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><tbody><tr><td>CGH microarray 8 x 60K</td><td>Agilent</td><td>G4126</td><td></td></tr><tr><td>Array CGH wash buffers</td><td>Agilent</td><td>5188-5226</td><td></td></tr><tr><td>Array CGH hybridisation buffer</td><td>Agilent</td><td>5188-5380</td><td></td></tr><tr><td>Minelute purification kit</td><td>Qiagen</td><td>28006</td><td></td></tr><tr><td>Array CGH labelling kit</td><td>Enzo</td><td>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 ...

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Research

JoVE Journal

Biology

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

19.5K 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

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detecção de Copy Number Alterações Usando Sequencing única célula

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Genética

FISH for Pre-implantation Genetic Diagnosis 

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people's hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting.

FISH for Pre-implantation Genetic Diagnosis

This article describes the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to pre-implantation genetic diagnosis (PGD) in a clinical setting.

FISH para diagnóstico pré-implantação Genética

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a ...

Medicina

Serum and Plasma Copy Number Detection Using Real-time PCR

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, and prediction of treatment resistance. Starting from the hypothesis that androgen receptor (AR) gene copy number (CN) gain is a frequent event in metastatic castration resistance prostate cancer (mCRPC), we propose to analyze this event in cfDNA as a potential predictive biomarker.
We evaluated AR CN in cfDNA using 2 different real-time PCR assays and 2 reference genes (RNaseP and AGO1). DNA amount of 60 ng was used for each assay combination. AR CN gain was confirmed using Digital PCR as a more accurate method. CN variation analysis has already been demonstrated to be informative for the prediction of treatment resistance in the setting of mCRPC, but it could be useful also for other purposes in different patient settings. CN analysis on cfDNA has several advantages: it is non-invasive, rapid and easy to perform, and it starts from a small volume of serum or plasma material.

This manuscript describes the copy number variation analysis performed in serum or plasma DNA using real-time PCR approach. This method is suitable for the prediction of drug resistance in castration resistant prostate cancer patients, but it could be informative also for other diseases.

Soro e Plasma cópia número deteção usando PCR em tempo real

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, ...

Pesquisa do câncer

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including biological such as T-DNA or transposon, chemical such as ethyl methanesulfonate (EMS), or physical such as ionization radiation. The type of mutation observed varies depending on the mutagen used. For ionization radiation induced mutants, mutations include deletion, duplication, or rearrangement. While T-DNA or transposon-based mutagenesis is limited to species that are susceptible to transformation, chemical or physical mutagenesis can be applied to a broad range of species. However, the characterization of mutations derived from chemical or physical mutagenesis traditionally relies on a map-based cloning approach, which is labor intensive and time consuming. Here, we show that a high-density genome array-based comparative genomic hybridization (aCGH) platform can be applied to efficiently detect and characterize copy number variations (CNVs) in mutants derived from fast neutron bombardment (FNB) mutagenesis in Medicago truncatula, a legume species. Whole genome sequence analysis shows that there are more than 50,000 genes or gene models in M. truncatula. At present, FNB-induced mutants in M. truncatula are derived from more than 150,000 M1 lines, representing invaluable genetic resources for functional studies of genes in the genome. The aCGH platform described here is an efficient tool for characterizing FNB-induced mutants in M. truncatula.

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

This protocol provides experimental steps and information about reagents, equipment, and analysis tools for researchers who are interested in carrying out whole genome array-based comparative genomic hybridization (CGH) analysis of copy number variations in plants.

Uma plataforma array de hibridização genômica comparativa para detecção eficiente de variações no número de cópias em Medicago truncatula mutantes induzidos por nêutrons rápidos

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including ...

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields. dPCR is capable of detecting rare targets; in addition, dPCR offers the possibility for absolute and precise quantification of nucleic acids without the need for calibrators and standard curves. In fact, the reaction partitioning enriches the target from the background, which improves amplification efficiency and tolerance to inhibitors. Such characteristics make dPCR an optimal tool for the detection of the CDH1a rare transcript.

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

The manuscript describes a chip-based digital PCR assay to detect a rare CDH1 transcript variant (CDH1a) in fresh-frozen normal and tumor tissues obtained from patients with gastric cancer.

Deteção de uma variante de transcrição raro CDH1 em tecidos de câncer gástrico fresco congelado pelo PCR Digital baseada em Chip

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective tag single nucleotide polymorphism (SNP) approach using a multiplexed genotyping assay with mass spectrometry, to investigate gene pathway associations in clinical cohorts. We investigate the food allergy candidate locus Interleukin13 (IL13) as an example. This method efficiently maximizes the coverage by taking advantage of shared linkage disequilibrium (LD) within a region. Selected LD SNPs are then designed into a multiplexed assay, enabling up to 40 different SNPs to be analyzed simultaneously, boosting cost-effectiveness. Polymerase chain reaction (PCR) is used to amplify the target loci, followed by single nucleotide extension, and the amplicons are then measured using matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrometry. The raw output is analyzed with the genotype calling software, using stringent quality control definitions and cut-offs, and high probability genotypes are determined and output for data analysis.

Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies.

Gene candidato teste em estudos de coorte clínica com genotipagem multiplexada e espectrometria de massa

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

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 ...

Biologia

Pre-Implantation Genetic Testing for Aneuploidy on a Semiconductor Based Next-Generation Sequencing Platform

Next-generation sequencing has gained increasing importance in the clinical application in the determination of genetic variants. In the pre-implantation genetic test, this technique has its unique advantages in scalability, throughput, and cost. For the pre-implantation genetic test for aneuploidy analysis, the semiconductor-based next-generation sequencing (NGS) system presented here provides a comprehensive approach to determine structural genetic variants at a minimum resolution of 8 Mb. From sample acquisition to the final report, the working process requires multiple steps with close adherence to protocols. Since various critical steps could determine the outcome of amplification, quality of the library, coverage of reads, and output of data, descriptive information with visual demonstration other than words could offer more detail to the operation and manipulation, which may have a great impact on the results of all critical steps. The methods presented herein will display the procedures involved in whole genome amplification (WGA) of biopsied Trophectoderm (TE) cells, genomic library construction, sequencer management, and finally, generating copy number variants' reports.

The protocol presents the overall in-lab procedures required in pre-implantation genetic testing for aneuploidy on a semiconductor-based next-generation sequencing platform. Here we present the detailed steps of whole genome amplification, DNA fragment selection, library construction, template preparation, and sequencing working flow with representative results.

Teste genético pré-implantação para aneuploidia em uma plataforma de sequenciamento de próxima geração baseada em semicondutores

Next-generation sequencing has gained increasing importance in the clinical application in the determination of genetic variants. In the pre-implantation ...

Comparative Lesions Analysis Through a Targeted Sequencing Approach

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective anti-tumor strategies. Although concerns are frequently raised due to differences in sample processing and depth of coverage, next-generation sequencing of solid tumors have unraveled a highly variable degree of ITH across tumor types. Capturing the genetic relatedness between primary and metastatic lesions through the identification of clonal and subclonal populations is critical to the design of therapies for advance-stage diseases. Here, we report a method for comparative lesions analysis that allows for the identification of clonal and subclonal populations among different specimens from the same patient. The experimental approach described here integrates three well-established approaches: histological analysis, high-coverage multi-lesion sequencing, and immunophenotypic analyses. In order to minimize the effects on the detection of subclonal events by inappropriate sample processing, we subjected tissues to careful pathological examination and neoplastic cell enrichment. Quality controlled DNA from neoplastic lesions and normal tissues was then subjected to high coverage sequencing, targeting the coding regions of 409 relevant cancer genes. While only looking at a limited genomic space, our approach enables evaluating the extent of heterogeneity among somatic alterations (single-nucleotide mutations and copy-number variations) in distinct lesions from a given patient. Through comparative analysis of sequencing data, we were able to distinguish clonal vs. subclonal alterations. The majority of ITH is often ascribed to passenger mutations; therefore, we also used immunohistochemistry to predict functional consequences of mutations. While this protocol has been applied to a specific tumor type, we anticipate that the methodology described here is broadly applicable to other solid tumor types.

This article describes a method to identify clonal and subclonal alterations among different specimens from a given patient. Although the experiments described here focus on a specific tumor type, the approach is broadly applicable to other solid tumors.

Análise comparativa de lesões por meio de uma abordagem de sequenciamento direcionada

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective ...

Comportamento

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

Simples massa de Leitura Digital de Ácido Nucleico Quantificação Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...