The authors have no acknowledgements.

1. L&#39;étiquetage Réaction <ol><li> Avant d&#39;utiliser, dUTP pré-aliquote cyanine 3 et 5 nucléotides marqués, non marqués et des amorces aléatoires aux concentrations recommandées par le fabricant et magasin en plaques à 96 puits à -20 C prêt à l&#39;emploi. Pour les fins de ce protocole, aliquote 10,5 pi de la dUTP marqué de appropriée et 10,5 ul nucléotides non marqués et des amorces aléatoires à chaque puits. </li><li> Décongeler la plaque prête à l&#39;emploi de 96 puits de nucléotides et des amorces à 4 ° C pendant environ 1 heure, à l&#39;abri de la lumière. </li><li> Une fois décongelé, équilibrer cette plaque à température ambiante pendant au moins 30 min, à l&#39;abri de la lumière. </li><li> Equilibrer échantillons d&#39;ADN à 60 ° C pendant au moins 15 min. </li><li> L&#39;utilisation d&#39;un robot de manipulation liquide, distribuer de l&#39;eau sans nucléase à chaque puits d&#39;une plaque de 96 puits. NOTE: Le volume d&#39;eau dépendra de la concentration de chaque échantillon d&#39;ADN d&#39;entrée et doit être suffisante pour donner une concentration finale de 50 ng / ul. </li><li> Utilisation d&#39;un liquide hanmani- robot, pipette 1 ug d&#39;ADN dans un puits de la plaque à 96 puits (voir 1.5) pour porter le volume total à 20 ul. NOTE: quantités d&#39;ADN inférieures peuvent être utilisés même si la qualité des données qui en résulte peut être compromise (pour les échantillons précieux, les quantités d&#39;ADN d&#39;entrée vers le bas à 400 ng peuvent être utilisés). </li><li> En utilisant un robot de manipulation de liquide, transférer 20 pi de nucleotides et des amorces équilibrée dans chaque puits de la plaque de l&#39;ADN dilué. </li><li> Sceller la plaque 96 puits avec des bouchons de bande (un joint étanche est essentiel). </li><li> Dénaturer l&#39;ADN à 99 ° C pendant 10 min dans une machine PCR avec un couvercle chauffé et puis enclenchez refroidir sur glace pendant 5 min à des amorces de recuit. </li><li> L&#39;utilisation d&#39;un robot de manipulation de liquide, ajouter 10 pi de l&#39;ADN Klenow Exo enzyme polymérase ADN à chaque pipette et mélanger soigneusement. NOTE: Le volume total devrait être de 50 pi. </li><li> Sceller la plaque à 96 puits et incuber à 37 ° C pendant 16 heures. REMARQUE: Une incubation plus courte de 4 heures est suffisante cependant un O / N danscubation peut être plus pratique. </li><li> En utilisant un robot de manipulation de liquide, ajouter 5 ul de tampon d&#39;arrêt à chaque puits. REMARQUE: Un résumé des réactifs utilisés pour la réaction de marquage est indiqué dans le Tableau 1. </li></ol> 2. Suppression non constituées en nucléotides <ol><li> Retirer nucléotides non constituées en société en utilisant la silice-membrane à base de colonnes de spin et un robot de spin de traitement de la colonne dédiée (les colonnes de spin peuvent également être traitées manuellement). Suivez les instructions du fabricant. <ol><li> Transférer le contenu de chaque puits dans un tube de 2 ml; pré-label-ci avec ID et de. REMARQUE: Cette étape peut être réalisée manuellement ou de préférence en utilisant un robot de manipulation de liquide. </li><li> Si l&#39;on utilise un robot de traitement de colonne de centrifugation, puis charger le robot avec 2 ml des tubes, des colonnes de centrifugation de purification de l&#39;ADN et des tampons associés. </li><li> Ajouter 250 ul de haute teneur en sel, tampon de liaison (tampon PB) l&#39;ADN à la réaction de marquage pour lier l&#39;ADN marqué à la silicemembrane. </li><li> Éliminer les impuretés en lavant la membrane deux fois avec 500 pi de tampon de lavage (tampon PE). </li><li> Ajouter 15 pi de tampon d&#39;élution faible de sel (tampon EB) à la membrane pour récupérer l&#39;ADN purifié, marqué dans un volume de ~ 12 ul. </li></ol></li></ol> 3. Mélanger Paires Hyb <ol><li> Combiner les paires appropriées de la cyanine 3 et 5 de l&#39;ADN marqué, COT-1 ADN, un mélange de blocage fourni par le fabricant et un tampon d&#39;hybridation fourni par le fabricant en utilisant un robot de manipulation de liquide. NOTE: Ces paires Hyb peut être un échantillon et une référence, ou un échantillon vs échantillon si CNV partagé ne sont pas un problème (par exemple, nous jumelons régulièrement des échantillons de phénotype correspondent pas, comme une dysplasie des patients rénale vs un patient de craniosténose, car ils sont très peu probable pour transporter le même cliniquement significative CNV). Si l&#39;on utilise un ADN de référence, une source d&#39;ADN du commerce est recommandée, et il doit être traité le long de l&#39;échantillon. <ol><li> Utilisation d&#39;une manipulation de liquides robot, ajouter COT-1 ADN (3333 ng / ul), le fabricant fournies blocage mélange et un tampon d&#39;hybridation à chaque puits d&#39;une plaque à 96 puits de sorte que chaque puits contient 1,1 ul COT-1 ADN, 4,95 ul de mélange de blocage et 24,75 ul du tampon d&#39;hybridation. </li><li> Ajouter 9,35 ul de l&#39;ADN approprié cyanine 3-marqué dans chaque puits. Pré-humide chaque pointe pour améliorer la précision du pipetage. </li><li> Ajouter 9,35 ul de l&#39;ADN approprié cyanine 5 marqué à chaque puits. Pré-humide chaque pointe pour améliorer la précision du pipetage. </li><li> Sceller la plaque à 96 puits et vortex pendant 1 min. Faites tourner la plaque à 96 puits pour recueillir le contenu au fond de chaque puits. </li></ol></li></ol> 4. Hybridation <ol><li> Préchauffer un four à hybridation à 65 ° C et préchauffer une diapositive de support et une chambre d&#39;hybridation pour chaque diapositive de tableau. <ol><li> Dénaturer l&#39;ADN marqué à une pré-incubation avant l&#39;hybridation étant à appliquer sur les tableaux. Effectuer une hybridation dans un four rotatif pendant 24 heures. </li><li> Incuber la plaque à 96 puits à 70 ° C pendant 30 minutes, puis laisser à 37 ° C pendant au moins 5 min. </li><li> Travailler sur une plate-forme chauffée à 42 ° C, charger la sauvegarde chaque diapositive dans une chambre d&#39;hybridation avant de l&#39;utiliser. Assurez-vous que la partie transparente de la lame de joint se aligne avec la partie «fenêtré» de la chambre d&#39;hybridation. </li><li> Pipet 42 ul du mélange d&#39;hybridation préparé précédemment (voir section 3) sur la position appropriée sur le réseau sauvegarde diapositive. Pré-humide chaque pointe pour améliorer la précision du pipetage. Pipet lentement sur le centre de chaque position, assurant que le liquide ne touche pas la limite de l&#39;anneau en caoutchouc. </li><li> Une fois toutes les positions sur la lame de support sont remplis, abaissez délicatement la lame de tableau sur elle et assembler la chambre d&#39;hybridation. Assurez-vous que le côté du tableau avec écrit sur elle est confrontée à la diapositive de support. Veillez à serrer l&#39;hybridation vis de chambre entièrement. </li><li> Inspectez la chambre d&#39;hybridation assemblé. ENSure qu&#39;il n&#39;y ait pas de fuite de mélange d&#39;hybridation à l&#39;extérieur des limites de la bague en caoutchouc et chaque bulle d&#39;air est d&#39;environ 4 mm de hauteur lorsque la chambre d&#39;hybridation est reposé sur son extrémité verticalement. Tournez la chambre d&#39;hybridation et vérifier qu&#39;il n&#39;y a pas de bulles d&#39;air qui sont coincés en position et ne bouge pas. Se il ya toutes ces choses, donner la chambre forte d&#39;un robinet sur le banc. Vérifiez que toutes les bulles d&#39;air se déplacent. </li><li> Placer la chambre d&#39;hybridation dans le four rotatif à 65 ° C pendant 24 heures. Assurez-vous que le rotor four est équilibrée; utiliser d&#39;autres chambres vides si nécessaire. </li></ol></li></ol> 5. Lavage et diapositives Numérisation <ol><li> Laver tableaux pour éliminer l&#39;ADN marqué excès et puis analyser pour mesurer la fluorescence de chaque sonde. <ol><li> Prenez chambres d&#39;hybridation sortie du four et à démonter. Les diapositives de tableaux et joints seront collées; plonger ceux-ci dans une tampon de lavage et utiliser une paire de pinces en plastique, plat tranchant pour les forcer à part. </li><li> DiSCard la diapositive de joint et placer la lame de tableau dans un rack immergée dans un tampon 1. </li><li> Laver la glissière de tableau dans ~ 700 ml de tampon de lavage fraîche 1 pendant 5 min, en remuant vigoureusement (utiliser une puce et d&#39;un agitateur magnétique). </li><li> Transférer la lame de tableau à ~ 700 ml tampon de lavage 2 et laver pendant 90 secondes, en remuant vigoureusement. Soulevez doucement les diapositives de tableaux sur tampon de lavage 2, ils devraient émerger sec. </li><li> Charger la lame de matrice dans un porte-lame du dispositif de balayage, en utilisant un protecteur de lame. Chargez la diapositive dans le scanner de réseau et de numérisation en suivant les instructions du fabricant du tableau. </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.

CGH array ne détectera pas remaniements équilibrés ou des anomalies de ploïdie tels que la triploïdie. En outre, les déséquilibres mosaïque de bas niveau ne peuvent pas être détectés. Cependant, CGH array a une résolution plus élevée pour la détection CNV que l&#39;analyse des chromosomes bandes G laquelle il a remplacé dans de nombreux laboratoires de cytogénétique. Ce est donc la norme de référence actuelle pour la détection du génome entier CNV. Elle peut être remplacée par des technologies de...

Il a été connu depuis de nombreuses années que les suppressions ou des copies supplémentaires de segments chromosomiques causent une déficience intellectuelle, une dysmorphie et des malformations congentical, et dans certains cas provoquer des syndromes génétiques 1. Cependant, la seule technologie disponible jusqu&#39;au milieu des années 2000 pour la détection du génome entier de ces changements était G-bandes analyse des chromosomes, qui a une résolution de l&#39;ordre 5-10Mb, selon la région et la nature du déséquilibre, et qui ne peut pas détecter chromosomes anormaux où la matière a été remplacé par une région d&#39;un chromosome différent avec le même motif de bandes. Techniques cytogénétiques auxiliaires tels que l&#39;hybridation fluorescente in situ et multiplex sonde amplification spécifique de ligature en ont été disponibles pour l&#39;interrogation des loci spécifiques, en cas de suspicion déséquilibre syndromique spécifique, mais ce ne est que l&#39;introduction d&#39;hybridation génomique comparative (CGH array) en s diagnostic clinique de routineervice 2-5 que la détection du génome entier du nombre de copies variantes (CNV) est devenu possible à une résolution considérablement augmenté (généralement autour de 120kb). Travaux de service clinique parallèlement à des études de recherche ont montré que CNV pour certaines régions sont très répandues dans la population normale 6-7, tandis que d&#39;autres CNV, auparavant indétectable, sont associés à neurodisabilities comme l&#39;autisme et l&#39;épilepsie 8-11. Le protocole décrit dans ce document est utilisé dans notre laboratoire de diagnostic clinique UK National Health Service (NHS); nous utilisons de nouvelles stratégies d&#39;hybridation, les tests par lots et de la robotique de minimiser les coûts de ce service financé par l&#39;Etat. Avant le protocole détaillé ci-dessous, l&#39;ADN de haute qualité doit être extraite de la matière de départ appropriée, habituellement du sang, des cellules cultivées ou des échantillons de tissus. Spectrophotométrie peut être utilisé pour mesurer la concentration (devrait être&gt; 50 ng / ul) et vérifier 260: 280 rapports d&#39;absorbance (b devraite 1,8 à 2,0). L&#39;électrophorèse sur gel peut être utilisée pour vérifier que l&#39;ADN soit de haut poids moléculaire sans dégradation significative. Ce protocole est conçu pour les laboratoires de débit plus élevés qui sont étiquetage 96 échantillons par exécution à l&#39;aide automatisés robotique de manipulation de liquides. Cependant, il peut être adapté pour les laboratoires de débit inférieurs sans automatisation.

<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>
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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.

Chaque sonde sur un réseau hybride est visualisée comme un mélange de fluorochromes rouge et vert (voir Figure 1). Le ratio de rouge à un signal vert fluorescent pour chaque sonde est quantifiée par le scanner et le logiciel associé graphiquement en ratios de log2 selon leur position génomique, et identifie les régions relevant de limites prédéfinies extérieur. Les traces de tableau résultant permettent interprétation de régions identifiées comme génomiquement déséquilibrée. Par exemp...

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 pour la détection des variantes du nombre de copies génomiques a remplacé l&#39;analyse du caryotype G-bandes. Cet article décrit la technologie et son application dans un laboratoire de services de diagnostic.

Tableau hybridation génomique comparative (CGH array) pour la détection des variantes génomiques du nombre de copies

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

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Research

JoVE Journal

Biology

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

19.4K vues.  Guy's & St Thomas' NHS Foundation Trust.  CGH array pour la détection des variantes du nombre de copies génomiques a remplacé l'analyse du caryotype G-bandes. Cet article décrit la technologie et son application dans un laboratoire de services de diagnostic. 

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

Isolation of Genomic DNA from Mouse Tails

Isolement de l&#39;ADN génomique de souris Tails

Recherche

Biologie

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.

Démonstration technique de l&#39;hybridation tableau Whole Genome génomique comparative

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-drive tableau pour les maladies chroniques In vivo Enregistrement: Disque de fabrication

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.

Grand Insérer production bibliothèque génomique de l&#39;environnement

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.

Cellules souches: le contrôle qualité des échantillons, hybridation Array et balayage

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.

Genomic IRM - une ressource publique pour l&#39;étude des schémas de séquence dans l&#39;ADN génomique

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.

Écrans concurrentiel des bibliothèques génomiques de levure Barcoded

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.

Détection de rares variantes génomiques à partir de séquençage regroupés à l&#39;aide 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.

Transformation génomique 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.

Capture d&#39;sélectif de 5-hydroxyméthylcytosine partir de l&#39;ADN génomique

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 ...