The authors have no acknowledgements.

Reação 1. Rotulagem <ol><li> Antes de usar, dUTPs pré-aliquota de cianina 3 e 5 nucleótidos marcados, não marcados e iniciadores aleatórios para as concentrações recomendadas pelo fabricante em placas e armazenamento de 96 poços a -20 ° C prontas para utilização. Para efeitos do presente protocolo, alíquota de 10,5 mL da dUTP marcado adequado e 10,5 ul nucleotídeos não marcados e iniciadores aleatórios a cada poço. </li><li> Descongelar o prato pronto-para-uso de 96 poços de nucleótidos e os iniciadores a 4 ° C durante cerca de 1 hora, protegidas da luz. </li><li> Uma vez descongelado, esta placa equilibrar à temperatura ambiente durante pelo menos 30 minutos, ao abrigo da luz. </li><li> Equilibrar as amostras de ADN a 60 ° C durante pelo menos 15 min. </li><li> Usando um robot de tratamento de líquidos, dispensar a água livre de nuclease a cada poço de uma placa de 96 poços. NOTA: O volume de água dependerá da concentração de cada amostra de DNA de entrada e deve ser suficiente para resultar numa concentração final de 50 ng / ul. </li><li> Usando um líquido hanrobô dling, pipeta de 1 ug de ADN em um poço da placa de 96 poços (ver 1.5) para levar o volume total de 20 ul. NOTA: Pequenas quantidades de ADN pode ser usado, embora a qualidade de dados resultante pode ser comprometida (por preciosas amostras, as quantidades de ADN de entrada para baixo para 400 ng pode ser utilizado). </li><li> Usando um robot de tratamento de líquidos, transferir 20 ul de nucleótidos equilibradas e iniciadores em cada poço da placa de ADN diluída. </li><li> Selar a placa de 96 poços com tampas de tira (uma vedação estanque é importante). </li><li> Desnaturar o ADN a 99 ° C durante 10 min, em uma máquina PCR com uma tampa aquecida e em seguida, encaixe fresco em gelo durante 5 min a iniciadores recozimento. </li><li> Usando um tratamento robô líquido, adicione 10 ml de enzima polimerase Klenow Exo DNA para cada DNAs e mix pipeta completamente. NOTA: O volume total deve ser de 50 mL. </li><li> Selar a placa de 96 poços e incuba-se a 37 ° C durante 16 horas. NOTA: A incubação mais curto de 4 horas é suficiente, contudo, uma O / N emcubation pode ser mais conveniente. </li><li> Usando um robot de tratamento de líquidos, adicionam-se 5 ul de tampão de paragem a cada poço. NOTA: Um resumo dos reagentes utilizados para a reacção de marcação é mostrada na Tabela 1. </li></ol> 2. Remoção de não incorporados Nucleotides <ol><li> Remover nucleótidos não incorporados utilizando sílica-membrana baseado colunas de giro e um robô rotação processamento coluna dedicada (as colunas de spin também podem ser processados ​​manualmente). Siga as instruções do fabricante. <ol><li> Transferir o conteúdo de cada cavidade para um tubo de 2 mL; pré-label estes com bem IDs. NOTA: Este passo pode ser realizado manualmente ou de preferência através de um robô tratamento líquido. </li><li> Se estiver usando um robô processamento coluna de rotação, em seguida, coloque o robô com 2 ml tubos, colunas de giro de purificação de DNA e buffers associados. </li><li> Adicionar 250 ul de sal elevado, de ADN tampão de ligação (tampão de PB) para a reacção de marcação para ligar o ADN marcado com a sílicamembrana. </li><li> Remover as impurezas por lavagem da membrana, duas vezes com 500 ul de tampão de lavagem (tampão PE). </li><li> Adicionar 15 ul de tampão de eluição de sal baixo (tampão EB) para a membrana para recuperar o ADN purificado, marcado num volume de ~ 12 mL. </li></ol></li></ol> 3. Combinar pares HYB <ol><li> Combine os pares adequados de cyanine 3 e 5 DNA marcado, COT-1 DNA, um mix de bloqueio fornecido pelo fabricante e um tampão de hibridação fornecido pelo fabricante usando um robô de manipulação de líquidos. NOTA: Estes pares hyb pode ser um exemplo e uma referência, ou amostra vs amostra se CNVs compartilhada não são um problema (por exemplo, nós rotineiramente emparelhar amostras fenótipo-incompatíveis, como um paciente displasia renal vs um paciente craniossinostose, como eles são muito Não deve levar o mesmo CNV clinicamente significativo). Se utilizar um ADN de referência, é recomendado um fornecimento de ADN comercial, e que deve ser processado juntamente com a amostra. <ol><li> Usando um tratamento líquido robot, adicionar COT-1 DNA (3333 ng / mL), a mistura de bloqueio e de tampão de hibridação para cada poço de uma placa de 96 poços de modo a que cada poço contém 1,1 uL de ADN Cot-1, 4,95 ul mistura bloqueio e 24.75 fornecido pelo fabricante ul de tampão de hibridação. </li><li> Adicionar 9,35 mL da apropriada 3 cianina ADN marcado a cada poço. Pre-wet cada ponta para melhorar a pipetagem precisão. </li><li> Adicionar 9,35 mL da ADN cianina apropriado 5-marcado a cada poço. Pre-wet cada ponta para melhorar a pipetagem precisão. </li><li> Selar a placa de 96 poços e vortex durante 1 min. Girar a placa de 96 poços para recolher o conteúdo na parte inferior de cada poço. </li></ol></li></ol> 4. Hibridização <ol><li> Pré-aqueça um forno de hibridação a 65 ° C e Pré-aquecer um slide de apoio e uma câmara de hibridação para cada slide matriz. <ol><li> Desnaturar o ADN marcado em uma incubação pré-hibridação, antes de ser aplicável às matrizes. Realizar hibridação num forno rotativo durante 24 horas. </li><li> Incubar a placa de 96 poços a 70 ° C durante 30 min e, em seguida, deixar a 37 ° C durante pelo menos 5 min. </li><li> Trabalhando numa plataforma aquecida a 42 ° C, carregar cada lâmina apoio para uma câmara de hibridação, antes da utilização. Certifique-se que a parte transparente do slide junta alinha com a peça &#39;janelas&#39; da câmara de hibridação. </li><li> Pipet 42 mL da mistura de hibridação previamente preparado (ver secção 3) na posição apropriada na matriz backing slide. Pre-wet cada ponta para melhorar a pipetagem precisão. Pipet lentamente sobre o centro de cada posição, certificando-se que o líquido não toca o limite anel de borracha. </li><li> Uma vez que todas as posições no slide backing são preenchidos, abaixe cuidadosamente o slide matriz nele e montar a câmara de hibridação. Certifique-se de que o lado da matriz com a escrita sobre ele está voltado para o slide de apoio. Certifique-se de apertar o parafuso de câmara de hibridação totalmente. </li><li> Inspecione a câmara de hibridação montado. ENSure que não há vazamento de mistura de hibridação fora dos limites do anel de borracha e cada bolha de ar é de aproximadamente 4 mm de altura quando a câmara de hibridação é descansou em sua vertical. Gire a câmara de hibridação e verifique se não há bolhas de ar que estão presas na posição e não em movimento. Se houver algum destes sintomas, a câmara de dar uma pancada no banco. Verifique se todas as bolhas de ar estão se movendo. </li><li> Coloque a câmara de hibridação para o forno rotativo, a 65 ° C durante 24 horas. Verifique se o rotor forno é equilibrada; utilizar outras câmaras vazias, se necessário. </li></ol></li></ol> 5. Lavar e Digitalização de Slides <ol><li> Lave matrizes para remover o excesso de DNA marcado e depois digitalizar para medir a fluorescência de cada sonda. <ol><li> Tome câmaras de hibridação fora do forno e desmontar. A matriz ea junta slides serão coladas; submergir estes em tampão de lavagem 1 e usar um par de plástico, pinça plana gumes para erguer-los. </li><li> DiScard o slide junta e coloque a lâmina matriz em um rack submerso em tampão 1. </li><li> Lava-se a lâmina de matriz em ~ 700 ml de tampão de lavagem fresca para 1 5 minutos, agitando vigorosamente (usar um pulgas e um agitador magnético). </li><li> Transferir as lâminas de matriz para tampão de lavagem ~ 700 ml 2 e lavar durante 90 segundos, mexendo vigorosamente. Levante cuidadosamente as lâminas de matriz fora de tampão de lavagem 2, eles devem emergir seco. </li><li> Carregue a lâmina matriz em um suporte de slides do scanner, usando um protetor de slide. Coloque o slide no scanner matriz e digitalizar seguindo as instruções do fabricante do array. </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.

Matriz CGH não vai detectar rearranjos equilibrados ou anormalidades ploidy como triploidy. Além disso, o baixo nível de desequilíbrios do mosaico não pode ser detectado. No entanto, array CGH tem uma resolução maior para a detecção CNV de análise cromossômica G-unida qual substituiu em muitos laboratórios de citogenética. É, por conseguinte, o padrão de ouro para detecção de corrente de CNV de todo o genoma. Ele pode ser substituído por tecnologias de sequenciamento de próxima geração no futuro, ma...

Ficou conhecido por muitos anos que deleções ou cópias extras de segmentos cromossômicos causam deficiência intelectual, dismorfismo e malformações congentical e, em alguns casos, causar síndromes genéticas 1. No entanto, a única tecnologia disponível até meados dos anos 2000 para a detecção de todo o genoma dessas mudanças foi G-unida análise cromossômica, que tem uma resolução de cerca de 5-10Mb, dependendo da região e da natureza do problema, e que não pode detectar cromossomas anormais onde o material foi substituída por uma região de um cromossoma diferente, com o mesmo padrão de bandas. Técnicas de citogenética auxiliares, tais como hibridização fluorescente in situ e multiplex amplificação sonda específica para a ligação ter sido disponível para o interrogatório de loci específico, em casos de suspeita de desequilíbrio sindrômica específico, mas não foi até a introdução da matriz de hibridização genômica comparativa (array CGH) na rotina clínica de diagnóstico service 2-5 que a detecção de todo o genoma do número de cópias variantes (CNVs) tornou-se possível a um grande aumento da resolução (normalmente em torno de 120kb). Trabalho de serviço clínico ao lado de estudos de pesquisa têm mostrado que CNVs para algumas regiões são mais prevalentes na população normal de 6-7, ao passo que outros CNVs, anteriormente indetectável, estão associados com neurodisabilities como o autismo e epilepsia 8-11. O protocolo descrito neste documento é utilizado em nosso UK National Health Service (NHS) laboratório de diagnóstico clínico; usamos novas estratégias de hibridização, ensaios de lotes e robótica para minimizar o custo neste serviço financiado pelo Estado. Antes de o protocolo detalhado abaixo, o ADN de alta qualidade devem ser extraídos a partir do material de partida apropriado, vulgarmente sangue, células de cultura ou amostras de tecido. Espectrofotometria pode ser utilizado para medir a concentração (deve ser&gt; 50 ng / ul) e vá 260: 280 razão de absorvância (b deveme 1,8-2,0). A electroforese em gel pode ser usado para verificar que o ADN é de elevado peso molecular sem degradação significativa. Este protocolo é projetado para laboratórios de maior rendimento que estão rotulando 96 amostras por execução usando a robótica automatizados de movimentação de líquidos. No entanto, ele pode ser adaptado para laboratórios de rendimento inferiores sem automação.

<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 numa matriz hibridado é visualizado como uma mistura de vermelho e verde fluorocromos (ver Figura 1). A relação do vermelho ao sinal verde fluorescente para cada sonda é quantificado pelo scanner e software associado traça estes como rácios log2 acordo com sua posição genômica, e identifica regiões abrangidas limites predefinidos fora. Os traços da matriz resultantes permitem a interpretação de regiões identificadas como genomicamente desequilibrado. Por exemplo, o rastreio a pa...

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

Matriz CGH para a detecção de variantes do número de cópia genômicas substituiu análise do cariótipo G-unida. Este artigo descreve a tecnologia e sua aplicação em um laboratório de serviço de diagnóstico.

Matriz Hibridização Genômica Comparativa (Array CGH) para a detecção de Genomic copiar o número de 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 visualizações.  Guy's & St Thomas' NHS Foundation Trust.  Matriz CGH para a detecção de variantes do número de cópia genômicas substituiu análise do cariótipo G-unida. Este artigo descreve a tecnologia e sua aplicação em um laboratório de serviço de diagnóstico. 

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

Isolation of Genomic DNA from Mouse Tails

Isolamento de DNA genômico de Tails rato

Biologia

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.

Demonstração de técnicas Whole Genome hibridização genômica comparativa de matriz

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 Array para Crônica In vivo</emGravação>: Fabricação Unidade

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.

Grandes Inserir Produção Ambiental Biblioteca Genômica

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 DNA: Controle de Qualidade da amostra, Hibridação Array e Digitalização

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.

Genômica MRI - um recurso público para estudar os padrões de seqüência dentro de DNA genômico

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.

Competitiva Screens Genomic de Bibliotecas Yeast 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.

Detecção de variantes raras genômico de Seqüenciamento combinados usando 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.

Transformação genômica do 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 selectivo de 5-hydroxymethylcytosine a partir de DNA 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 ...