Name: methyl-binding dna capture sequencing for patient tissues
Uploaded: 2016-10-31T12:30:00.000+00:00
Duration: 8 min 40 s
Description: Watch this Scientific Journal Video about Methyl-binding DNA capture Sequencing for Patient Tissues at JoVE.com

El trabajo es apoyado por CPRIT Investigación Formación Premio RP140105, así como parcialmente apoyado por los Institutos Nacionales de Estados Unidos de Salud (NIH) subvenciones R01 GM114142 y por William Owens &amp; Ella Fundación de Investigación Médica.

Todos los tejidos se obtienen después de la aprobación del Comité de Revisión Institucional y cuando todos los participantes dieron su consentimiento para ambos análisis moleculares y estudios de seguimiento. Los protocolos son aprobados por el Comité de Estudios Humanos de la Universidad de Texas Health Science Center en San Antonio. 1. unión Metil-de captura de ADN (MBDCap) <ol><li> Recogida de muestras y el aislamiento de ADN <ol><li> Recoger tumor mayor o muestras de tejido normal a partir de muestras de tejido embebidas en parafina de pacientes. </li><li> Utilice un kit comercial de mini ADN para aislar el ADN genómico de las muestras de tejido embebidas en parafina de acuerdo con el protocolo del fabricante. </li><li> Utilice un kit de enriquecimiento de ADN metilado con el procedimiento descrito aquí de acuerdo con el protocolo del fabricante. </li></ol></li><li> Lavado del grano inicial Nota: Las perlas se utilizan para acoplar con proteínas MBD-biotina, que detecta la metilación del ADN en el genoma. Las cuentas se suspenden generalmenteen una solución de reserva. Utilice estos pasos para lavar este líquido. <ol><li> Volver a suspender las acciones de perlas de estreptavidina (Ver Tabla de Materiales) pipeteando suavemente hacia arriba y hacia abajo para obtener una suspensión homogénea. No mezclar los granos con el vórtex. </li><li> Por un g de ADN de entrada, ADD10 l de perlas a un tubo con 90 l de 1x bind / tampón de lavado. Rotar durante 1 min. No mezclar mediante agitación. </li><li> Se coloca el tubo (s) sobre una rejilla magnética durante 1 min. </li><li> Retire el líquido con una pipeta y descartar el líquido. </li><li> Añadir 250 l de 1x bind / tampón de lavado hasta el talón y girar durante 1 min. </li><li> Repita los pasos 1.2.3 - 1.2.5 dos veces. </li></ol></li><li> El acoplamiento de la proteína MBD-biotina a las perlas <ol><li> Para 1,2 g de ADN de entrada, añadir 7 l (3,5 g) de la proteína MBD-biotina a cada tubo. </li><li> Añadir 93 l de 1x bind / tampón de lavado a la proteína para hacer un volumen final de 100 mL. </li><li> Mezclar el grano-proteínamezcla en un mezclador giratorio a temperatura ambiente durante 1 h. </li></ol></li><li> La fragmentación de ADN (de entrada) <ol><li> Someter a ultrasonidos ADN mediante la adición de 1,2 g de ADN genómico total en 96 l de tampón TE y 24 l de 5x tampón / lavado de enlace para hacer una solución de 120 mL. Utilice 30 s de potencia ON y OFF 30 s de energía durante la sonicación, 20 - 25 veces. </li><li> Ejecutar 1 l de solución de ADN se sometió a ultrasonidos utilizando un sistema Bioanalyzer con un kit de ADN de alta sensibilidad comercial para comprobar el tamaño de los fragmentos a estar en el rango de 150 a 350 pb de acuerdo con el protocolo del fabricante. </li></ol></li><li> Se lavan las MBD-cuentas <ol><li> Se coloca el tubo que contiene MBD-cuentas sobre una rejilla magnética durante 1 min. </li><li> Retire y deseche el líquido con una pipeta sin tocar las bolas. </li><li> Volver a suspender las perlas con 250 l de 1x bind / tampón de lavado. </li><li> Mezclar las perlas en un mezclador giratorio a temperatura ambiente durante 5 min. </li><li> Repita los pasos 1.5.1 - 1.5.4 dos veces. </li&gt; </ol></li><li> La reacción de captura de ADN fragmentado (1 g de ADN de entrada) <ol><li> Transferir la mezcla de ADN / Buffer al tubo que contiene los MBD-perlas. </li><li> Mezclar los MBD-perlas con el ADN en un mezclador giratorio durante 1 h a TA. Alternativamente, la mezcla de O / N a 4 ° C. </li></ol></li><li> Extracción de ADN no capturado de las perlas <ol><li> Después de mezclar el ADN y MBD-perlas, colocar el tubo en la gradilla magnética durante 1 minuto para concentran todas las perlas en la pared interior del tubo. </li><li> Eliminar el líquido sobrenadante con una pipeta y guardarlo en un tubo de microcentrífuga libre de DNasa limpio como el ADN no metilado. Guarde esta muestra en hielo. </li><li> Añadir 200 l de 1x bind / tampón de lavado a las perlas para lavar los granos. </li><li> Mezclar las perlas en un mezclador giratorio para 3 min. </li><li> Se coloca el tubo en la gradilla magnética durante 1 min. </li><li> Retire el líquido con una pipeta y guardarlo en un tubo de microcentrífuga. </li><li> para la tapatura reacciones de ≤1 g de ADN de entrada, repita los pasos 1.7.3 - 1.7.6 para 2 más fracciones de lavado en total. </li></ol></li><li> Elución sola fracción <ol><li> Mezclar tampón bajo en sal con tampón de alta salinidad (1: 1) para obtener tampón de elución (NaCl 1,000 mM). </li><li> Resuspender las perlas en 200 l de tampón de elución (NaCl 1,000 mM). </li><li> Se incuban las cuentas de un mezclador rotativo durante 3 min. </li><li> Se coloca el tubo en la gradilla magnética durante 1 min. </li><li> Con el tubo en su lugar en la rejilla magnética, eliminar el líquido con una pipeta sin tocar las bolas con la punta de la pipeta, y guardarlo en un tubo de microcentrífuga de 1,5 ml libre de DNasa limpia. Guarde esta muestra en hielo. </li><li> Repetir los pasos 1.8.1 - 1.8.4 una vez, la recogida de la segunda muestra en el mismo tubo (volumen total será de 400 l). Almacenar la muestra agrupada en hielo. </li></ol></li><li> Precipitación con etanol (limpieza ADN) <ol><li> Para cada uno, de lavado y elución fr fracción noncapturedom los pasos anteriores, añadir 1 l de glucógeno (20 mg / l, incluido en el kit), el volumen de un décimo de la muestra de acetato de sodio 3M, pH 5,2 (por ejemplo, 40 l por 400 l de muestra) y 2 volúmenes de muestra de 100% etanol (por ejemplo, 800 l por 400 l de muestra). El volumen total es de 1.241 l. </li><li> Mezclar bien e incubar a -80 ° C durante al menos 2 h. </li><li> Centrifugar el tubo durante 15 min a 11.363 xg a 4 ° C. </li><li> deseche con cuidado el sobrenadante sin perturbar el sedimento. </li><li> Añadir 500 l de etanol frío al 70%. </li><li> Centrifugar el tubo durante 5 minutos a 11.363 xga 4 ° C </li><li> deseche con cuidado el sobrenadante sin perturbar el sedimento. </li><li> Repita los pasos 1.9.6 - 1.9.7 vez y eliminar cualquier sobrenadante residual que queda. </li><li> El aire seco de la pastilla de ~ 5 min. No seque completamente el sedimento. </li><li> Resuspender el sedimento de ADN en 37,5 l de agua libre de ADNasa u otrovolumen apropiado de tampón. </li><li> Coloque el ADN en hielo o almacenar el ADN a -20 ° C o por debajo hasta su uso posterior. </li><li> Compruebe que la cantidad total de ADN es más de 20 ng, (por ejemplo, utilizar el sistema de cuantificación fluorométrica, ver Tabla de Materiales). </li></ol></li></ol> 2. Secuenciación <ol><li> Usa los fragmentos de ADN recuperados del procedimiento MBDCap para la secuenciación. Para cada muestra que se secuenció, una cantidad total de 20 - Se requiere 40 ng de ADN para la preparación de la biblioteca. </li><li> Para la preparación de la biblioteca de ADN-seq utilizar un sistema de construcción de la biblioteca totalmente automatizado (Ver Tabla de Materiales) y seguir el protocolo estándar suministrado por el fabricante. Seleccione fragmentos de ADN de tamaño 200 a 400 pb para la preparación de la biblioteca. El sistema de preparación de la biblioteca de automatización permite estandarizar el procedimiento de preparación de la biblioteca y minimizar la variación entre las muestras debido a la preparación manual. </li><li> primer uso del adaptadorRS y diluir a la concentración de 100x. Por ello, utilice 10 l para cada muestra. </li><li> Después de la etapa 2.2, llevará a cabo la reacción, y cuantificar la biblioteca de ADN-ss usando un sistema de cuantificación fluorométrico (Ver Tabla de Materiales). <ol><li> Establecer procedimiento de amplificación por PCR. La elección de la mezcla de PCR principal es mejorar la eficiencia de la PCR de GC enriquecido región del genoma. En un tubo de PCR, añadir 15 l de la biblioteca de ADN-ss, 25 l de PCR Master Mix (Ver Tabla de Materiales), 2 l de PCR mezcla de cebadores (Ver Tabla de Materiales), y 8 l de H2O </li></ol></li><li> Seguir las recomendaciones PCR del fabricante de la mezcla maestra de PCR, la alteración de los tiempos de ciclo y temperaturas para diferentes materiales de partida: 98 ° C durante 45 s, los ciclos X de 98 ° C durante 15 s, 65 ° C durante 30 s, 72 ° C durante 30 s, y luego 72 ° C durante 1 min. Mantener a 4 ° C <ol><li> perfORM suficientes ciclos para terminar con un 2 - 10 nM de ADN de la librería (8 - 10 ciclos). </li></ol></li><li> Limpiar reacción de PCR con 1: relación de perlas de purificación de PCR de acuerdo con el protocolo del fabricante 1 (Ver Tabla de Materiales). </li><li> Eluir con 20 - tampón de elución 30 l. </li><li> Código de barras de cada muestra y piscina 4 muestras juntas en un carril de una celda de flujo. Utilizar el protocolo estándar de 50 pb lectura única para la secuenciación (Ver Tabla de Materiales). </li></ol> 3. Análisis de Bioinformática Nota: Además de procesar los archivos FASTQ primas obtenidas de la secuenciación para llevar a cabo el control de calidad y el mapeo de las secuencias cortas de ADN (lee) en el genoma. <ol><li> El uso de Bowtie alineador de lectura corta, herramienta de mapeo del genoma en un archivo de muestra FASTQ para mapear las lecturas al genoma de referencia. Muchas herramientas de mapeo de lectura están disponibles para llevar a cabo este paso y se pueden utilizar en base a las preferencias individuales. <ol><li> Permitir hasta 2 desajustes en el whole lee, mientras que la cartografía al genoma de referencia. Reportar hasta 20 mejores alineaciones para cada lectura. Por ejemplo, utilizar pajarita -v -k 2 --el 20 --chunkmbs 200 &lt;EBWT&gt; &lt;fastqFile&gt; &lt;alignFile&gt;. </li></ol></li><li> Utilice LONUT 9 para recuperar el mapeado multiplican lee para mejorar la detección de las regiones enriquecidas. Para cada lectura, a lo sumo una alineación se recuperaría cuando está lo suficientemente cerca de cualquiera de los anteriormente llamados picos. Por ejemplo, utilice: Perl lonut.pl -g hg19 -w -p 250 99 &lt;alignFile&gt; &lt;OutputPath&gt;. LONUT llevará a cabo los siguientes pasos. <ol><li> alineaciones divididos en un asignan únicamente lee y se multiplican asignan lee. Pico de llamada asignada de forma única en la persona que llama se lee usando pico CORREA 11. La opción -w -p 250 y 99 en el comando de ejemplo son para el cinturón. </li><li> Combinar asignada de forma única lee con el recuperado lee obtenido de LONUT, y el combinado lee, en formato BED, se utilizará en el análisis adicional. Calcular el número de Combined lee para la futura normalización. </li></ol></li><li> Bin la lee. Extracto lee en la región de interés mediante secuencias de comandos Perl: Perl methyPipeline.pl &lt;sampleInfoFile&gt; &lt;refGeneFile&gt; -up &lt;longitud que se extiende aguas arriba&gt; dn &lt;longitud que se extiende aguas abajo&gt;. Para cada archivo que aparece en la cama sampleInfoFile, el script de perl realiza las tareas siguientes. Nota: Consulte el archivo de codificación complementaria para el script Perl. <ol><li> Bin lee en el tamaño de ubicación fija, por ejemplo, de 100 pb, de 24 cromosomas. </li><li> Para cada gen se especifica en refGeneFile, extraer los contenedores alrededor del sitio de inicio de transcripción (TSS), hasta la longitud que se extiende especificado por la opción -up y -dn. Por ejemplo, extraer los datos de la región que está arriba y aguas abajo de 4 kb de TSS. En bin tamaño de 100 pb, este extrajeron los datos tienen 81 contenedores, y el cubo central corresponde a la SAT. </li><li> Para el gen de la cadena antisentido, voltear la región extraída de izquierda a derecha de manera que los contenedores de aguas arriba son siempre placed a la izquierda con el script de perl. </li><li> Además dividir la región de ± 4 kb TSS en tres subregiones: A la izquierda, aguas arriba a aguas arriba de 4 kb 2 kb; Medio, aguas arriba y abajo de 2 kb; Derecha, aguas abajo de 2 kb a 4 kb. </li><li> Para cada muestra, se divide el recuento de lecturas en cada intervalo por el número total de lecturas combinadas asignada calculada en 3.3.3. La normalización eliminará la muestra específica leer las diferencias y permite comparar la lee a través de diferentes muestras. </li></ol></li><li> Ejecutar el script bash (como se indica en el archivo de script) para realizar la prueba estadística de la normalizado lecturas obtenida de la región a la izquierda, para detectar la metilación diferencial entre el grupo normal y ocurre en las regiones descritas como en la región del flanco. Nota: Consulte el archivo de codificación adicional para la escritura del golpe. Sobre la base de las regiones que están metilados diferencialmente, hay siete combinaciones: Toda la región: la metilación diferencial en toda la región de ± 4 kb TSS middlmetilación diferencial tanto en la región centro y la izquierda: eLeft metilación diferencial tanto en la región central y derecho: MiddleRight metilación diferencial tanto en la región a la izquierda y la derecha: el flanco A la izquierda: la metilación diferencial en la región izquierda solamente metilación diferencial en la región de la derecha solamente: Derecho metilación diferencial en la región media solamente: Medio </li><li> Utilice la misma escritura del golpe para visualizar la metilación diferencial en una parcela tornado. Representar gráficamente los hiper e hipo genes metilados en un panel separado, y ordenarlos en el orden de la combinación de regiones como se describe en el apartado 3.4, respectivamente. </li></ol>

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Este protocolo se desarrolló en los laboratorios del Dr. Tim Huang y el Dr. Victor Jin de la Universidad de Texas Health Science Center en San Antonio.

La técnica MBDCap-seq es un enfoque de enriquecimiento de afinidad 3, considerado como una alternativa rentable al investigar cohortes con un gran número de pacientes 15. El oleoducto que aquí se presenta describe un enfoque integral, desde la adquisición de la muestra para el análisis y la interpretación de los datos. Uno de los pasos más importantes es la creación de un procedimiento de amplificación por PCR para mejorar la eficiencia de la PCR de las regiones GC enriquecido en el genoma ya que es donde se p...

Regulación epigenética de genes a través de la metilación del ADN es uno de los mecanismos esenciales requeridos para determinar el destino de la célula por la diferenciación estable de diferentes tipos de tejidos en el cuerpo 1. La desregulación de este proceso ha sido conocido por causar varias enfermedades incluyendo el cáncer 2. Este proceso implica principalmente la adición de grupos metilo en el residuo de citosina en los dinucleótidos CpG de ADN 3. Hay algunas técnicas diferentes que actualmente se utilizan para investigar este mecanismo, cada uno con sus propias ventajas tal como se describe en muchos estudios 2-8. Aquí vamos a discutir una de estas técnicas llamadas Metil-Binding secuenciación de ADN de captura (MBDCap-ss), donde utilizamos una técnica de enriquecimiento de afinidad para identificar regiones del ADN metilado. Esta técnica se basa en la capacidad de unión metil-de la proteína MBD2 para enriquecer los fragmentos de ADN genómicos que contienen sitios CpG metilados. Utilizamos un kit de enriquecimiento de ADN metilado comercialpara el aislamiento de estas regiones metiladas. Nuestro laboratorio ha proyectado cientos de muestras de pacientes utilizando esta técnica y aquí proporcionar un protocolo optimizado integral, que se puede utilizar para investigar grandes cohortes de pacientes. Como es evidente con cualquier tecnología de secuenciación de nueva generación, MBDCap-seq también requiere un enfoque de la bioinformática específicos con el fin de cuantificar con precisión los niveles de metilación a través de las muestras. Ha habido muchos estudios recientes, en un esfuerzo para optimizar el proceso de normalización y el análisis de los datos de secuenciación de 9, 10. En este protocolo, se demuestra uno de estos métodos de aplicación de un enfoque de recuperación de lectura única - LONUT - seguido de la normalización lineal de cada muestra con el fin de permitir las comparaciones imparciales través de gran número de muestras de pacientes.

<table><tbody><tr><td>Methylminer DNA enrichment Kit</td><td>Invitrogen</td><td>ME10025</td><td></td></tr><tr><td>Dynabeads M-280 Streptavidin</td><td>Invitrogen</td><td>112-05D</td><td></td></tr><tr><td>Bioruptor Plus Sonication Device</td><td>diagenode</td><td>B01020001</td><td></td></tr><tr><td>3 M sodium acetate, pH 5.2</td><td>Sigma</td><td>S7899</td><td>100 mL</td></tr><tr><td>SPRIworks Fragment Library System I</td><td>Beckman Coulter</td><td>A50100</td><td>Fully automated library construction system</td></tr><tr><td>Adapter Primers</td><td>Bioo Scientific</td><td>514104</td><td>PCR primer mix</td></tr><tr><td>Qubit</td><td>Invitrogen</td><td>Q32854</td><td>Fluorometric Quantitation System</td></tr><tr><td>PCR master mix</td><td>KAPA scientific</td><td>KK2621</td><td>PCR master mix</td></tr><tr><td>AMPure XP</td><td>Beckman Coulter</td><td>A63881</td><td>PCR Purification beads</td></tr><tr><td>EB Buffer</td><td>Qiagen</td><td>19086</td><td></td></tr><tr><td>HiSeq 2000 Sequencing System</td><td>Illumina</td><td></td><td></td></tr></tbody></table>

methyl-binding dna capture sequencing for patient tissues

La metilación es una de las modificaciones epigenéticas esenciales en el ADN, que es responsable de la regulación precisa de los genes necesarios para el desarrollo estable y la diferenciación de diferentes tipos de tejidos. La desregulación de este proceso es a menudo el sello de diversas enfermedades como el cáncer. Aquí, se describe una de las técnicas de secuenciación recientes, Metil-Binding secuenciación de captura de ADN (MBDCap-seq), que se utiliza para cuantificar la metilación en diversos tejidos normales y de enfermedad para grandes cohortes de pacientes. Se describe un protocolo detallado de este enfoque enriquecimiento por afinidad junto con una tubería de bioinformática para lograr la cuantificación óptima. Esta técnica se ha utilizado para secuenciar cientos de pacientes a través de diversos tipos de cáncer como parte de la metiloma proyecto 1000 (Sistema de metiloma Cáncer).

Hemos utilizado MBDCap-seq para estudiar alteraciones de metilación del ADN en un gran número de pacientes de diversos tipos de cáncer incluyendo cáncer de mama 12, endometrial 13, de la próstata 14, y los cánceres de hígado, entre otros. Aquí se demuestra alguna información del cáncer de mama estudio publicado recientemente 12. En este caso, se utilizó todo el enfoque de secuenciación del genoma para identificar las islas CpG que están metilados diferencialmente en el tumor con respe...

Watch this Scientific Journal Video about Methyl-binding DNA capture Sequencing for Patient Tissues at JoVE.com

Methyl-binding DNA capture Sequencing for Patient Tissues

Aquí se presenta un protocolo para investigar la metilación del ADN del genoma de ancho en estudios de cribado de pacientes clínicos a gran escala utilizando la secuenciación Metil-unión de captura de ADN (SEC-MBDCap o MBD-ss) la tecnología y el análisis de tuberías bioinformática posteriores.

Secuenciación de captura de ADN de unión a metil-para tejidos del paciente

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

methyl-binding-dna-capture-sequencing-for-patient-tissues

Research

JoVE Journal

Genetics

8.6K vistas.  University of Texas Health Science Center at San Antonio.  Aquí se presenta un protocolo para investigar la metilación del ADN del genoma de ancho en estudios de cribado de pacientes clínicos a gran escala utilizando la secuenciación Metil-unión de captura de ADN (SEC-MBDCap o MBD-ss) la tecnología y el análisis de tuberías bioinformática posteriores. 

Video: Methyl-binding DNA capture Sequencing for Patient Tissues

Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during cancer development and progression. Despite this, the global epigenetic regulation of lncRNAs and repetitive sequences in cancer has not been well investigated, particularly in chronic lymphocytic leukemia (CLL). This study focuses on a unique approach: the immunoprecipitation-based capture of double-stranded, methylated DNA fragments using methyl-binding domain (MBD) proteins, followed by next-generation sequencing (MBD-seq). CLL patient samples belonging to two prognostic subgroups (5 IGVH mutated samples + 5 IGVH unmutated samples) were used in this study. Analysis revealed 5,800 hypermethylated and 12,570 hypomethylated CLL-specific differentially methylated genes (cllDMGs) compared to normal healthy controls. Importantly, these results identified several CLL-specific, differentially methylated lncRNAs, repetitive elements, and protein-coding genes with potential prognostic value. This work outlines a detailed protocol for an MBD-seq and bioinformatics pipeline developed for the comprehensive analysis of global methylation profiles in highly CpG-rich regions using CLL patient samples. Finally, a protein-coding gene and an lncRNA were validated using pyrosequencing, which is a highly quantitative method to analyze CpG methylation levels to further corroborate the findings from the MBD-seq protocol.

This work describes an optimized methyl-CpG-binding domain (MBD) sequencing protocol and a computational pipeline to identify differentially methylated CpG-rich regions in chronic lymphocytic leukemia (CLL) patients.

Análisis de metilación de ADN completo usando un método de captura de dominio de unión a metil-CpG en pacientes con leucemia linfocítica crónica

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during ...

Investigación

Genética

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

Metodología para la detección exacta de la metilación del ADN mitocondrial

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

Inmunoprecipitación de ADN metilado

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

Biología

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases 3,4.
DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming 5. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides.
CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample.
Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.

Análisis Optimizado de la metilación del ADN y la expresión génica de áreas pequeñas, anatómicamente definidas del cerebro

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to ...

Neurociencias

Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is a useful measure to provide an accurate analysis of the methylation status of gastrointestinal (GI) malignancies. Given the multiple potential translational uses of DNA methylation analysis, practicing clinicians and others new to DNA methylation studies need to be able to understand step by step how these genome-wide analyses are performed. The goal of this protocol is to provide a detailed description of how this method is used for the biomarker identification in GI malignancies. Importantly, we describe three critical steps that are needed to obtain accurate results during genome-wide analysis. Clearly and concisely written, these three methods are often lacking and not noticeable to those new to epigenetic studies. We used 48 samples of a GI malignancy (gastric cancer) to highlight practically how genome-wide DNA methylation analysis can be performed for GI malignancies.

Herein, we describe a procedure for genome-wide analysis of DNA methylation in gastrointestinal cancers. The procedure is of relevance to studies that investigate relationships between methylation patterns of genes and factors contributing to carcinogenesis in gastrointestinal cancers.

Análisis del genoma en general de la metilación del ADN en el cáncer gastrointestinal

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is ...

Investigación sobre el cáncer

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 ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

Captura selectiva de 5-hidroximetilcitosina a partir de ADN genómico

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

In vitro Selección de represores transcripcionales diseñados para el silenciamiento epigenético dirigido

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among ...

Bioingeniería

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 ...

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 ...