Los autores agradecen a la Sra. Jennifer M. Lommel y Tara N. Rognan para el mantenimiento de plantas de Arabidopsis. Este trabajo fue apoyado por la puesta en marcha de fondos de la Universidad de Saint Louis y los Institutos Nacionales de Salud subvenciones 1R15GM086846-01 y 3R15GM086846-01S1 a W. Xiao.

I. genética Cruce 1. Castrando el progenitor femenino Con el fin de distinguir los alelos maternos y paternos mediante el uso de polimorfismo de secuencia de ADN, dos ecotipos diferentes, por ejemplo, Columbia-0 (Col-0) y Ler, será elegido como padres de familia femenino y masculino. Las plantas deben ser joven y saludable. Se puede castrar el progenitor femenino mediante el uso de un microscopio de disección, de aumento visor, o el ojo desnudo. Localizar 12 etapa flores (Smyth et al., 1990) y quitar cualquier flor o silicuas por encima y por debajo de ellos por el recorte de la base del pedúnculo con las tijeras. Esterilizar pinzas por inmersión de la base de la punta suavemente en un vaso de etanol al 95%, lo que eliminará los granos de polen en las pinzas y matar al polen también. Haciendo palanca con cuidado, aparte las yemas florales con unas pinzas y retire con cuidado 4 sépalos, pétalos 4, y 6 estambres, los pistilos dejando al descubierto e intacta. Trate de evitar el daño a los carpelos durante este proceso. 2. La elección del donante de polen y llevar a cabo la polinización Los donantes de polen son más anthesed flores se abren en la etapa 14, con los pétalos que se extiende en un ángulo de 90 ° para el pistilo (Smyth et al., 1990), en el que una gran cantidad de polen está derramando. Coge la flor en la base y justo por encima del pedúnculo, lo que hará que la flor de difusión abierta. Polvo el estigma del pistilo preparado con la antera. Después de la polinización, el estigma que serán cubiertos con el polen amarillo, que puede ser fácilmente observado bajo un microscopio. 3. Etiquetado de la cruz Después de la polinización todos los pistilos castrado en una planta, que marca la cruz con una etiqueta de la joyería y la información de los padres de ambos sexos y la fecha. Coloque una estaca en el suelo cerca de la planta, utilice una cadena para atar el tallo de la inflorescencia de la hoguera, y cubren los pistilos de polinización con una bolsa de plástico. II. El aislamiento de los tejidos del endospermo en Arabidopsis 1. Preparación de materiales Por lo general, obtener el material necesario listo antes de la cosecha del endospermo y los tejidos del embrión: un tanque de nitrógeno líquido, nitrógeno líquido, un microscopio de disección, dos nuevos pares de pinzas de punta fina (5 INOX FST por Dumont biología, Suiza.), Portaobjetos de vidrio ( 3 &quot;x 1&quot; x 1.0 mm), un pH de 5,7 solución de 0,3 M sorbitol y 5 mM MES. 2. Silicuas recolección A las 7 - y 8 días después de la polinización (DAP), las semillas están listas para ser cosechadas a mediados de la última etapa de torpedo de la embriogénesis y la disección de endospermo y el embrión. A veces, puede tardar 9-10 DAP si las flores están desvirtuadas demasiado joven. 3. Endospermo y el embrión de aislamiento Ya que las semillas de Arabidopsis son pequeños, se utiliza un microscopio de disección para aislar el endospermo y el embrión. Ponga una silicua 8-DAP con un microscopio, use un par de pinzas para sostener el pedicelo silicua y el uso de la punta del otro par de pinzas para abra la silicua en el margen donde se fusionan dos carpelos. Use un par de pinzas para recoger una semilla en soluciones de pH 5,7 de 0,3 M sorbitol y 5 mM MES, hacer un pequeño corte en el extremo micrópilo se deslice fuera del embrión, apriete el extremo sin cortar para expulsar el endospermo y el endosperma separado de cubierta de la semilla (Kinoshita et al., 2004). Puesto que el embrión y el endospermo en microtubos separado en nitrógeno líquido. Continúe hasta que se acumulan embrión o endospermo 10 a 15 silicuas y luego guarde el tubo en un congelador de -80 º C. En algunos casos, el endospermo no tiene que ser separado de cubierta de la semilla, es decir, se puede aislar el ARN total a partir del endospermo y la mezcla de cáscara de la semilla para el análisis de los genes la impronta. III. La secuenciación de bisulfito 1. Reactivos necesarios Bromuro de amonio cetiltrimetil (CTAB) para la preparación de ADN genómico, las enzimas de restricción, 3 M NaOH (recién hecho), 6,42 M de urea / 4 bisulfito de sodio M (2 metabisulfito de sodio M, Sigma-Aldrich, S9000, Na2S2O5, peso molecular: 190), 10 mM hidroquinona, un kit de purificación de ADN (Promega ADN Asistente de limpieza del sistema, cat. # A7280), tampón TE, 6,3 M NaOH (recién hecha), 10 M NH 4 OAc, 20 mg / l tRNA, y el 100% de etanol . 2. Los detalles del protocolo de tratamiento de bisulfito <ol><li> Aislar ADN genómico o del endospermo del embrión mediante un procedimiento de CTAB (Rogers y Bendich, 1988). </li><li> Recopilación de 100 ng - 2 mg de ADN genómico en 20 l de volumen de 100 l total con enzimas de restricción que cortan fuera de la región para ser analizados. Para el promotor de MEA, usamos XhoI, NdeI y PstI o HindIII. </li><li> Desnaturalizar las enzimas de restricción mediante la ebullición del ADN de cinco minutos y luego apagar el hielo. </li><li> Añadir un volumen 1 / 9 (2,2 l de 20 l de ADN digerido), de 3 M de NaOH y se incuba a 37 ° C durante 15 minutos. </li><li> Transferir la solución a un tubo de 250 l de PCR. </li><li> Disolver 7,5 g de urea en 10 ml de agua destilada estéril; Poco a poco añadir 7,6 g de metabisulfito de sodio durante 1-2 horas y la calefacción por lo general ayuda a la disolución, ajustar el pH a 5 con NaOH recién hecho 10 M, añadir agua destilada estéril a una final volumen de 20 ml. Se trata de 6,24 M de urea / 4 solución de sodio bisulfito M. </li><li> Añadir 6,24 M de urea / 4 M solución de bisulfito de sodio a una concentración final de 5,36 M y M 3,44, respectivamente (Paulin et al., 1998). Por ejemplo, añadir 208 l de 6,42 M de urea / 4 M solución de bisulfito de sodio a los anteriores 22,2 l de ADN genómico desnaturalizado (Xiao et al., 2003). </li><li> Añadir 10 mM hidroquinona al ADN a una concentración final de 0,5 mM (12 l para la digestión 20 l). </li><li> Realizar el tratamiento de bisulfito en una máquina de PCR: 30 ciclos de 55 ° C durante 15 minutos y los segundos 95 ° C durante 30. </li><li> Desalar el ADN bisulfito tratados con el ADN Asistente de Limpieza del sistema de Promega y el seguimiento del protocolo (Jacobsen et al., 2000). </li><li> Medir el volumen exacto de TE se recuperó de la columna después de la desalación y añadir 6,3 M de NaOH a una concentración final de 0,3 M. Se incuba a 37 ° C durante 15 minutos. </li><li> Añadir 10 M NH 4 OAc (pH 7,0) a una concentración final de 3 M, 2 l de 20 ug / l tRNA, y 3 volúmenes de etanol al 100% y luego mezclar. Centrifugar durante 15 minutos a 14.000 rpm. </li><li> Lavar el sedimento una vez con etanol al 70%, hacer un centrifugado corto, y retirar el etanol extra. </li><li> Seco en pelets en un speedvac durante 5-10 minutos y resuspender en 25 hasta 100 l de buffer TE en función de la cantidad de ADN de partida. El bisulfito sódico tratados con ADN está listo para el análisis de PCR. </li></ol> 3. Amplificación por PCR <ol><li> Desde citosinas no metiladas se convierten en uracilo, es difícil para amplificar un fragmento de gran tamaño utilizando el ADN tratado con bisulfito como una plantilla. Por lo tanto, suelen diseñar primers para amplificar un producto de no más de 500 pb. Para la secuencia del promotor de 4 kb MEA, hemos diseñado varios conjuntos de cebadores y la superposición de fragmentos amplificados 14 para cubrir toda la región (Xiao et al., 2003). </li><li> A la secuencia de la parte superior de hebra, en el diseño de una cartilla con interés, I) elegir una G (guanina)-rica región con el fin de tener una temperatura más alta de recocido sin nucleótidos de largo adicionales en las cartillas, II) el cambio C (citosina) a Y ( pirimidina) en el CG y los contextos de GNC y cambiar el resto de C a T (timina). En el diseño de un cebador inverso, I) elija una región rica en C, II) el cambio de G R (purina) en CG y los contextos de GNC y cambiar el resto de G a A (adenina). </li><li> A la secuencia de la parte inferior-cadena, en el diseño de una cartilla con interés, I) elija una región rica en C, II) el cambio G a R en CG y los contextos de GNC y el cambio del G restantes a A. En el diseño de un cebador inverso, I) elegir una región rica en G; II) El cambio C a Y (pirimidina) en el CG y los contextos de GNC y cambiar el resto de C para T. </li><li> Por lo general, el uso 1.2 l de sodio bisulfito tratados con ADN como una plantilla para cada amplificación por PCR (Xiao et al., 2003). El producto de PCR debe ser analizado por electroforesis en gel para confirmar el tamaño correcto del fragmento, luego de ser gel purificado y clonado en el vector TOPO TA clonación pCR2.1 (Invitrogen) como una inserción. Una sola colonia se recoge y se cultivan; ADN plásmido extraído y enviado para su secuenciación. </li></ol> 4. Análisis de secuencias El principio de la secuenciación de bisulfito es que la citosina no metilados se convertirá en uracilo debido a la desaminación hidrolítica por la alta concentración de bisulfito de sodio a pH 5,0, que se amplifica a medida que la timina en el producto de PCR, mientras que el 5-metil citosina no será modificado por bisulfito de sodio y aún no se han citosina después de la amplificación por PCR (Clark et al, 1994;. Frommer et al, 1992).. Después de obtener el resultado de la secuencia, se la compara con la plantilla capítulo específico que se utiliza para la amplificación por PCR. Si un residuo de citosina en la plantilla se lee como una timina en el resultado de la secuencia, indica que la citosina no está metilado. Si un residuo de citosina en la plantilla sigue siendo una citosina en la secuencia, significa que la citosina está metilado. IV. Resultados representante <img src="/files/ftp_upload/2327/2327fig1.jpg" alt="Figura 1 /" /> Figura 1.

<ol>
	<li>Clark, S. J., Harrison, J., Paul, C. L., Frommer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+sensitivity+mapping+of+methylated+cytosines.">High sensitivity mapping of methylated cytosines.</a> Nucleic Acids Res. 22, 2990-2997 (1994).</li><li>Cokus, S. J., Feng, S., Zhang, X., Chen, Z., Merriman, B., Haudenschild, C. D., Sriharsa Pradhan, S., Nelson, S. F., Pellegrini, M., Jacobsen, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shotgun+bisulphite+sequencing+of+the+Arabidopsis+genome+reveals+DNA+methylation+patterning.">Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning.</a> Nature. 452, 215-219 (2008).</li><li>Frommer, M., McDonald, L. E., Millar, D. S., Collis, C. M., Watt, F., Grigg, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genomic+sequencing+protocol+that+yields+a+positive+display+of+5-methylcytosine+residues+in+individual+DNA+strands.">A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.</a> Proc Natl Acad Sci USA. 89, 1827-1831 (1992).</li><li>Gehring, M., Huh, J. H., Hsieh, T. F., Penterman, J., Choi, Y., Harada, J. J., Goldberg, R. B., Fischer, R. L. D. E. M. E. T. E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+glycosylase+establishes+MEDEA+polycomb+gene+self-imprinting+by+allele-specific+demethylation.">DNA glycosylase establishes MEDEA polycomb gene self-imprinting by allele-specific demethylation.</a> Cell. 124, 495-506 (2006).</li><li>Henderson, I. R., Chan, S. R., Cao, X., Johnson, L., Jacobsen, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+sodium+bisulfite+sequencing+in+plants.">Accurate sodium bisulfite sequencing in plants.</a> Epigenetics. 5, 47-49 (2010).</li><li>Hsieh, T. F., Ibarra, C. A., Silva, P., Zemach, A., Eshed-Williams, L., Fischer, R. L., Zilberman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+demethylation+of+Arabidopsis+endosperm.">Genome-wide demethylation of Arabidopsis endosperm.</a> Science. 324, 1451-1454 (2009).</li><li>Jacobsen, S. E., Sakai, H., Finnegan, E. J., Cao, X., Meyerowitz, E. 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Es relativamente fácil de separar del embrión y endospermo cubierta de la semilla, pero es tedioso para separar el endospermo de la cáscara de la semilla, especialmente para las semillas en la etapa de principios o mediados de torpedo de la embriogénesis. Desde cubierta de la semilla sólo aporta una pequeña cantidad de tejido, para algunos genes, por ejemplo, MEA y FWA, no hay que separar el endospermo de la cáscara de la semilla. Esto significa que podemos aislar ARN a partir de una mezcla de e...

Suministros <ul><li> Microscopio de disección </li><li> Tijeras </li><li> Pinzas de punta fina </li><li> Joyas de la etiqueta </li><li> Las estacas de plantas </li><li> Cuerdas o cintas de cierre </li><li> 4 &quot;X 2&quot; X 8 &quot;Bolsas de Polietileno </li><li> 3 &quot;x 1&quot; x 1.0 mm Micropreparados </li><li> El nitrógeno líquido </li><li> Los contenedores de nitrógeno líquido </li><li> Bloquear el calor </li><li> Tubos para PCR </li><li> Termociclador </li><li> Tubos de microcentrífuga </li><li> Microcentrífuga </li><li> Gel de electroforesis de instalación </li><li> Arabidopsis thaliana Columbia-0 plantas </li><li> Arabidopsis thaliana Landsberg erecta plantas </li></ul> Reactivos <ul><li> 70% de etanol </li><li> Etanol al 95% </li><li> 100% de etanol </li><li> 0,3 M sorbitol y 5 mM MES, pH 5,7 </li><li> Bromuro de amonio cetiltrimetil (CTAB) </li><li> 100% de etanol </li><li> Cholorform </li><li> Las enzimas de restricción </li><li> 3 M NaOH </li><li> 6,3 M NaOH </li><li> 6,24 M de urea / 4 bisulfito de sodio M </li><li> Destilada estéril H 2 O </li><li> 10 mM hidroquinona </li><li> Asistente de ADN Clean-Up System (Promega) </li><li> 10 M NH 4 OAc </li><li> 20 mg / l tRNA </li><li> Tampón TE </li><li> El kit de clonación TOPO TA (Invitrogen) </li></ul>

determination of dna methylation of imprinted genes in arabidopsis endosperm

Arabidopsis thaliana es un organismo modelo excelente para estudiar los mecanismos epigenéticos. Una de las razones es el mutante nulo de pérdida de función de ADN metiltransferasa es viable, proporcionando así un sistema para estudiar cómo la pérdida de metilación del ADN en el genoma afecta el crecimiento y desarrollo. La impronta se refiere a la expresión diferencial de los alelos maternos y paternos, y juega un papel importante en el desarrollo de la reproducción, tanto en mamíferos y plantas. Metilación del ADN es fundamental para determinar si los alelos maternos y paternos de un gen impreso se expresa o se silencian. En las plantas con flores, hay un evento de doble fertilización en la reproducción: un espermatozoide fertiliza el óvulo para formar un embrión y el espermatozoide se fusiona con la célula de segundo central para dar lugar a endospermo. Endospermo es el tejido donde se produce en las plantas de impresión. MEDEA, un dominio establecido Polycomb gen del grupo, y la FWA, un factor de transcripción que regulan la floración, son los dos primeros genes demostrado ser impreso en el endospermo y su expresión está controlada por la metilación del ADN y desmetilación en las plantas. Con el fin de determinar el estado de impresión de un gen y el patrón de metilación en el endospermo, tenemos que ser capaces de aislar primero endospermo. Desde la semilla es muy pequeña en Arabidopsis, que sigue siendo un reto para aislar endospermo Arabidopsis y examinar su metilación. En este protocolo de vídeo, se presenta la manera de realizar un cruce genético, para aislar el tejido del endospermo de las semillas, y para determinar el estado de metilación de la secuenciación de bisulfito.

Watch this Scientific Journal Video about Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm at JoVE.com

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

La impresión es un fenómeno en la planta y reproducción de los mamíferos. Metilación del ADN juega un papel importante en los mecanismos de impresión. Aislar endospermo y determinar el estado de metilación de los genes impresos en<em&gt; Arabidopsis</em&gt; Puede ser difícil. En este protocolo, se describe cómo aislar endospermo y determinar la metilación de la secuenciación de bisulfito.

Determinación de la metilación del ADN de los genes impresos en Arabidopsis Endospermo

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

determination-dna-methylation-imprinted-genes-arabidopsis

Research

JoVE Journal

Biology

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

17.4K vistas.  Saint Louis University. La impresión es un fenómeno en la planta y reproducción de los mamíferos. Metilación del ADN juega un papel importante en los mecanismos de impresión. Aislar endospermo y determinar el estado de metilación de los genes impresos en Arabidopsis Puede ser difícil. En este protocolo, se describe cómo aislar endospermo y determinar la metilación de la secuenciación de bisulfito.

Video: Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

La metilación del ADN: Bisulfito de modificación y análisis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

Investigación

Biología

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

Chromosomics: Detección de alteraciones numéricas y estructurales en todos los 24 cromosomas humanos de manera simultánea utilizando un nuevo ensayo FISH OctoChrome

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a &#34;seed coat bedding&#34; assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

We present the procedure to assemble a seed coat bedding assay (SCBA) from Arabidopsis thaliana seeds. The SCBA was shown to be a powerful tool to explore genetically and in vitro how the endosperm controls seed germination in dormant seeds and in response to light cues. The SCBA is in principle applicable under any situation where the endosperm is suspected to influence embryonic growth.

Un Abrigo Ropa de cama de Ensayo de Semillas para explorar Genéticamente<em&gt; In Vitro</em&gt; ¿Cómo la germinación de semillas en Controles Endosperma<em&gt; Arabidopsis thaliana</em

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

Imágenes en tiempo real de Soltero Engineered ARN transcritos en células vivas utilizando Ratiometric bimoleculares Balizas

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

gDNA Enriquecimiento por una tecnología basada en la transposasa de Análisis NGS de la secuencia entera de<em&gt; BRCA1</em&gt;,<em&gt; BRCA2</em&gt;, y 9 genes involucrados en la reparación del ADN daños

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

In vitro metilación de ensayo para estudiar la proteína arginina metilación

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

Enhanced reducido Representación bisulfito secuenciación para la Evaluación de la metilación del ADN en la Resolución Base Par

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

La correlación de Gene-específica de ADN de metilación cambios con la expresión y actividad transcripcional de astrocíticos<em&gt; KCNJ10</em&gt; (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

Dirigida análisis de metilación del ADN mediante secuenciación de próxima generación

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Ensayo de inmunoprecipitación de la cromatina para la Identificación de Arabidopsis proteína-DNA<em&gt; En Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...