante. No utilizar pipetas desechables de plástico para transferir el cloroformo, el cloroformo se derrita el plástico. Use una pipeta de vidrio, o un vaso cilindro graduado, o verter cloroformo directamente en tubos de 50 ml cónicos. 10. Extracto de las pastillas de suelo 2 veces más; 10.1. Añadir 5 ml de tampón de extracción de los sedimentos del suelo, mezcle suavemente con una punta ml seguido de agitación breve a baja velocidad. Nota: Agitar y mezclar el búfer antes de su uso. 10.2. Se incuba a 65 ° C durante 10 minutos en horno de hibridación. Centrifugar a 1800 x g, durante 10 minutos a 10-14 ° C. Transferir el sobrenadante en el tubo en el paso 9. Mantener el tubo con sobrenadante recogido y el alcohol cloroformo-isoamílico en hielo durante la extracción. Nota: Asegúrese de agregar el sobrenadante al mismo tubo, sin cruzar la mezcla entre las muestras. 10.3. Repetir 9.1 y 9.2 una vez más. 11. El uso de un plato giratorio, con mucha suavidad oscilar el tubo que contiene el sobrenadante recogido (14-19 ml) y cloroformo-alcohol isoamílico a 1,25 rpm durante 10 minutos. Nota: Cierre la tapa muy bien para evitar fugas. Coloque una toalla de papel en la placa oscilante antes de colocar el tubo de manera alguna fuga se puede detectar fácilmente. 12. Centrifugar a 1800 x g, durante 20 minutos a 10-14 ° C. 13. Transferencia de fase acuosa a un tubo nuevo. Nota: No molestar, la interfase entre la fase acuosa (capa superior) y organizacionesnic (capa inferior) cuando se transfiera el sobrenadante. Deje aproximadamente 1,5-2 ml de sobrenadante para garantizar la recogida de sólo sobrenadante limpio. 14. Recuperar el ADN con alcohol isopropílico (paso 15.a - 21.a). Por otra parte, el uso Amicon ® Ultra-15 dispositivos centrífugos de filtro (paso 15.b - 21.b) si espera muy baja cantidad de biomasa en la muestra, que se produce apenas visible precipitado por precipitación alcohol isopropílico que es difícil de recuperar. 15.a. Coloque el sobrenadante a un tubo de ultracentrífuga nuevo. 16.a. Agregar el alcohol isopropílico al sobrenadante recogido en una proporción de volumen de 0,6:1. Invertir los tubos suavemente varias veces para mezclar. 17.a incuba durante 30 min a temperatura ambiente. 18.a. Centrifugar a 16.000 x g, durante 20 minutos a 20 - 25 ° C. Asegúrese de que los pesos de todos los tubos están bien equilibradas antes de la centrifugación. Nota: Marque la dirección de la fuerza centrífuga en el tubo, para que pueda detectar el pellet de ADN con mayor facilidad cuando el rendimiento de ADN es baja. 19.A. Eliminar el alcohol isopropílico por decantación o la aspiración al vacío. Tenga cuidado de no alterar el pellet de ADN. Nota: Eliminar el alcohol isopropílico, tan pronto como la centrifugación se ha completado. Tenga mucho cuidado de no perder el pellet de ADN. El tamaño del pellet de ADN puede variar ampliamente dependiendo del tipo de suelo, de muy visible (~ 9 mm de diámetro) que apenas se ve. Se recomienda para eliminar los residuos de alcohol isopropílico por la aspiración a lo largo de la pared del tubo que no cuidado para que al succionar el pellet de ADN. 20.A. Seco en pelets de ADN a temperatura ambiente durante 5 - 20 min. Nota: No más seco en pelets de ADN, de lo contrario será difícil de disolver el ADN. 21.A. Resuspender el pellet de ADN en 200 l de TE. Se mezcla con unos golpecitos suaves. La transferencia de la solución de ADN en un tubo de 1,5 ml. Mantener el tubo a 4 ° C durante la noche con el fin de disolver completamente el ADN. Nota: Dependiendo del tamaño del pellet, es posible ajustar el volumen de TE que añadir. Por lo general, 200 a 400 l de TE funciona bien. * Como alternativa, siga los pasos 15.b - 21.b. 15.b. Prelavado un Amicon ® Ultra-15 dispositivos de filtro centrífugo, agregar 15 ml de TE en el dispositivo y se centrifuga a 3500 x g, durante 10 minutos. Deseche el flujo continuo. 16.b. Coloque la solución de ADN a partir del paso 13 al filtro Amicon pre-lavado. Centrifugar a 3500 x g hasta que el volumen del ADN se reduce a 200 a 500 l. Deseche el flujo continuo. 17.B. Lavar el ADN con el doble de TE, agregar 10 ml de TE a los dispositivos de Amicon filtro. Centrifugar a 3500 x g hasta que el volumen del ADN se reduce a aproximadamente 200 l. Deseche el flujo continuo. Repetir una vez más. 18.b. La transferencia de la solución de ADN en el filtro a un tubo de 1,5 ml nuevo. 19.b. Añadir 40 ml TE al filtro Amicon. Lave ambos lados de la membrana de filtro pipeteando arriba y abajo con el fin de recuperar los residuos de ADN en el filtro. Añadir esta solución de lavado en el tubo de paso 18.b. 20.b. Si es necesario, el ADN puede ser más concentrado por Microcon ® Ultracel YM-30 Unidad de filtro centrífugo, pre-lavado del filtro mediante la adición de 200 l de TE y centrifugar a 10.000 x g durante 7 minutos. Añadir la solución de ADN al filtro y centrifugar a 5000 x g durante 1 min. Repetir la centrifugación hasta que la cantidad de solución de ADN en el filtro reduce a 50 l aproximadamente. Nota: El volumen máximo inicial de la muestra para Microcon filtro es de 500 l. No centrifugar a velocidades de más de 14.000 x g. No más de centrífuga hasta que se seque completamente la membrana. Asegúrese de que algo de líquido todavía cubre el filtro después de la centrifugación. Si más de centrifugado y la membrana se seca, a continuación, añadir 15 l TE, se agita suavemente durante 30 segundos, y continúe con el paso 21.b. 21.b. Coloque la unidad del filtro boca abajo en un tubo de Microcon nuevas y centrifugar a 1000 xg durante 3 minutos para recuperar el ADN. 22. Cuantificar el ADN de gel de TAE (0,8%, 0.5xTAE, 50 V de 4 horas), y por espectrofotómetro Nanodrop o Pico-verde de ensayo y lector de placas. Nota: la cantidad de ADN se puede estimar mediante la comparación de la banda de la muestra a la banda con marcador de concentración conocida (marcadores moleculares de ADN de alta o λHindIII) en el gel. 23. Compruebe la integridad del ADN de alto peso molecular mediante electroforesis en campo pulsado (PFGE) con un 1%, 1xTAE gel (Para más detalles vea el Paso II, part2 en <a href="https://www.jove.com/index/details.stp?id=1387">https://www.jove.com/index/details.stp?id = 1387</a> , doi: 10.3791/1387 por Taupp et al 2009).. Nota: Se puede utilizar gel de agarosa normal para este propósito de control de calidad en lugar de agarosa de baja fusión. En electropho breve,las condiciones de resistencia son los siguientes: 2-250 kb, 6 voltios, el tiempo inicial de conexión: 5 segundos, tiempo final de conmutación: 15 segundos, una duración de 12 horas a 14 ° C. SYBR Gold mancha durante una hora para visualizar el ADN en el gel. 24. Si los pesos moleculares de ADN extraído son satisfactorios, a continuación, proceder a la siguiente etapa CsCl centrifugación de gradiente. Por otra parte, el ADN puede ser almacenado a -20 ° C o -80 º C antes de la centrífuga CsCl. El tamaño medio del ADN debe ser igual o superior a 36 Kb, si usted quiere construir una gran inserción de la biblioteca metagenómica ADN. Parte II. De purificación de ADN usando ultracentrifugación gradiente de cloruro de cesio Preparación de la centrífuga: 1,5 - 2 horas Centrifugación: 18 horas La eliminación de bromuro de etidio (EtBr) y la concentración de ADN después de la centrifugación: 3 horas Procedimiento Para más información, consulte Wright et al. . 2.009 <a href="https://www.jove.com/index/details.stp?id=1352">https://www.jove.com/index/details.stp?id=1352</a> , doi: 10.3791/1352 Resultados representante / Resultados La cantidad total de ADN genómico aislado de 2 g de bosque orgánica del suelo (O horizonte) y horizontes minerales (AE, AB, y los horizontes Bt) (la muestra de la productividad del suelo a largo plazo (LTSP) sitio ubicado en Skulow lago, BC, Canadá y gestionados por BC Ministerio de Bosques y rango) fue de 10 mg a 180 mg. El tamaño medio de ADN fue aislado por lo general entre 40 a 60 kb (Figura 1). La relación de absorbancia a 260 nm/280 nm entre 1.3 a 1.6 y se produjo un pico de 230 nm, lo que puede indicar contaminación de ácidos húmicos observa con frecuencia en las muestras de suelo. La centrifugación en gradiente de CsCl reduce drásticamente la contaminación y también aumentó la proporción de 260/280 a 1,8 a 1,9 como se muestra en la Figura 2a y 2b. <img src="files/ftp_upload/1569/1569fig1.jpg" alt="Figura 1" /> Figura 1. PFGE imágenes del ADN genómico extraído antes de ultracentrifugación CsCl. lane1: 1 kb marcador de 100 ng, carril 2: λHind III marcador de 100 ng, carril 3: λHind III marcador de 250 ng, carril 4: λHind III marcador de 500 ng, lane5: Fosmid 36 kb marcador de 100 ng, calle 6 y 7: genómica ADN aislado de las Ae horizonte mineral del suelo, carril 8 y 9: ADN genómico aislado de la AB horizonte mineral del suelo en el sitio LTSP encuentra Skulow lago, BC <a href="http://jove.com/files/ftp_upload/1569/1569fig2.jpg"><img src="/files/ftp_upload/1569/1569fig2tmb.jpg" alt="Figura 2" border="0" /></a> Chromograms la figura 2. De ADN genómico detectado por espectrofotometría antes (A) y después de la ultracentrifugación CsCl (B). Tenga en cuenta la reducción en el valor de absorbancia a la longitud de onda 230 nm después de la ultracentrifugación CsCl, lo que indica una mejora en la calidad del ADN genómico.

<ol>
	<li>Hurt, R. A., Qiu, X., Wu, L., Roh, Y., Palumbo, A. V., Tiedje, J. M., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+recovery+of+RNA+and+DNA+from+soils+and+sediments.">Simultaneous recovery of RNA and DNA from soils and sediments.</a> AEM. 67 (10), 4495-4503 (2001).</li><li>Wright, J. J., Lee, S., Zaikova, E., Walsh, D. A., Hallam, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+extraction+from+0.22+&#956;M+Sterivex+filters+and+cesium+chloride+density+gradient+centrifugation.">DNA extraction from 0.22 &#956;M Sterivex filters and cesium chloride density gradient centrifugation.</a> JoVE. , (2009).</li><li>Taupp, M., Lee, S., Hawley, A., Yang, J., Hallam, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+insert+environmental+genomic+library+production.">Large insert environmental genomic library production.</a> J Vis Exp. , (2009).</li></ol>

Receta * La desnaturalización solución 4 M guanidina isotiocianato 10 mM Tris-HCl (pH 7,0) 1 mM EDTA 0,5% 2-mercaptoetanol <table border="0" style="font-size:9pt; width:400px;"><tr><td colspan="2"> Solución de desnaturalización: 10 ml en total </td></tr><tr><td> Guanidina isotiocianato (118,16 MW) </td><td> 4,73 g </td></tr><tr><td> 1M Tris-HCl (pH 7,0) </td><td> 100 l </td></tr><tr><td> 0,5 M EDTA </td><td> 20 l </td></tr><tr><td colspan="2"> Agregue el agua a 9,95 ml en total. </td></tr><tr><td colspan="2"> Autoclave. </td></tr><tr><td colspan="2"> Añadir 50 l de 2-mercaptoetanol justo antes del uso. (5 l de 2-mercaptoetanol por 1 ml de solución desnaturalizante) Nota: Mantenga la solución de desnaturalización a 4 ° C. No utilice buffer más de una semana. Si es posible, una solución tampón fresca para su uso. </td></tr></table> ** Tampón de extracción 100 mM tampón fosfato de sodio [pH 7.0] 100 mM Tris-HCl [pH 7.0] 100 mM EDTA [pH 8.0] 1,5 M NaCl 1% hexadeciltrimetilamonio (CTAB) SDS al 2% <table border="0" style="font-size:9pt; width:400px;"><tr><td colspan="2"> Buffer de extracción de una L en el total de </td></tr><tr><td> De sodio 1M tampón fosfato [pH 7.0] * </td><td> 100 ml </td></tr><tr><td> 1M Tris-HCl [pH 7.0] </td><td> 100 ml </td></tr><tr><td> 0,5 M EDTA [pH 8.0] </td><td> 200 ml </td></tr><tr><td> 5 M NaCl </td><td> 300 ml </td></tr><tr><td colspan="2"> Autoclave y conservar a temperatura ambiente. </td></tr><tr><td colspan="2"> Añadir 200 ml, el bromuro de 5% hexadeciltrimetilamonio (CTAB, autoclave) y 100 ml, 20% SDS (autoclave), justo antes de su uso. Si CTAB se cristalizó, se derrite a 60 ° C. * 1M tampón fosfato de sodio: 57,7 ml, de sodio 1M fosfato monobásico (NaH 2 PO 4) y 42,3 ml, 1 M de fosfato sódico dibásico (NaH 2 PO 4). Ajustar el pH a 7.0 Nota: No use el 20% SDS si tiene la precipitación. Es normal ver suspensión lechosa cuando se agrega a la solución de SDS. Una vez que se agrega el desarrollo sostenible, colocar el tampón de extracción a 60 ° C para asegurar la SDS está bien suspendido. </td></tr></table>

extraction of high molecular weight genomic dna from soils and sediments

El microbioma del suelo es un reservorio inmenso y relativamente inexplorado de la diversidad genómica y la innovación metabólica que está íntimamente asociado a los nutrientes y el flujo de energía dentro de los ecosistemas terrestres. Cultivo independiente de genómica ambiental, también se conoce como metagenómica, enfoques promesa de un acceso sin precedentes a esta información genética con respecto a la reconstrucción de la vía y el cribado funcional de alto valor terapéutico y los procesos de conversión de biomasa. Sin embargo, el microbioma de la tierra sigue siendo un reto en gran parte debido a la dificultad en la obtención de ADN de alto peso molecular de una calidad suficiente para la producción de grandes bibliotecas de inserción. Aquí presentamos un protocolo para la extracción de alto peso molecular, el ADN genómico de la comunidad microbiana de los suelos y sedimentos. La calidad del ADN genómico aislado es ideal para la construcción de grandes bibliotecas de insertar genómica ambiental para las aplicaciones posteriores de secuenciación y detección. El procedimiento se inicia con la lisis celular. Paredes y membranas celulares de los microorganismos se lisan por fuerzas mecánicas (trituración) y químicos (β-mercaptoetanol). ADN genómico se aísla con tampón de extracción, el alcohol cloroformo-isoamílico y alcohol isopropílico. Los tampones utilizados para la lisis y etapas de extracción incluyen isotiocianato de guanidina y el bromuro de hexadeciltrimetilamonio (CTAB) para preservar la integridad de la alta molecular del ADN genómico de peso. Dependiendo de su aplicación posterior, el ADN genómico aislado puede ser purificado con cloruro de cesio (CsCl) ultracentrifugación gradiente, lo que reduce las impurezas tales como los ácidos húmicos. El primer procedimiento, la extracción, dura aproximadamente 8 horas, sin incluir la cuantificación de ADN paso. El gradiente de CsCl ultracentrifugación, es un proceso de dos días. Durante todo el procedimiento, el ADN genómico debe ser tratada con cuidado para evitar la esquila, evitar la agitación grave y dura pipeteo repetitivo.

Watch this Scientific Journal Video about Extraction of High Molecular Weight Genomic DNA from Soils and Sediments at JoVE.com

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

Una metodología para aislar de alto peso molecular y ADN genómico de alta calidad de la comunidad microbiana del suelo se describe.

La extracción de ADN de alta molecular genómico de peso de los suelos y los sedimentos

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

extraction-high-molecular-weight-genomic-dna-from-soils

Research

JoVE Journal

Biology

25.5K vistas.  University of British Columbia - UBC. Una metodología para aislar de alto peso molecular y ADN genómico de alta calidad de la comunidad microbiana del suelo se describe.

Video: Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

Isolation of Genomic DNA from Mouse Tails

El aislamiento de ADN genómico de colas de ratón

Investigación

Biología

DNA Extraction from 0.22 &mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been treated with storage/lysis buffer and archived at -80&deg;C, and to purify this DNA using a cesium chloride density gradient.  The protocol begins with two one-hour incubation steps to liberate DNA from cells and remove RNA.  Next, a series of Phenol:Chloroform and Chloroform extractions are performed followed by centrifugation to remove proteins and cell membrane components, collection of the aqueous DNA extract, and several buffer exchange steps to wash and concentrate the extract.  Part Five describes the optional purification via cesium chloride density gradient.  It is recommended to work with less than 15 samples at one time to avoid confusion and cut down protocol time. The total time required for this protocol depends on the number of samples to be extracted. For 10-15 samples and assuming the proper centrifugation equipment is available, this entire protocol should take 3 days. Make sure you have the hybridization ovens set to temperature at the outset of the process.

DNA Extraction from 0.22 &amp;mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

We describe a method for extraction of high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;m Sterivex filters, followed by cesium chloride density gradient centrifugation for purification.

Extracción de ADN de 0,22 M Filtros Sterivex y centrifugación de densidad de cloruro de cesio Gradient

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

Genómica resonancia magnética - un recurso público para el estudio de patrones de secuencia en el ADN genómico

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

Extracción de ADN a partir de material incluido en parafina para análisis genéticos y epigenéticos

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

La extracción de ADN de alto peso molecular a partir de tapetes microbianos

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.
The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes1, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.
We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.

We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.

Un método práctico y novela para extraer el ADN genómico de los kits de extracción de sangre para la Preservación Plasma Protein

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

Captura selectiva de 5-hidroximetilcitosina a partir de ADN genómico

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Amplificación, secuenciación de próxima generación, y Genómica Mapeo de ADN retrovirales sitios de integración

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

La mucina en gel de agarosa de electroforesis: Western Blotting para glicoproteínas de alta peso molecular


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, host characteristics, and broader ecosystem functions. With this rapid increase in breadth and depth of sequencing capabilities have come methods to extract, amplify, analyze, and interpret environmental DNA successfully with maximum efficiency. Unfortunately, performing DNA extractions quickly can come at the cost of increasing the risk of contamination among samples. In particular, high-throughput extractions that are based on samples contained in a 96-well plate offer a relatively quick method, compared to single-tube extractions, but also increase opportunities for well-to-well cross-contamination. To minimize the risk of cross-contamination among samples, while retaining the benefits of high-throughput extraction techniques, we developed a new method for loading environmental samples into 96-well plates. We used pierceable PCR sealing films to cover each plate while loading samples and added samples first to PCR tubes before moving them into wells; together, these practices reduce the risk of sample drift and unintended double loading of wells. The method outlined in this paper provides researchers with an approach to maximize available high-throughput extraction techniques while reducing the risk of cross-contamination inherent to 96-well plates. We provide a detailed step by step outline of how to move from sample collection to DNA extraction while minimizing the risk of unwanted cross-contamination.

Current methods for loading 96-well plates for DNA extractions can be prone to contamination. We detail a new method for loading 96-well plates that reduces risk of cross-contamination among wells. Our method will help other researchers capitalize on the efficiency of high-throughput DNA extraction techniques and minimize risk of contamination.

Los métodos modificados para la carga de placas de extracción de ADN de alto rendimiento reducen el potencial de contaminación

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, ...