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

O trabalho é apoiado pelo CPRIT Research Training Award RP140105, bem como parcialmente suportado pelo US National Institutes of Health (NIH) concede R01 GM114142 e por William &amp; Ella Owens Medical Research Foundation.

Todos os tecidos são obtidos após a aprovação da comissão Institutional Review Board e quando todos os participantes consentiram em ambas as análises moleculares e estudos de acompanhamento. Os protocolos são aprovados pela Comissão de Estudos Humanos da Universidade do Texas Health Science Center em San Antonio. 1. Captação de ligação ao ADN de metilo (MBDCap) <ol><li> Coleta de amostras e isolamento de ADN <ol><li> Recolha tumor granel ou amostras de tecidos normais a partir de amostras de tecido embebidos em parafina de pacientes. </li><li> Usar um DNA Mini Kit comercial para isolar o ADN genómico a partir das amostras de tecido embebidos em parafina de acordo com o protocolo do fabricante. </li><li> Use um kit de enriquecimento de DNA metilado com o processo aqui descrito de acordo com o protocolo do fabricante. </li></ol></li><li> Lavagem talão inicial Nota: Os grânulos são utilizados para acoplar com a proteína MBD-Biotina, que detecta a metilação de ADN no genoma. Grânulos são usualmente suspensoem uma solução de estoque. Use estas etapas para lavar este líquido. <ol><li> Ressuspender o estoque de contas de estreptavidina (Veja a Tabela de Materiais) cuidado pipetando para cima e para baixo para obter uma suspensão homogénea. Não misture as contas em vórtice. </li><li> Para um ug de ADN de entrada, ADD10 uL de esferas para um tubo com 90 ul de 1x Bind Buffer / lavagem. Gire durante 1 min. Não misturar em vórtice. </li><li> Colocar o tubo (s) em uma cremalheira magnético durante 1 minuto. </li><li> Remover o líquido com uma pipeta e eliminar o líquido. </li><li> Adicionar 250 ul de 1x tampão de Bind / lavagem para os grânulos e girar durante 1 min. </li><li> Repita os passos 1.2.3 - 1.2.5 duas vezes. </li></ol></li><li> O acoplamento da proteína MBD-Biotina para os grânulos <ol><li> Para 1,2 ug de ADN de entrada, adicionar 7 ul (3,5 ug) de proteína MBD-Biotina a cada tubo. </li><li> Adicionar 93 ul de 1x Bind Buffer / lavagem à proteína para fazer um volume final de 100 uL. </li><li> Misturar a proteína de grânulos-mistura em um misturador rotativo à temperatura ambiente durante 1 h. </li></ol></li><li> DNA fragmentado (entrada) <ol><li> Sonicar ADN pela adição de 1,2 ug de ADN genómico total em 96 uL de tampão TE e 24? L de 5x tampão de Bind / lavagem para fazer uma solução de 120 uL. Use 30 s ON poder e 30 s OFF energia durante sonicação, 20 - 25 vezes. </li><li> Corra 1 uL de solução de ADN sonicada usando um sistema Bioanalyzer com um kit de ADN comercial de alta sensibilidade para verificar o tamanho dos fragmentos de estar na gama de 150-350 pb de acordo com o protocolo do fabricante. </li></ol></li><li> Lave as MBD-esferas <ol><li> Colocar o tubo contendo MBD-esferas em uma cremalheira magnético durante 1 minuto. </li><li> Retirar e descartar o líquido com uma pipeta sem tocar as esferas. </li><li> Ressuspender as esferas com 250 ul de 1x tampão de Bind / lavagem. </li><li> Misturar os grânulos num misturador rotativo à temperatura ambiente durante 5 min. </li><li> Repita os passos 1.5.1 - 1.5.4 duas vezes. </li&gt; </ol></li><li> Reação de captura de DNA fragmentado (1 ug de DNA de entrada) <ol><li> Transferir a mistura de ADN / tampão ao tubo contendo os MBD-grânulos. </li><li> Misturar os grânulos com MBD-ADN em um misturador rotativo durante 1 h à TA. Como alternativa, misture O / N a 4 o C. </li></ol></li><li> A remoção de DNA não-capturado a partir das pérolas <ol><li> Depois de misturar o ADN e MBD-grânulos, colocar o tubo na cremalheira magnética durante 1 min a concentrar-se todas as esferas na parede interior do tubo. </li><li> Remover o líquido sobrenadante com uma pipeta e salve-o em um tubo de microcentrífuga livre de DNase limpo como DNA não metilado. Armazenar esta amostra em gelo. </li><li> Adicionar 200 ul de 1x tampão de Bind / lavagem para os grânulos para lavar as pérolas. </li><li> Misturar os grânulos num misturador rotativo durante 3 min. </li><li> Colocar o tubo na cremalheira magnético durante 1 minuto. </li><li> Retirar o líquido com uma pipeta e salve-o em um tubo de microcentrífuga. </li><li> para capture reações de ≤1 mg de DNA de entrada, repita os passos 1.7.3 - 1.7.6 para mais 2 frações de lavagem no total. </li></ol></li><li> Eluição em dose única <ol><li> Misture solução salina diluída com tampão de alto teor salino (1: 1) para obter tampão de eluição (NaCl 1,000 mM). </li><li> Ressuspender as pérolas em 200 ul de tampão de eluição (NaCl 1,000 mM). </li><li> Incubar os grânulos num misturador rotativo durante 3 min. </li><li> Colocar o tubo na cremalheira magnético durante 1 minuto. </li><li> Com o tubo no lugar na prateleira magnética, remover o líquido com uma pipeta sem tocar as contas com a ponta da pipeta, e guardá-lo em um tubo de microcentrífuga limpo livre de DNase de 1,5 mL. Armazenar esta amostra em gelo. </li><li> Repita os passos 1.8.1 - 1.8.4 uma vez, a segunda coleta no mesmo tubo (volume total será de 400 mL). Armazenar a amostra colectiva no gelo. </li></ol></li><li> Precipitação com etanol (limpeza do ADN) <ol><li> Para cada noncaptured, lavagem e eluição fração from os passos anteriores, adicionar 1 ul de glicogénio (20 mg / mL, incluídos no kit), Volume 10/1 amostra de acetato de sódio 3M, pH 5,2 (por exemplo, 40 ul por 400 ul de amostra) e 2 volumes de amostra de 100% etanol (por exemplo, 800 mL por 400 mL de amostra). O volume total é de 1.241 mL. </li><li> Misturar bem e incubar à temperatura de -80 ° C durante pelo menos 2 h. </li><li> Centrifuga-se o tubo durante 15 minutos a 11363 xg, a 4 o C. </li><li> Cuidadosamente descartar o sobrenadante sem perturbar o sedimento. </li><li> Adicionar 500 uL de etanol frio a 70%. </li><li> Centrifugue o tubo por 5 min a 11.363 xg a 4 o C. </li><li> Cuidadosamente descartar o sobrenadante sem perturbar o sedimento. </li><li> Repita os passos 1.9.6 - 1.9.7 uma vez e remover qualquer sobrenadante residual. </li><li> Ar seco o sedimento para ~ 5 min. Não secar completamente o sedimento. </li><li> Ressuspender o sedimento de DNA em 37,5 uL de água sem ADNase ou outravolume apropriado de tampão. </li><li> Coloque o DNA em gelo ou armazenar o DNA a -20 ° C ou inferior até à sua utilização. </li><li> Verifique se a quantidade total de DNA é superior a 20 ng, (por exemplo, usar o sistema de quantificação fluorimétrica, veja Materiais Tabela). </li></ol></li></ol> 2. Sequencing <ol><li> Use os fragmentos de DNA recuperados de procedimento MBDCap para o sequenciamento. Para cada amostra a ser sequenciada, uma quantidade total de 20 - 40 ng de ADN é necessária para a preparação da biblioteca. </li><li> Para a preparação da biblioteca de ADN-SEQ usar um sistema de construção da biblioteca totalmente automatizado (Ver Tabela Materiais) e seguir o protocolo padrão fornecido pelo fabricante. Seleccione fragmentos de DNA de tamanho 200-400 bp para a preparação da biblioteca. O sistema de preparação biblioteca de automação permite padronizar o procedimento de preparação da biblioteca e minimizar a variação entre as amostras devido à preparação manual. </li><li> privilegiada uso do adaptadorrs e diluir a concentração de 100x. Deste, use 10 mL para cada amostra. </li><li> Seguindo o passo 2.2, levar a cabo a reacção, e quantificar a biblioteca de ADN-SEQ usando um sistema de quantificação fluorométrico (Ver Tabela Materiais). <ol><li> Configurar o procedimento de amplificação PCR. A escolha de PCR Master Mix é melhorar a eficiência da PCR da região CG enriquecida do genoma. Num tubo de PCR, adicionar biblioteca 15? L DNA-SEQ, 25 pL de PCR mestre de mistura (Ver Tabela Materiais), 2 uL do iniciador de PCR mix (ver Materiais Tabela), e 8 mL de H 2 O. </li></ol></li><li> Seguir a recomendação PCR do fabricante da PCR Master Mix, alterando vezes ciclismo e temperaturas para diferentes matérias-primas: 98 o C durante 45 s, ciclos X de 98 o C durante 15 s, 65 ° C durante 30 s, 72 ° C durante 30 s, e em seguida 72 ° C durante 1 min. Segure a 4 o C. <ol><li> perform ciclos suficientes para acabar com 2-10 nM de DNA biblioteca (8 - 10 ciclos). </li></ol></li><li> Limpar reacção de PCR com 1: 1 de PCR grânulos de purificação de acordo com o protocolo do fabricante (Ver Tabela Materiais). </li><li> Eluir com 20 - 30 ul de tampão de eluição. </li><li> Código de barras de cada amostra e piscina 4 amostras juntos em uma pista de uma célula de fluxo. Use o protocolo de leitura padrão de 50 bp única para sequenciação (Ver Tabela de Materiais). </li></ol> 3. Análise Bioinformatics Nota: Além disso processar os arquivos fastq brutos obtidos a partir do sequenciamento para realizar o controle de qualidade e mapear as sequências de DNA curtas (lê) para o genoma. <ol><li> Use Bowtie alinhador curto ler, ferramenta de mapeamento do genoma em cada arquivo de amostra fastq para mapear a lê para o genoma de referência. Muitas ferramentas de mapeamento de leitura estão disponíveis para realizar esta etapa e pode ser usado com base na preferência individual. <ol><li> Permitir até 2 inadequações no whole lê enquanto mapeamento para o genoma de referência. Relatar até 20 melhores alinhamentos para cada leitura. Por exemplo, usar gravata borboleta -v 2 --best -k 20 --chunkmbs 200 &lt;ebwt&gt; &lt;fastqFile&gt; &lt;alignFile&gt;. </li></ol></li><li> Use LONUT 9 para recuperar a multiplicar lê mapeado para melhorar a detecção das regiões enriquecidas. Para cada leitura, no máximo, um alinhamento seria recuperado quando se está perto o suficiente para qualquer um dos picos acima chamada. Por exemplo, use: perl lonut.pl -g hg19 -w 250 -p 99 &lt;alignFile&gt; &lt;OutputPath&gt;. LONUT irá realizar os seguintes passos. <ol><li> alinhamentos dividido em mapeados exclusivamente lê e se multiplicam mapeados lê. Pico de chamadas na mapeados exclusivamente lê usando chamador pico CORREIA 11. A opção -w 250 e -p 99 no comando exemplo são para cinto. </li><li> Combine exclusivamente mapeado lê com o recuperado leituras obtidas a partir de LONUT, e o combinado lê, em formato CAMA, vai ser utilizado em análises posteriores. Calcule o número de Combined lê para a normalização futuro. </li></ol></li><li> Bin as leituras. Extrato lê na região de interesse usando script perl: perl methyPipeline.pl &lt;sampleInfoFile&gt; &lt;refGeneFile&gt; -up &lt;comprimento que se estende a montante&gt; -dn &lt;comprimento que se estende a jusante&gt;. Para cada arquivo de cama listados na sampleInfoFile, o script perl executa as tarefas abaixo. Nota: Ver o arquivo de codificação complementar para o script perl. <ol><li> Bin as leituras em tamanho de posição fixa, por exemplo, de 100 pb, por 24 cromossomos. </li><li> Para cada gene especificado em refGeneFile, extrair os recipientes em torno do local de início da transcrição (SST), até ao comprimento que se estende especificado por -up opção e -dn. Por exemplo, extrair os dados da região que é a montante ea jusante 4 KB de TSS. No bin tamanho de 100 pb, este extraíram os dados têm 81 caixas, ea bin centro corresponde ao TSS. </li><li> Para o gene da cadeia anti-sentido, inverter a região extraído da esquerda para a direita, para que caixas de upstream são sempre placed na esquerda com o script perl. </li><li> Além disso dividir TSS ± ​​região de 4 kb em três sub-regiões: esquerda, a montante 4 kb a montante de 2 kb; Oriente, a montante ea jusante 2 kb; Direita, a jusante 2 kb a 4 kb. </li><li> Para cada amostra, dividir a contagem de leituras em cada bin pelo número total de Combinado mapeados lê calculada em 3.3.3. A normalização vai eliminar a amostra específica ler diferenças e permite comparar as leituras entre amostras diferentes. </li></ol></li><li> Executar o script de festa (como indicado no ficheiro de script) para executar o teste estatístico na normalizada leituras obtidas a partir da região esquerda, para detectar a metilação diferencial entre o grupo normal e o caso nas regiões descritas como na região do flanco. Nota: Consulte o arquivo de codificação complementar para o script bash. Com base nas regiões que são diferencialmente metilados, existem sete combinações: Toda a região: metilação diferencial ao longo ± região 4 kb do TSS middlmetilação diferencial tanto em região média e esquerda: eLeft metilação diferencial tanto em meio e direita região: MiddleRight metilação diferencial em ambas as regiões esquerda e direita: flanco Esquerda: metilação diferencial na região deixou apenas metilação diferencial na região direito só: Direito metilação diferencial na região média apenas: Médio </li><li> Use o mesmo script bash para visualizar a metilação diferencial em uma trama tornado. Traçar os hiper e hipo genes metilados em um painel separado, e classificar na ordem da combinação região, conforme descrito em 3.4, respectivamente. </li></ol>

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Este protocolo é desenvolvido nos laboratórios do Dr. Tim Huang e Dr. Victor Jin da Universidade do Texas Health Science Center em San Antonio.

A técnica MBDCap-seq é uma abordagem de enriquecimento de afinidade 3, considerada como uma alternativa de custo eficaz quando se investiga coortes com um grande número de pacientes 15. O gasoduto apresentado aqui descreve uma abordagem abrangente, desde a colheita de amostra para análise de dados e interpretação. Um dos passos mais importantes é a criação de um procedimento de amplificação por PCR para melhorar a eficiência da PCR das regiões GC enriquecido no genoma como este é onde ocorre a metilação...

Regulação epigenética de genes por meio de metilação do ADN é um dos mecanismos essenciais necessários para determinar o destino da célula por diferenciação estável de diferentes tipos de tecidos do corpo 1. A desregulação deste processo tem sido conhecido por causar diversas doenças, incluindo o cancro 2. Este processo envolve, principalmente, a adição de grupos metilo no resíduo de citosina nos dinucleótidos CpG de ADN 3. Existem algumas técnicas diferentes, actualmente utilizados para investigar este mecanismo, tendo cada um as suas próprias vantagens, conforme descrito em muitos estudos 2-8. Aqui vamos discutir uma destas técnicas chamados metil-Binding Captura de sequenciamento de DNA (MBDCap-seq), onde usamos uma técnica de enriquecimento de afinidade para identificar regiões metiladas do DNA. Esta técnica baseia-se na capacidade de ligação de metilo da proteína MBD2 para enriquecer os fragmentos de DNA genómico contendo locais CpG metilados. Nós utilizamos um kit de enriquecimento de DNA metilado comercialpara o isolamento destas regiões metiladas. O nosso laboratório fez a triagem centenas de amostras de pacientes, utilizando esta técnica e aqui nós fornecemos um protocolo optimizado abrangente, que pode ser usado para investigar grandes grupos de pacientes. Como é evidente, com qualquer tecnologia de sequenciamento de próxima geração, MBDCap-seq também requer uma abordagem bioinformática específicos, a fim de quantificar com precisão os níveis de metilação através das amostras. Tem havido muitos estudos recentes em um esforço para otimizar o processo de normalização e análise dos dados de sequenciamento 9, 10. Neste protocolo, demonstramos um destes métodos de aplicação de uma abordagem única de recuperação de leitura - LONUT - seguido pela normalização linear de cada amostra, a fim de permitir comparações imparciais toda grande número de amostras de doentes.

<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

A metilação é uma das modificações epigenética essenciais para o DNA, o qual é responsável pela regulação precisa dos genes necessários para o desenvolvimento e diferenciação estável de diferentes tipos de tecidos. A desregulação deste processo é frequentemente a característica de diversas doenças como o cancro. Aqui, nós descrevemos uma das técnicas de sequenciação recentes, Metil-Binding sequenciação de ADN de captura (MBDCap-SEQ), usada para quantificar a metilação em vários tecidos normais e de doença para grandes grupos de pacientes. Descreve-se um protocolo detalhado desta abordagem de enriquecimento por afinidade ao longo de uma conduta bioinformática para alcançar quantificação ideal. Esta técnica tem sido utilizada para sequenciar centenas de pacientes em vários tipos de câncer, como parte do projeto methylome 1.000 (Sistema Methylome Câncer).

Usámos MBDCap-SEQ de estudar as alterações de metilação do DNA em um grande número de pacientes de diversos tipos de cancro incluindo cancro da mama 12, 13 do endométrio, próstata 14, e cancros do fígado, entre outros. Aqui demonstramos algumas informações a partir do estudo do cancro da mama publicou recentemente 12. Neste exemplo, utilizou-se toda a abordagem de sequenciação do genoma para identificar ilhas CpG que são diferencialmente metilados no tumor em relação à perpendicul...

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

Aqui apresentamos um protocolo para investigar a metilação do DNA do genoma ampla em grande escala estudos de triagem de pacientes clínicos utilizando o Metil-Binding tecnologia de sequenciamento de DNA Captura (MBDCap-seq ou MBD-seq) eo gasoduto análise bioinformática subsequentes.

Sequenciamento de captura de DNA de ligação de metilo em tecidos de pacientes

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 visualizações.  University of Texas Health Science Center at San Antonio.  Aqui apresentamos um protocolo para investigar a metilação do DNA do genoma ampla em grande escala estudos de triagem de pacientes clínicos utilizando o Metil-Binding tecnologia de sequenciamento de DNA Captura (MBDCap-seq ou MBD-seq) eo gasoduto análise bioinformática subsequentes. 

Vídeo: 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.

Análise abrangente de metilação de DNA usando um método baseado em captação de domínio de ligação de metil-CpG em pacientes com leucemia linfocítica crônica

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

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.

Metodologia para detecção precisa de metilação do DNA 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).

Imunoprecipitação DNA 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 ...

Biologia

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.

Análise otimizado de metilação do DNA e expressão gênica de pequenas áreas anatomicamente definidos do Cérebro

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 ...

Neurociência

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.

Análise genoma-em todo o DNA da metilação de DNA no câncer gastrointestinal

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

Pesquisa do câncer

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.

A metilação do DNA: Modificação e Análise de bissulfito

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 selectivo de 5-hydroxymethylcytosine a partir de DNA genómico

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

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 Seleção de Repressores Transcricionais Projetados para Silenciamento Epigenético Direcionado

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

Bioengenharia

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.

Aprimorada Redução Representação Bisulfite Sequencing para a Avaliação de metilação de DNA na resolução de pares de bases

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.

Alvejado DNA análise de metilação por Sequenciação de próxima geração

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 ...