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

Il lavoro è sostenuto da CPRIT Research Training Award RP140105, così come in parte sostenuto da Stati Uniti National Institutes of Health (NIH) sovvenzioni R01 GM114142 e da William &amp; Ella Owens Medical Research Foundation.

Tutti i tessuti sono ottenuti a seguito approvazione del comitato Institutional Review Board e quando tutti i partecipanti acconsentito ad entrambe le analisi molecolari e studi di follow-up. I protocolli sono approvati dal Comitato studi sugli esseri umani presso l&#39;Università del Texas Health Science Center a San Antonio. 1. Metil-legame al DNA di cattura (MBDCap) <ol><li> Raccolta dei campioni e l&#39;isolamento del DNA <ol><li> Raccogliere tumore alla rinfusa o normali campioni di tessuto da campioni di tessuto inclusi in paraffina paziente. </li><li> Utilizzare un mini DNA kit commerciale per isolare DNA genomico da campioni di tessuto inclusi in paraffina secondo il protocollo del produttore. </li><li> Utilizzare un kit di arricchimento DNA metilato con la procedura qui descritta secondo il protocollo del produttore. </li></ol></li><li> Lavaggio tallone iniziale Nota: Le perle sono impiegati per accoppiare con MBD-biotina proteine, che rileva la metilazione del DNA sul genoma. Perle di solito sono sospesiin una soluzione madre. Utilizzare questa procedura per lavare questo liquido. <ol><li> Risospendere il magazzino di perline streptavidina (vedi Materiali Tavolo) pipettando gentilmente su e giù per ottenere una sospensione omogenea. Non mescolare le perline nel vortex. </li><li> Per uno mg di DNA di ingresso, Add10 ml di perline per un tubo con 90 ml di 1x Bind / tampone di lavaggio. Ruotare per 1 min. Non mescolare nel vortex. </li><li> Posizionare il tubo (s) su un sostegno magnetico per 1 min. </li><li> Rimuovere il liquido con una pipetta e scartare il liquido. </li><li> Aggiungere 250 ml di 1x Bind / tampone di lavaggio ai talloni e ruotare per 1 min. </li><li> Ripetere i punti 1.2.3 - 1.2.5 due volte. </li></ol></li><li> Accoppiamento la proteina MBD-biotina alle perline <ol><li> Per 1.2 mg di DNA di input, aggiungere 7 ml (3,5 mg) di proteine ​​MBD-biotina ad ogni provetta. </li><li> Aggiungere 93 ml di 1x Bind / tampone di lavaggio alla proteina di fare un volume finale di 100 ml. </li><li> Mescolare il perline-proteinacomposto su un agitatore rotante a temperatura ambiente per 1 h. </li></ol></li><li> Frammentato DNA (ingresso) <ol><li> DNA Sonicare con l&#39;aggiunta di 1,2 mg di DNA genomico totale in 96 ml di tampone TE e 24 ml di tampone di 5x / lavaggio Bind per ottenere una soluzione di 120 ml. Utilizzare 30 s ON potenza e 30 s OFF alimentazione durante sonicazione, 20 - 25 volte. </li><li> Eseguire 1 ml di soluzione di DNA sonicato utilizzando un sistema bioanalyzer con un kit commerciale DNA alta sensibilità per controllare la dimensione dei frammenti per essere nell&#39;intervallo di: 150 - 350 bp secondo il protocollo del produttore. </li></ol></li><li> Lavare le MBD-perline <ol><li> Posizionare il tubo contenente MBD-perline su un sostegno magnetico per 1 minuto. </li><li> Rimuovere e gettare il liquido con una pipetta senza toccare le perline. </li><li> Risospendere le sfere con 250 ml di 1x Bind / tampone di lavaggio. </li><li> Mescolare le perline su un agitatore rotante a temperatura ambiente per 5 min. </li><li> Ripetere i punti 1.5.1 - 1.5.4 due volte. </li&gt; </ol></li><li> Reazione di cattura del DNA frammentato (1 mg DNA di ingresso) <ol><li> Trasferire la miscela di DNA / Buffer alla provetta contenente le MBD-perline. </li><li> Mescolare le MBD-perline con il DNA su un agitatore rotante per 1 ora a RT. In alternativa, mescolare O / N a 4 ° C. </li></ol></li><li> Rimozione di DNA non catturato dalle perline <ol><li> Dopo la miscelazione il DNA e MBD-perline, posizionare il tubo sul sostegno magnetico per 1 min di concentrare tutte le perline sulla parete interna del tubo. </li><li> Rimuovere il surnatante con una pipetta e salvarlo in un free-DNasi provetta pulita microcentrifuga come DNA nonmethylated. Conservare questo campione sul ghiaccio. </li><li> Aggiungere 200 ml di 1x Bind / tampone di lavaggio per le perline per lavare le perline. </li><li> Mescolare le perline su un agitatore rotante per 3 min. </li><li> Posizionare il tubo sul sostegno magnetico per 1 min. </li><li> Rimuovere il liquido con una pipetta e salvarlo in una provetta. </li><li> per capture reazioni di ≤1 mg di DNA di ingresso, ripetere i punti 1.7.3 - 1.7.6 per altre 2 frazioni di lavaggio in totale. </li></ol></li><li> Eluizione frazione singolo <ol><li> Mescolare buffer di basso contenuto di sale con tampone alto contenuto di sale (1: 1) per ottenere tampone di eluizione (1.000 mm NaCl). </li><li> Risospendere le sfere in 200 microlitri di tampone di eluizione (1.000 mm NaCl). </li><li> Incubare le perline su un miscelatore rotante per 3 min. </li><li> Posizionare il tubo sul sostegno magnetico per 1 min. </li><li> Con il tubo in posizione sulla griglia magnetica, rimuovere il liquido con una pipetta senza toccare le perle con la punta della pipetta, e salvarla in un 1,5 ml provetta senza DNasi pulito. Conservare questo campione sul ghiaccio. </li><li> Ripetere i punti 1.8.1 - 1.8.4 volta, raccogliendo il secondo campione nella stessa provetta (volume totale sarà di 400 microlitri). Conservare il campione in pool sul ghiaccio. </li></ol></li><li> Etanolo precipitazioni (cleanup DNA) <ol><li> Per ogni noncaptured, lavaggio, e eluizione frazione from i passaggi precedenti, aggiungere 1 ml di glicogeno (20 mg / mL, incluso nel kit), il volume 1/10 campione di sodio acetato 3M, pH 5.2 (ad esempio, 40 ml per 400 ml di campione) e 2 volumi di campione di 100% etanolo (ad esempio, 800 microlitri per 400 microlitri di campione). Il volume totale è di 1.241 ml. </li><li> Mescolare bene e incubare a -80 ° C per almeno 2 ore. </li><li> Centrifugare la provetta per 15 min a 11.363 xg a 4 ° C. </li><li> Eliminare attentamente il surnatante senza disturbare il pellet. </li><li> Aggiungere 500 ml di freddo il 70% di etanolo. </li><li> Centrifugare la provetta per 5 minuti a 11.363 xg a 4 ° C. </li><li> Eliminare attentamente il surnatante senza disturbare il pellet. </li><li> Ripetere i punti 1.9.6 - 1.9.7 volta e rimuovere ogni residuo di surnatante rimanente. </li><li> Far asciugare il pellet per ~ 5 min. Non asciugare completamente il pellet. </li><li> Risospendere il pellet di DNA in 37,5 ml di acqua priva di DNasi o altroadeguato volume di tampone. </li><li> Posizionare il DNA su ghiaccio o conservare il DNA a -20 ° C o al di sotto fino a nuovo uso. </li><li> Verificare che la quantità totale di DNA è superiore a 20 ng, (ad esempio, usare il sistema di quantificazione fluorimetrica, vedi Materiali Tavolo). </li></ol></li></ol> 2. Sequencing <ol><li> Utilizzare i frammenti di DNA recuperati dalla procedura MBDCap per il sequenziamento. Per ogni campione da sequenziato, un importo totale di 20 - 40 ng DNA è necessario per la preparazione biblioteca. </li><li> Per la preparazione biblioteca DNA-Seq usare un sistema di costruzione di biblioteche completamente automatizzato (vedi Materiali Tavolo) e seguire il protocollo standard fornito dal produttore. Selezionare i frammenti di DNA di dimensioni 200-400 bp per la preparazione biblioteca. Il sistema di preparazione biblioteca di automazione permette di standardizzare la procedura di preparazione biblioteca e riducendo al minimo la variazione tra i campioni a causa di preparazione manuale. </li><li> Utilizzare l&#39;adattatore Primers e portare a concentrazione 100x. Di questo, utilizzare 10 microlitri per ogni campione. </li><li> Seguendo passo 2.2, prendere la reazione, e quantificare la libreria di DNA-ss utilizzando un sistema di quantificazione fluorimetrico (vedi Materiali Tavolo). <ol><li> Impostare procedura di amplificazione PCR. La scelta di PCR master mix è di migliorare l&#39;efficienza della PCR di GC arricchito regione del genoma. In un tubo di PCR, aggiungere biblioteca 15 microlitri di DNA-ss, 25 microlitri PCR Master Mix (vedi Materiali Tavolo), 2 microlitri PCR Primer Mix (vedi Materiali Tavolo), e 8 ml H 2 O. </li></ol></li><li> Seguire raccomandazione PCR dal produttore del master mix PCR, alterando i tempi di ciclismo e le temperature per diversi materiali di partenza: 98 ° C per 45 s, X cicli di 98 ° C per 15 s, 65 ° C per 30 s, 72 ° C per 30 s, quindi 72 ° C per 1 min. Tenere a 4 ° C. <ol><li> Perform abbastanza cicli di finire con 2-10 nM di DNA biblioteca (8 - 10 cicli). </li></ol></li><li> Pulire reazione PCR con rapporto 1: 1 di PCR perline purificazione secondo il protocollo del produttore (vedi Materiali Tavolo). </li><li> Eluire con 20 - 30 microlitri di tampone di eluizione. </li><li> Codice a barre ogni campione e piscina 4 campioni insieme in una corsia di una cella di flusso. Utilizzare il 50 bp unico protocollo lettura standard per il sequenziamento (vedi Materiali Tavolo). </li></ol> 3. Analisi Bioinformatica Nota: ulteriore processo i file FASTQ grezzi ottenuti dal sequenziamento per eseguire il controllo qualità e la mappatura delle brevi sequenze di DNA (legge) al genoma. <ol><li> Usa Bowtie breve lettura di allineamento, strumento di mappatura del genoma su ciascun file di esempio FASTQ per mappare la legge per il genoma di riferimento. Molti strumenti di mappatura di lettura sono a disposizione per eseguire questo passaggio e può essere utilizzato in base alle preferenze individuali. <ol><li> Attendere fino a 2 disallineamenti nel da te legge mentre la mappatura del genoma di riferimento. Segnala fino a 20 migliori allineamenti per ogni lettura. Ad esempio, utilizzare papillon -v 2 --migliore -k 20 --chunkmbs 200 &lt;ebwt&gt; &lt;fastqFile&gt; &lt;alignFile&gt;. </li></ol></li><li> Utilizzare LONUT 9 di recuperare il moltiplicano mappato legge per migliorare l&#39;individuazione delle regioni arricchite. Per ogni lettura, al più un allineamento potrebbe essere recuperato quando è abbastanza vicino a qualsiasi delle sopra dette picchi. Ad esempio, utilizzare: perl lonut.pl -g hg19 -w 250 -p 99 &lt;alignFile&gt; &lt;OutputPath&gt;. LONUT eseguirà le seguenti operazioni. <ol><li> allineamenti diviso in mappati in modo univoco legge e si moltiplicano mappati legge. Picco di chiamate sulla mappata univocamente legge utilizzando picco chiamante nastro 11. L&#39;opzione -w 250 e -p 99 nel comando esempio sono per la cintura. </li><li> Combina in modo univoco mappato legge con il recuperato letture ottenuto da LONUT, e il combinato legge, in formato LETTO, verranno utilizzati in ulteriori analisi. Calcolare il numero di CombinEd legge per il futuro la normalizzazione. </li></ol></li><li> Bin la legge. Estratto di legge nella regione di interesse utilizzando script perl: perl methyPipeline.pl &lt;sampleInfoFile&gt; &lt;refGeneFile&gt; up &lt;estende a monte lunghezza&gt; -dn &lt;valle lunghezza si estende&gt;. Per ogni file letto elencato nel sampleInfoFile, lo script perl esegue i compiti di seguito. Nota: Vedere file di codifica supplementare per lo script Perl. <ol><li> Bin la legge in formato bin fisso, ad esempio, 100 bp, per 24 cromosomi. </li><li> Per ogni gene specificato nel refGeneFile, estrarre i cassonetti intorno sito di inizio della trascrizione (TSS), fino alla lunghezza estensione specificata da un&#39;opzione up e -dn. Ad esempio, estrarre i dati nella regione che è a monte ea valle 4 kb di TSS. A bin dimensioni 100 bp, questo estratto i dati hanno 81 bidoni, e il bidone centrale corrisponde al TSS. </li><li> Per il gene sul filamento antisenso, capovolgere la regione estratta da sinistra a destra in modo che bidoni monte sono sempre placed sulla sinistra con lo script Perl. </li><li> Ulteriori dividere il TSS ± ​​regione 4 kb in tre regioni sub: a sinistra, a monte 4 kb a monte 2 kb; Medio, a monte ea valle 2 kb; A destra, a valle 2 kb a 4 KB. </li><li> Per ogni campione, dividere il conteggio di legge in ogni bin per il numero totale di combinati mappato letture calcolato 3.3.3. La normalizzazione eliminerà la specifica del campione leggere differenze e permette di confrontare la legge attraverso diversi campioni. </li></ol></li><li> Eseguire lo script bash (come indicato nel file script) per eseguire test statistico sul normalizzato letture ottenuti nella regione sinistra, per individuare metilazione differenziale tra normale e cassa gruppo nelle regioni descritti come nella regione del fianco. Nota: Vedere il file di codifica supplementare per lo script bash. Sulla base delle regioni che sono differenzialmente metilati, vi sono sette combinazioni: Tutta la regione: metilazione differenziale in tutta la TSS ± ​​regione 4 kb middleLeft: differenziale metilazione sia in regione centrale e di sinistra MiddleRight: differenziale metilazione nella regione sia centro e destra Fianco: differenziale metilazione sia in regione a destra ea sinistra A sinistra: metilazione differenziale nella regione solo a sinistra A destra: differenziale metilazione nella regione solo a destra Medio: differenziale metilazione nella regione centrale solo </li><li> Utilizzare lo stesso script bash per visualizzare la metilazione differenziale in una trama tornado. Tracciare le iper e ipo geni metilati su un pannello separato, e ordinare nell&#39;ordine della combinazione regione come descritto in 3.4, rispettivamente. </li></ol>

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Questo protocollo è stato sviluppato nei laboratori del Dr. Tim Huang e il Dr. Victor Jin presso l&#39;Università del Texas Health Science Center a San Antonio.

La tecnica MBDCap-seq è un approccio arricchimento affinità 3, considerata un&#39;alternativa economica nell&#39;ambito delle indagini coorti con un grande numero di pazienti 15. Il gasdotto presentato qui descrive un approccio globale da approvvigionamento di esempio per l&#39;analisi e l&#39;interpretazione dei dati. Uno dei passi più importanti è la creazione di una procedura di amplificazione PCR per migliorare l&#39;efficienza della PCR delle regioni GC arricchito nel genoma come questo è dove si verifica la ...

Regolazione epigenetica di geni attraverso metilazione del DNA è uno dei meccanismi essenziali necessari per determinare il destino della cellula dalla differenziazione stabile di diversi tipi di tessuto nel corpo 1. Disregolazione di questo processo è stato conosciuto per causare varie malattie tra cui il cancro 2. Questo processo comporta principalmente l&#39;aggiunta di gruppi metilici sul residuo citosina in dinucleotidi CpG del DNA 3. Esistono alcune tecniche diverse attualmente utilizzate per indagare questo meccanismo, ciascuno con i propri vantaggi come indicato in molti studi 2-8. Qui discuteremo una di queste tecniche chiamati Metil-Binding DNA Capture sequenziamento (MBDCap-ss), dove utilizziamo una tecnica di affinità arricchimento per identificare le regioni metilato del DNA. Questa tecnica si basa sulla capacità metil-legame della proteina MBD2 per arricchire i frammenti di DNA genomico contenenti denaturato siti CpG. Utilizziamo un kit di arricchimento DNA metilato commercialeper l&#39;isolamento di tali regioni metilati. Il nostro laboratorio ha proiettato in centinaia di campioni di pazienti con questa tecnica e qui mettiamo a disposizione un protocollo ottimizzato completo, che può essere utilizzato per studiare grandi coorti di pazienti. Come evidente con qualsiasi tecnologia di sequenziamento di prossima generazione, MBDCap-ss richiede anche un approccio bioinformatico specifiche al fine di quantificare con precisione i livelli di metilazione attraverso i campioni. Ci sono stati molti studi recenti, nel tentativo di ottimizzare il processo di normalizzazione e l&#39;analisi dei dati di sequenziamento 9, 10. In questo protocollo, dimostriamo uno di questi metodi di attuazione di un approccio unico di recupero lettura - LONUT - seguita dalla normalizzazione lineare di ogni campione, al fine di consentire il confronto imparziali attraverso gran numero di campioni di pazienti.

<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 metilazione è una delle modificazioni epigenetiche essenziali al DNA, che è responsabile per la precisa regolazione dei geni necessari per lo sviluppo stabile e differenziazione dei diversi tipi di tessuto. Disregolazione di questo processo è spesso il segno distintivo di varie malattie come il cancro. Qui, si delineano uno dei recenti tecniche di sequenziamento, Metil-Binding DNA Capture sequenziamento (MBDCap-ss), utilizzato per quantificare la metilazione in vari tessuti normali e di malattia per i grandi coorti di pazienti. Descriviamo un protocollo dettagliato di questo approccio arricchimento affinità con un oleodotto bioinformatica per raggiungere quantificazione ottimale. Questa tecnica è stata utilizzata per sequenziare centinaia di pazienti in vari tipi di cancro come parte del progetto 1.000 methylome (Cancer Methylome System).

Abbiamo usato MBDCap-seq per studiare alterazioni di metilazione del DNA in un gran numero di pazienti da diversi tipi di cancro compresi seno 12, dell&#39;endometrio 13, prostata 14, e tumori del fegato, tra gli altri. Qui mostriamo alcune informazioni dallo studio del cancro al seno pubblicato di recente 12. In questo caso, abbiamo utilizzato l&#39;intero approccio di sequenziamento del genoma per identificare isole CpG che sono differenziale metilato nei tumori rispetto al normale nelle diverse...

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

Qui vi presentiamo un protocollo per indagare genoma metilazione del DNA in studi clinici di screening dei pazienti su larga scala utilizzando il metil-Binding sequenziamento del DNA di cattura (MBDCap-ss o MBD-ss), la tecnologia e la successiva analisi bioinformatica pipeline.

Metil-binding cattura sequenziamento del DNA per tessuti del paziente

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 Views.  University of Texas Health Science Center at San Antonio.  Qui vi presentiamo un protocollo per indagare genoma metilazione del DNA in studi clinici di screening dei pazienti su larga scala utilizzando il metil-Binding sequenziamento del DNA di cattura (MBDCap-ss o MBD-ss), la tecnologia e la successiva analisi bioinformatica pipeline. 

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.

Analisi completa della metilazione del DNA usando un metodo basato sulla cattura di dominio di metile-CpG in pazienti con leucemia linfocitica cronica

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

Ricerca

Genetica

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 per la rilevazione accurata di metilazione del DNA mitocondriale

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).

Immunoprecipitazione DNA metilato

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.

Ottimizzato Analisi della metilazione del DNA e l&#39;espressione genica da piccole aree anatomicamente definiti del cervello

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 ...

Neuroscienze

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.

Analisi a livello genomico della metilazione del DNA nel cancro gastrointestinale

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

Ricerca sul cancro

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.

Metilazione del DNA: Modifica bisolfito e analisi

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.

Cattura selettiva di 5 hydroxymethylcytosine dal DNA genomico

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 Selezione di repressori trascrizionali ingegnerizzati per il silenziamento epigenetico mirato

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

Bioingegneria

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 ridotto Rappresentanza bisolfito sequenziamento per la valutazione della metilazione del DNA in Risoluzione coppia di basi

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.

Targeted DNA metilazione Analisi per sequenziamento di prossima generazione

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 ...