The authors have no acknowledgements.

Reacción 1. El etiquetado <ol><li> Antes de su uso, dUTPs pre-alícuota cianina 3 y 5-etiquetados, nucleótidos no marcados y cebadores aleatorios en las concentraciones recomendadas por el fabricante en placas y tienda de 96 pocillos a -20 C listo para su uso. A los fines de este protocolo, alícuota 10,5 l de la dUTP etiquetado apropiado y 10,5 l nucleótidos no marcados y cebadores aleatorios a cada pocillo. </li><li> Descongelar la placa lista para el uso de 96 pocillos de nucleótidos y cebadores a 4 ° C durante aproximadamente 1 hora, protegido de la luz. </li><li> Una vez descongelado, esta placa de equilibrar a temperatura ambiente durante al menos 30 min, protegido de la luz. </li><li> Equilibrar muestras de ADN a 60 ° C durante al menos 15 min. </li><li> El uso de un robot de manejo de líquidos, dispensar agua libre de nucleasa a cada pocillo de una placa de 96 pocillos. NOTA: El volumen de agua dependerá de la concentración de cada muestra de ADN de entrada y debe ser suficiente para dar lugar a una concentración final de 50 ng / ul. </li><li> El uso de un líquido de hanrobot dling, pipeta de 1 g de ADN en un pocillo de la placa de 96 pocillos (véase 1.5) para llevar el volumen total a 20 l. NOTA: Pequeñas cantidades de ADN se pueden usar aunque la calidad de los datos resultantes puede verse comprometida (para muestras preciosas, cantidades de ADN de entrada abajo a 400 ng pueden ser utilizados). </li><li> El uso de un robot de manejo de líquidos, transferir 20 l de los nucleótidos y cebadores equilibradas en cada pocillo de la placa de DNA diluido. </li><li> Sellar la placa de 96 pocillos con tapas de la tira (un sello hermético es crucial). </li><li> Desnaturalizar el ADN a 99 ° C durante 10 min en una máquina de PCR con una tapa calentada y luego haga presión fría en hielo durante 5 min a cebadores de recocido. </li><li> El uso de un robot de manipulación de líquidos, añadir 10 l de ADN Exo Klenow enzima polimerasa de ADN y cada mezcla pipeta a fondo. NOTA: El volumen total debe ser de 50 l. </li><li> Sellar la placa de 96 pocillos y se incuba a 37 ° C durante 16 horas. NOTA: Una incubación más corto de 4 horas es suficiente, sin embargo un O / N enincubación puede ser más conveniente. </li><li> El uso de un robot de manejo de líquidos, añadir 5 l de tampón de parada a cada pocillo. NOTA: Un resumen de los reactivos utilizados para la reacción de marcaje se muestra en la Tabla 1. </li></ol> 2. La eliminación de nucleótidos no incorporados <ol><li> Retire los nucleótidos no incorporados utilizando sílice-membrana basada columnas giratorias y un robot de procesamiento spin columna dedicada (las columnas de giro también se pueden procesar de forma manual). Siga las instrucciones del fabricante. <ol><li> Transferir el contenido de cada pocillo a un tubo de 2 ml; pre-etiqueta de estos con las identificaciones así. NOTA: Este paso se puede realizar manualmente o preferiblemente usando un robot de manejo de líquidos. </li><li> Si se utiliza un robot de procesamiento columna de centrifugación, a continuación, cargar el robot con tubos de 2 ml, spin columnas de purificación de ADN y tampones asociados. </li><li> Añadir 250 l de sal alta, el ADN de tampón de unión (tampón PB) para la reacción de marcaje para unir el ADN marcado a la sílicemembrana. </li><li> Eliminar las impurezas por lavado de la membrana dos veces con 500 l de tampón de lavado (tampón PE). </li><li> Añadir 15 l de tampón de elución de baja sal (tampón EB) a la membrana para recuperar el, ADN marcado purificado en un volumen de ~ 12 l. </li></ol></li></ol> 3. Combine Pairs Hyb <ol><li> Combine los pares apropiados de cianina 3 y 5 de ADN marcado, COT-1 DNA, una mezcla de bloqueo suministrado por el fabricante y un tampón de hibridación suministrado por el fabricante con un robot de manejo de líquidos. NOTA: Estos pares Hyb pueden ser una muestra y una referencia o muestra vs muestra si CNVs compartido no son un problema (por ejemplo, emparejamos rutinariamente muestras-fenotipo no coincidentes, como un paciente displasia renal vs un paciente craneosinostosis, ya que son muy poco probable que llevar la misma clínicamente significativa CNV). Si se utiliza un ADN de referencia, se recomienda un suministro de ADN comercial, y debe ser procesado junto con la muestra. <ol><li> El uso de un tratamiento de líquidos robot, añadir COT-1 DNA (3333 ng / l), el suministrado por el fabricante de la mezcla y el bloqueo de tampón de hibridación a cada pocillo de una placa de 96 pocillos de manera que cada pocillo contiene 1,1 l COT-1 DNA, 4,95 l de la mezcla de bloqueo y 24,75 tampón de hibridación l. </li><li> Añadir 9,35 l de la DNA de cianina apropiado marcado con 3 a cada pocillo. Pre-húmedo cada punta para mejorar la precisión de pipeteo. </li><li> Añadir 9,35 l de la DNA de cianina apropiado marcado con 5 a cada pocillo. Pre-húmedo cada punta para mejorar la precisión de pipeteo. </li><li> Sellar la placa de 96 pocillos y agitar durante 1 min. Girar la placa de 96 pocillos para recoger contenidos en la parte inferior de cada pocillo. </li></ol></li></ol> 4. La hibridación <ol><li> Precaliente un horno de hibridación a 65 ° C y precalentar una diapositiva de respaldo y una cámara de hibridación para cada diapositiva matriz. <ol><li> Desnaturalizar el ADN marcado en una incubación de pre-hibridación antes de ser aplicable a los arrays. Realizar la hibridación en un horno giratorio durante 24 horas. </li><li> Incubar la placa de 96 pocillos a 70 ° C durante 30 min y luego salir a 37 ° C durante al menos 5 min. </li><li> Trabajando en una plataforma se calentó a 42 ° C, cargar copias de cada diapositiva en una cámara de hibridación antes de su uso. Asegúrese de que la parte transparente de la diapositiva junta se alinea con la parte &#39;con ventanas&#39; de la cámara de hibridación. </li><li> Pipetear 42 l de la mezcla de hibridación preparado previamente (ver sección 3) en la posición adecuada en la matriz de diapositivas respaldo. Pre-húmedo cada punta para mejorar la precisión de pipeteo. Pipeta lentamente hacia el centro de cada posición, asegurándose de que el líquido no toca el límite del anillo de goma. </li><li> Una vez que todas las posiciones de la diapositiva respaldo están llenos, cuidadosamente baje la diapositiva matriz en él y montar la cámara de hibridación. Asegúrese de que el lado de la matriz con la escritura en él se enfrenta a la diapositiva respaldo. Asegúrese de apretar el tornillo de cámara de hibridación totalmente. </li><li> Inspeccione la cámara de hibridación montado. Esegurar que no hay fugas de mezcla de hibridación fuera de los límites de los anillos de goma y cada burbuja de aire es de ~ 4 mm de altura cuando la cámara de hibridación se apoya en su extremo verticalmente. Gire la cámara de hibridación y compruebe que no haya burbujas de aire que están atascados en posición y no se mueve. Si hay alguno de estos, dar la cámara de un golpe seco en el banquillo. Compruebe que todas las burbujas de aire se mueven. </li><li> Coloque la cámara de hibridación en el horno giratorio a 65 ° C durante 24 hr. Asegúrese de que el rotor del horno está equilibrado; utilizar otras cámaras vacías si es necesario. </li></ol></li></ol> 5. Lavado y Escaneo de diapositivas <ol><li> Lávese las matrices para eliminar ADN marcado exceso y luego escanear para medir la fluorescencia de cada sonda. <ol><li> Tome cámaras de hibridación fuera del horno y desmontar. Las diapositivas de la matriz y de la junta serán pegadas; sumergir estos en tampón de lavado 1 y utilizar un par de pinzas, de plástico plana filo para separarlas. </li><li> Discard la diapositiva junta y colocar la placa matriz en un rack sumergido en tampón 1. </li><li> Lavar el portaobjetos matriz en ~ 700 ml de tampón de lavado fresca 1 durante 5 min, se agita vigorosamente (utilizar una pulga y un agitador magnético). </li><li> Transferir el portaobjetos matriz a ~ 700 ml de tampón de lavado 2 y lavar durante 90 segundos, revolviendo vigorosamente. Levante suavemente las diapositivas de matriz fuera de tampón de lavado 2, deben surgir seco. </li><li> Cargar la diapositiva matriz en un soporte de diapositivas del escáner, utilizando un protector de diapositivas. Cargue la diapositiva en el escáner matriz y escanear siguiendo las instrucciones del fabricante matriz. </li></ol></li></ol>

<ol>
	<li>Miller, D. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consensus+statement:+chromosomal+microarray+is+a+first-tier+clinical+diagnostic+test+for+individuals+with+developmental+disabilities+or+congenital+anomalies.">Consensus statement: chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies.</a> Am. J. Hum. Genet. 86, 749-764 (2010).</li><li>Ahn, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Array+CGH+as+a+first+line+diagnostic+test+in+place+of+karyotyping+for+postnatal+referrals+-+results+from+four+years'+clinical+application+for+over+8,700+patients.">Array CGH as a first line diagnostic test in place of karyotyping for postnatal referrals - results from four years' clinical application for over 8,700 patients.</a> Mol. Cytogenet. 6 (16), 1755-8166 (2013).</li><li>Ahn, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+and+implementation+of+array+comparative+genomic+hybridisation+as+a+first+line+test+in+place+of+postnatal+karyotyping+for+genome+imbalance.">Validation and implementation of array comparative genomic hybridisation as a first line test in place of postnatal karyotyping for genome imbalance.</a> Mol. Cytogenet. 3 (9), 1755-8166 (2010).</li><li>Beaudet, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utility+of+chromosomal+microarray+analysis+in+developmental+and+behavioral+pediatrics.">The utility of chromosomal microarray analysis in developmental and behavioral pediatrics.</a> Child Dev. 84, 121-132 (2013).</li><li>Park, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+implementation+of+whole-genome+array+CGH+as+a+first-tier+test+in+5080+pre+and+postnatal+cases.">Clinical implementation of whole-genome array CGH as a first-tier test in 5080 pre and postnatal cases.</a> Mol. Cytogenet. 4, 12 (2011).</li><li>Iafrate, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+large-scale+variation+in+the+human+genome.">Detection of large-scale variation in the human genome.</a> Nat. Genet. 36, 949-951 (2004).</li><li>Redon, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+variation+in+copy+number+in+the+human+genome.">Global variation in copy number in the human genome.</a> Nature. 444, 444-454 (2006).</li><li>Cafferkey, M., Ahn, J. W., Flinter, F., Ogilvie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+features+in+patients+with+15q11.2(BP1-BP2)+deletion:+Further+delineation+of+an+emerging+syndrome.">Phenotypic features in patients with 15q11.2(BP1-BP2) deletion: Further delineation of an emerging syndrome.</a> Am. J. Med. Genet. A. 164, 1916-1922 (2014).</li><li>Molin, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+microdeletion+syndrome+at+3q13.31+characterised+by+developmental+delay,+postnatal+overgrowth,+hypoplastic+male+genitals,+and+characteristic+facial+features.">A novel microdeletion syndrome at 3q13.31 characterised by developmental delay, postnatal overgrowth, hypoplastic male genitals, and characteristic facial features.</a> J. Med. Genet. 49, 104-109 (2012).</li><li>Tropeano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male-biased+autosomal+effect+of+16p13.11+copy+number+variation+in+neurodevelopmental+disorders.">Male-biased autosomal effect of 16p13.11 copy number variation in neurodevelopmental disorders.</a> PLoS One. 8, e61365 (2013).</li><li>Bon, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+delineation+of+the+15q13+microdeletion+and+duplication+syndromes:+a+clinical+spectrum+varying+from+non-pathogenic+to+a+severe+outcome.">Further delineation of the 15q13 microdeletion and duplication syndromes: a clinical spectrum varying from non-pathogenic to a severe outcome.</a> J. Med. Genet. 46, 511-523 (2009).</li></ol>

The authors have nothing to disclose.

Array CGH no detectará reordenamientos balanceados o anormalidades ploidía como triploidía. Además, los desequilibrios de mosaico de bajo nivel no se pueden detectar. Sin embargo, array CGH tiene una mayor resolución para la detección de la CNV de análisis cromosómico bandas-G del cual ha sustituido en muchos laboratorios de citogenética. Por lo tanto, es el actual estándar de oro para la detección de la CNV en todo el genoma. Puede ser reemplazado por las tecnologías de secuenciación de próxima generació...

Se ha sabido durante muchos años que las supresiones o copias extra de segmentos cromosómicos provocan discapacidad intelectual, dismorfia y malformaciones congentical, y en algunos casos causar síndromes genéticos 1. Sin embargo, la única tecnología disponible hasta mediados de la década de 2000 para la detección de todo el genoma de estos cambios fue G-bandas cromosoma análisis, que tiene una resolución de alrededor de 5-10 MB, dependiendo de la región y la naturaleza de los desequilibrios, y que no puede detectar cromosomas anormales donde el material ha sido reemplazado por una región de un cromosoma diferente con el mismo patrón de bandas. Técnicas citogenéticas auxiliares tales como la hibridación fluorescente in situ y la sonda de amplificación ligadura específica multiplex han estado disponibles durante el interrogatorio de loci específicos, en los casos de sospecha de desequilibrio sindrómico específico, pero no fue hasta la introducción de la gama de hibridación genómica comparada (CGH array) en s de diagnóstico clínico de rutinaervicio 2-5 que la detección de todo el genoma de las variantes de número de copias (CNV) se hizo posible en un gran aumento de resolución (por lo general alrededor de 120kb). Trabajos de servicio clínico junto estudios de investigación han demostrado que la CNV para algunas regiones se han generalizado en la población normal 6-7, mientras que otro CNV, previamente indetectables, están asociados con neurodisabilities como el autismo y la epilepsia 8-11. El protocolo descrito en este documento se utiliza en nuestro laboratorio de diagnóstico clínico UK National Health Service (NHS); utilizamos estrategias de hibridación novela, ensayo de lotes y la robótica para minimizar el costo de este servicio financiado por el estado. Antes de el protocolo detallado a continuación, el ADN de alta calidad debe ser extraído del material de partida apropiado, comúnmente sangre, células cultivadas o muestras de tejido. Espectrofotometría se puede utilizar para medir la concentración (debe ser&gt; 50 ng / ul) y comprobar 260: 280 razón de absorbancia (b debee 1,8-2,0). La electroforesis en gel se puede utilizar para verificar que el ADN es de alto peso molecular sin degradación significativa. Este protocolo está diseñado para los laboratorios de mayor rendimiento que está etiquetando 96 muestras por corrida utilizando la robótica de manipulación de líquidos automatizados. Sin embargo, puede ser adaptado para laboratorios de rendimiento inferiores sin automatización.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CGH microarray 8 x 60K</td>
			<td style="width:71px;">Agilent</td>
			<td style="width:119px;">G4126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Array CGH wash buffers</td>
			<td style="width:71px;">Agilent</td>
			<td style="width:119px;">5188-5226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Array CGH hybridisation buffer</td>
			<td style="width:71px;">Agilent</td>
			<td style="width:119px;">5188-5380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Minelute purification kit</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">28006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Array CGH labelling kit</td>
			<td style="width:71px;">Enzo</td>
			<td style="width:119px;">ENZ-42672-0000</td>
			<td></td>
		</tr>
	</tbody>
</table>

array comparative genomic hybridization (array cgh) for detection of genomic copy number variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient&#8217;s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.

Cada sonda en una matriz hibridada se visualiza como una mezcla de fluorocromos rojo y verde (véase la Figura 1). La proporción de rojo a la señal fluorescente verde para cada sonda se cuantifica por el escáner y el software asociado traza en log2 ratios de acuerdo a su posición genómica, e identifica las regiones incluidas límites preestablecidos fuera. Las huellas de la matriz resultantes permiten la interpretación de las regiones identificadas como genómicamente desequilibrada. Por ejemplo, ...

Watch this Scientific Journal Video about Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants at JoVE.com

Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants

CGH array para la detección de variantes de número de copias genómicas ha reemplazado análisis de cariotipo de bandas-G. Este artículo describe la tecnología y su aplicación en un laboratorio de servicio de diagnóstico.

Matriz de hibridación genómica comparada (CGH array) para la detección de número de copias genómicas variantes

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its ...

array-comparative-genomic-hybridization-array-cgh-for-detection

Research

JoVE Journal

Biology

Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants

19.4K vistas.  Guy's & St Thomas' NHS Foundation Trust.  CGH array para la detección de variantes de número de copias genómicas ha reemplazado análisis de cariotipo de bandas-G. Este artículo describe la tecnología y su aplicación en un laboratorio de servicio de diagnóstico. 

Video: Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants

Isolation of Genomic DNA from Mouse Tails

El aislamiento de ADN genómico de colas de ratón

Investigación

Biología

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1. Recent advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases 2. The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in ~100 kilobase pair (kb) segments 2. These BAC targets are individually synthesized and spotted in duplicate on a single glass slide 2-4. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected - a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position 5,6. The SMRT array can detect alterations as small as 50 kb in size 7. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100ng) this allows profiling of precious samples such as those produced by microdissection 7,8. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection 8. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.

This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.

Demostración de técnicas de hibridación del genoma entero matriz Genómica Comparativa

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1.  Recent ...

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. Lightweight recording devices carrying a high density array of tetrodes allow for the simultaneous monitoring of the activity of tens to hundreds of individual neurons. Here we describe a protocol for the fabrication of a micro-drive array with twenty one independently movable micro-drives. This device has been used successfully to record from hippocampal and cortical neurons in our lab. We show how to prepare a custom designed, 3-D printed plastic base that will hold the micro-drives. We demonstrate how to construct the individual micro-drives and how to assemble the complete micro-drive array. Further preparation of the drive array for surgical implantation, such as the fabrication of tetrodes, loading of tetrodes into the drive array and gold-plating, is covered in a subsequent video article.

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

In this protocol we demonstrate how to fabricate a micro-drive array for chronic electrophysiological recordings in rats.

Micro-array de unidades de crónicos In vivo Grabación: Unidad de Fabricación

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. ...

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

Inserte grandes Ambiental Genómica Producción Biblioteca

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

Microarrays de ADN: Control de calidad de la muestra, la hibridación de la matriz y de escaneo

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

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 ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Pantallas competitiva Genómica de Bibliotecas de levadura con código de barras

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (<a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a>). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

La detección de raras variantes genómicas de secuenciación agruparon mediante SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

Transformación de Genómica de la Picoeukaryote<em&gt; Ostreococcus tauri</em

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

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 ...