1. Recogida de muestras <ol><li> Recoger muestras de sangre de individuos con el consentimiento informado adecuado. Para cada individuo, dibuja 4 a 5 ml de sangre en un tubo P100 (BD Diagnostics, Franklin Lake, NY, EE.UU.). Para propósitos de comparación, al mismo tiempo recoger la sangre en un tubo con EDTA. </li><li> Los tubos P100 BD contienen 15,8 ml K2EDTA secado por pulverización y un amplio espectro de cóctel patentada liofilizado de inhibidores de la proteasa para evitar la coagulación y estabilizar las proteínas del plasma. También contienen un separador mecánico. Después de recoger la sangre entera, mantener los dos tubos a temperatura ambiente durante 60 minutos a toda la noche hasta que se produce el procesamiento. </li><li> Centrifugar los tubos BD P100 a 2500 g durante quince minutos a temperatura ambiente. Transferir el plasma recogido por encima del separador mecánico en tubos de micro centrífuga. </li><li> Guarde los tubos P100, que contiene la sangre compactado debajo del separador, a -80 ° C hasta que esté listo para comenzar la ext ADNraction. </li></ol> 2.1 A partir de la sangre del tubo P100 (P100_DNA) <ol><li> Los tubos de P100 se almacenan a -80 ° C después de la extracción de plasma. Dentro de los 3 a 4 meses, recuperar el tubo que contiene la sangre P100 compactado del congelador y descongelar a 37 ° en un baño de agua durante 5 minutos (Figura 1). Retire cualquier plasma residual que se encuentra por encima del separador. Añadir 2 ml de solución de sacarosa al 17% (solución de gradiente probado en casa - datos no publicados) en los tubos de P100 por encima del separador (Figura 1B). Centrifugar los tubos a temperatura ambiente durante 20 min a 2500 g; este proceso empuja el separador mecánico a la parte superior del tubo y produce tres capas de solución que incluye la capa leucocitaria (Figura 1C). Extraer manualmente el separador utilizando un clip de metal gancho steriled (Figura 1D). </li><li> Después de recuperar el separador mecánico de cada tubo (Figura 1D), retire y discard de la capa de sacarosa superior. Transferir la capa media, que representa la capa de la capa leucocitaria (BC), en un tubo cónico de 15 ml estéril. Añadir tampón fosfato salino (PBS) para aumentar el volumen de la capa leucocitaria a 3 ml. Extraer el ADN a partir de la capa leucocitaria utilizando reactivos de la Blood Kit Qiagen Puregene según la recomendación del fabricante, a excepción de una velocidad de centrifugación de 2500 g (en lugar de 2000 g). </li><li> Para lisar y eliminar las células rojas de la sangre (RBC), añadir 9 ml de RBC lisis a 3 ml de la capa leucocitaria. Invertir cada tubo varias veces y se incuba a temperatura ambiente durante 10 min con inversión ocasional durante la incubación. Girar cada mezcla hacia abajo a 2.500 g durante 5 minutos y desechar el sobrenadante. Tratar el sedimento restante en cada tubo con 3 ml de solución de lisis celular y agitar durante 15 seg. Se trata el lisado con 1 ml de solución de precipitación de la proteína y agitar durante 15 seg. </li><li> Se centrifuga a 2.500 g durante 5 min y transferir el sobrenadante a un tubo cónico de 15 ml que contiene fresca3 ml de isopropanol al 100%. Invertir los nuevos tubos de 10 veces y centrifugar a 2500 g durante 3 min. Eliminar el sobrenadante y añadir 1 ml de etanol al 70%. Invertir los tubos cónicos de un par de veces para desalojar y lavar el sedimento de ADN. Centrifugar a 2500 g durante 1 min. Dejar que el precipitado se seque durante 3 min (colocar el tubo boca abajo) a temperatura ambiente y luego suspender el ADN con 250 ml de solución de rehidratación a 37 ° C durante 10 min y la transferencia a un pre-marcado con 1,7 ml microtubo. Guarde los tubos a -80 ° C hasta su uso. </li></ol> 2.2 De Sangre EDTA Ttube (EDTA_DNA) <ol><li> La sangre entera para las pruebas de inmunohematología rutina se recoge en tubos de plástico vaccutainer revisten mediante pulverización con 10,8 mg de K 2 EDTA y se almacenaron a -80 ° C. Debido a que los tubos no contienen un separador mecánico, la extracción de ADN no incluye las etapas que implican el separador mecánico (2.1.1 y 2.1.2). </li><li> Dentro de los 3 a 4 meses de almacenamiento de la sangre, el DNA se extrae mediante el KingFisher Flex (Thermo Fisher Scientific, Waltham, MA, EE.UU.). Se trata de un sistema de extracción de ADN basado en la tecnología de automatización de partículas magnéticas y se compone de células de lisis / unión a ADN, varias etapas de lavado y elución de ADN. </li><li> El kit utilizado en la extracción de ADN es el Flex 24 kit de ADN de sangre KingFisher (Qiagen, Alemania). </li></ol> 3. ADN Cantidad y Evaluación de la Calidad La cantidad, la calidad y la integridad del ADN genómico fueron evaluados por una combinación de método que incluye PicoGreen, la espectroscopia y la electroforesis. <ol><li> La concentración del ADN genómico se determina mediante la cuantificación Nanodrop y PicoGreen. </li><li> La pureza se determina a partir de la medición de 260/280 en relación el espectrofotómetro Nanodrop. </li><li> La muestra (500 ng) también está cargado en gel de agarosa al 1% para evaluar la integridad (degradación) del ADN. </li></ol> 4. GenotypinLos ensayos g y Control de Calidad <ol><li> Cinco P100_DNA y sus correspondientes muestras EDTA_DNA son seleccionados al azar para su posterior análisis mediante el Immunochip personalizado (Inmuno Análisis de ADN BeadChip). Immunochip es un chip de genotipado Infinium contiene 196.524 polimorfismos y está diseñado para los estudios de inmunogenética 5. </li><li> Para cada muestra, 200 ng de ADN se amplifica, fragmentada, se precipitó y se resuspendió en el tampón de hibridación apropiada. </li><li> Las muestras desnaturalizadas se hibridan en Immunochips a 48 ° C durante un mínimo de 16 horas. </li><li> Después de la hibridación, los Immunochips se procesan para reacciones de extensión de una sola base y se tiñen. </li><li> Los immunochips a continuación, se obtuvieron imágenes utilizando un lector de matriz Illumina Hiscan bolas y los datos de intensidad de talón normalizados se cargan en el software Illumina GenomeStudio y se convierten en genotipos. Los genotipos se denominan utilizando el algoritmo autocalling de GenomeStudio. El control de calidad eninvolucra la exclusión de los polimorfismos de un solo nucleótido (SNPs) con una tasa de llamada &lt;95%. </li><li> La tasa de llamada SNP se utiliza como una medida de la eficacia de la prueba immunochip. Se calcula como el porcentaje del número total de ensayos en el chip (196.524 SNPs codificado en cada chip) que lograr suficiente intensidad de la señal y la calidad para recibir una asignación automatizada genotipo. Se espera que el ADN de alta calidad (260/280 proporción de 1,8 o superior y en ausencia de degradación) para lograr al menos la misma tasa de llamada como el ADN de control HapMap incluido en el ensayo immunochip. El control de calidad implica la exclusión de SNPs con una tasa de llamada &lt;95% en cada conjunto de datos. </li></ol>

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+rapid+method+for+extraction+of+DNA+from+fresh+and+cryopreserved+clotted+human+blood.">Simple and rapid method for extraction of DNA from fresh and cryopreserved clotted human blood.</a> Clin. Chem. 42, 647-648 (1996).</li><li>Salazar, L. A., Hirata, M. H., Cavalli, S. A., Machado, M. O., Hirata, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+procedure+for+DNA+isolation+from+fresh+and+cryopreserved+clotted+human+blood+useful+in+clinical+molecular+testing.">Optimized procedure for DNA isolation from fresh and cryopreserved clotted human blood useful in clinical molecular testing.</a> Clin. Chem. 44, 1748-1750 (1998).</li><li>Matheson, L. A., Duong, T. T., Rosenberg, A. M., Yeung, R. 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Biotechnol. 46, 258-264 (2010).</li><li>Polychronakos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+points+in+mapping+autoimmunity.">Fine points in mapping autoimmunity.</a> Nature. 43, 1173-1174 (2011).</li><li>Cooper, J. D., Simmonds, M. J., Walker, N. M., Burren, O., Brand, O. J., Guo, H., Wallace, C., Stevens, H., Coleman, G., Franklyn, J. A., Todd, J. A., Gough, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seven+newly+identified+loci+for+autoimmune+thyroid+disease.">Seven newly identified loci for autoimmune thyroid disease.</a> Human Molecular Genetics. , (2012).</li></ol>

No hay conflictos de interés declarado.

Muchas enfermedades humanas tienen causas genéticas y exploración de la base genética de la enfermedad humana exige un ADN de alta calidad cada vez mayor. La fuente más común de ADN utilizado en los estudios genéticos se sangre entera anticoagulada. Debido a que la mayoría de laboratorios clínicos realizan las pruebas bioquímicas, serológicas, y virales como un primer paso en la investigación resultado fenotípico, la sangre se recoge a menudo en un tubo de plasma. Después de la recogida del plasma, la sangr...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent/equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>BD P100 tubes</td>
 <td>BD Diagnostics</td>
 <td>366448</td>
 </tr>
 <tr>
 <td>EDTA tubes</td>
 <td>BD Diagnostics</td>
 <td>367863</td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>Sigma</td>
 <td>S9378</td>
 </tr>
 <tr>
 <td>Paper clip</td>
 <td>Office product</td>
 <td></td>
 </tr>
 <tr>
 <td>15 ml tube</td>
 <td>Corning</td>
 <td>430052</td>
 </tr>
 <tr>
 <td>Red blood cells (RBC)</td>
 <td>Qiagen</td>
 <td>158389 (kit)</td>
 </tr>
 <tr>
 <td>Phosphate Buffer Saline (PBS)</td>
 <td>Thermo Scientific</td>
 <td>SH30256.01</td>
 </tr>
 <tr>
 <td>BioSprint 96 DNA Blood Kit (48) </td>
 <td>Qiagen</td>
 <td>940054</td>
 </tr>
 <tr>
 <td>1.7 ml microtubes</td>
 <td>Axygen</td>
 <td>MCT-175-C</td>
 </tr>
 <tr>
 <td>Human Immuno DNA Analysis BeadChip Kit</td>
 <td>Illumina</td>
 <td>WG-352-1001</td>
 </tr>
 <tr>
 <td>Bead array reader</td>
 <td>Illumina</td>
 <td>NA</td>
 </tr>
 <tr>
 <td>GenomeStudio Software</td>
 <td>Illumina</td>
 <td>N/A</td>
 </tr>
 </tbody>
</table>

a practical and novel method to extract genomic dna from blood collection kits for plasma protein preservation

Los exámenes de laboratorio pueden realizarse en las partes móviles o fluido de la sangre. El uso de diferentes tubos de recogida de sangre determina la porción de la sangre que puede ser analizada (sangre entera, plasma o suero). Los laboratorios que participan en el estudio de la base genética de los trastornos humanos se basan en la sangre entera anticoagulada recogido en vacutainer con EDTA como la fuente de ADN para el análisis genético / genómico. Debido a que la mayoría de laboratorios clínicos realizan las pruebas bioquímicas, serológicas y virales como un primer paso en la investigación resultado fenotípico, también se recogió sangre anticoagulada en el tubo que contiene heparina (tubo de plasma). Por lo tanto cuando el ADN y el plasma se necesitan para análisis simultáneos y paralelos de datos genómicos y proteómicos tanto, es habitual para recoger la sangre tanto en tubos de heparina y EDTA. Si la sangre se puede recoger en un solo tubo y servir como una fuente tanto para el plasma y de ADN, que el método sería considerado un avance a la metanfetamina existentesods. El uso de la sangre compactada después de la extracción de plasma representa una fuente alternativa para el ADN genómico, minimizando así la cantidad de muestras de sangre procesadas y reducir el número de muestras requeridas de cada paciente. Esto en última instancia, ahorrar tiempo y recursos. El sistema de recogida de sangre P100 BD para la conservación de las proteínas del plasma se crea como un método mejorado más de plasma anterior o recogida de suero tubos 1, para estabilizar el contenido de proteína de la sangre, permitiendo un mejor descubrimiento de biomarcadores de proteínas y proteómica experimentación a partir de sangre humana. Los tubos P100 BD contienen 15,8 ml de K2EDTA secado por pulverización y un amplio espectro de cóctel patentada liofilizado de inhibidores de la proteasa para evitar la coagulación y estabilizar las proteínas del plasma. También incluyen un separador mecánico, que proporciona una barrera física entre el plasma y glóbulos de células después de la centrifugación. Algunos métodos se han ideado para extraer el ADN de la sangre coagulada samples recogieron en tubos de plasma viejos 2-4. Desafíos de estos métodos se asocian principalmente con el tipo de separador dentro de los tubos (gel separador) y se incluyen dificultad en la recuperación de la sangre coagulada, la inconveniencia de fragmentar o dispersar el coágulo, y obstrucción de la extracción del coágulo por el gel de separación. Se presenta el primer método que extrae y purifica el ADN genómico de la extracción de sangre en los nuevos tubos P100 BD. Se compara la calidad de la muestra de ADN de P100 tubos para que a partir de tubos de EDTA. Nuestro enfoque es simple y eficiente. Se trata de cuatro pasos principales de la siguiente manera: 1) el uso de una BD P100 plasma (Diagnósticos de BD, Sparks, MD, EE.UU.) con el tubo separador mecánico de extracción de sangre, 2) la eliminación de la separación mecánica mediante una combinación de sacarosa y una estéril gancho de clip de papel metálico, 3) la separación de la capa de la capa leucocitaria que contiene los glóbulos blancos y 4) el aislamiento del ADN genómico a partir de lacapa leucocitaria usando un kit de extracción de ADN comercial regular o un protocolo estándar similar.

Evaluación de la calidad del ADN y medición de la concentración Los rendimientos de P100_DNAs fueron significativamente más bajos que los de EDTA_DNAs (Tablas 1 y 2). La pureza del ADN representada por su valor de 260/280 proporción es similar tanto para el conjunto de la muestra y P100_DNA EDTA_DNA (Tablas 1 y 2). La calidad del ADN es bastante uniforme dentro de cada conjunto de la muestra a pesar de cierta degradación ...

Watch this Scientific Journal Video about A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation at JoVE.com

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

Estamos describiendo un nuevo método de aislamiento de ADN genómico a partir de sangre entera recogida para el plasma / serología. Después de la recogida de plasma, la sangre compactado se normalmente se descarta. Nuestro nuevo método representa una mejora significativa sobre los métodos existentes y hace que el ADN y el plasma disponible a partir de una sola colección, sin solicitar la sangre adicional.

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 ...

a-practical-novel-method-to-extract-genomic-dna-from-blood-collection

Research

JoVE Journal

Biology

41.9K vistas.  Emory University School of Medicine and Children's Health Care of Atlanta. Estamos describiendo un nuevo método de aislamiento de ADN genómico a partir de sangre entera recogida para el plasma / serología. Después de la recogida de plasma, la sangre compactado se normalmente se descarta. Nuestro nuevo método representa una mejora significativa sobre los métodos existentes y hace que el ADN y el plasma disponible a partir de una sola colección, sin solicitar la sangre adicional.

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

Isolation of Genomic DNA from Mouse Tails

El aislamiento de ADN genómico de colas de ratón

Investigación

Biología

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

Extracción de sangre del cangrejo herradura de América, Limulus polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

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 ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Lípidos vesículas mediada por cromatografía de afinidad utilizando magnética clasificación de células activadas (LIMACS): un nuevo método para analizar la interacción de proteínas y lípidos

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

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 ...

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 ...

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

Una guía práctica para Phylogenetics para no expertos

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

Un método para la detección y validación de mutaciones de resistencia contra la quinasa Inhibidores

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

Repetida extracción de sangre para pruebas de sangre en adultos de pez cebra

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

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 ...