Name: selective capture of 5-hydroxymethylcytosine from genomic dna
Uploaded: 2012-10-05T13:00:00
Description: Watch this Scientific Journal Video about Selective Capture of 5-hydroxymethylcytosine from Genomic DNA at JoVE.com

Questo studio è stato sostenuto in parte dal National Institutes of Health (GM071440 a CH e NS051630/MH076090/MH078972 a PJ).

1. Frammentazione del DNA genomico Frammento di DNA genomico mediante ultrasuoni ad una vasta dimensione desiderata adatto per il genoma a livello di piattaforma di sequenziamento. (Di solito sonicazione a ~ 300 bp.) Verificare la distribuzione dimensionale del DNA genomico frammentato 1% gel (Figura 1). 2. Estrazione del DNA Determinare le quantità di DNA di partenza in base alla abbondanza di 5-HMC nel DNA genomico. Da...

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5-hydroxymethylcytosine (5-HMC) è un regalo recentemente identificato modificazione epigenetica in quantità notevoli in alcuni tipi di cellule di mammifero. Il metodo qui presentato è per la determinazione del genoma distribuzione a livello di 5-HMC. Usiamo batteriofago T4 β-glucosil trasferire una porzione multistrato glucosio contenente un gruppo azide sul gruppo ossidrile di 5-HMC. Il gruppo azide può essere chimicamente modificato con biotina per il rilevamento, l&#39;arricchimento di affinità, e sequenziament...

<table border="1"><tbody><tr><td> Nome </td><td> Azienda </td><td> Catalogare # </td><td> Commento </td></tr><tr><td></td><td></td><td></td><td> Reagenti </td></tr><tr><td> Cloruro di sodio 5M (NaCl) </td><td> Promega </td><td> V4221 </td><td></td></tr><tr><td> 0.5M pH8.0 Etilendiammintetraacetato (EDTA) </td><td> Promega </td><td> V4231 </td><td></td></tr><tr><td> 1M Trizma base (Tris) pH7.5 </td><td> Invitrogen </td><td> 15567-027) </td><td></td></tr><tr><td> HEPES 1M, pH 7,4 </td><td> Invitrogen </td><td> 15630 </td><td></td></tr><tr><td> Cloruro di magnesio (MgCl2) 1M </td><td> Ambion </td><td> AM9530G </td><td></td></tr><tr><td> Dimetilsolfossido (DMSO) </td><td> Sigma </td><td> D8418 </td><td></td></tr><tr><td> Tween 20 </td><td> Fisher BioReagents </td><td> BP337-100 </td><td></td></tr><tr><td> DBCO-SS-PEG3-biotina coniugato </td><td> Fare clic su Strumenti Chimica </td><td> A112P3 </td><td></td></tr><tr><td> 1,4-Dithiothreitol, ultrapura (DTT) superpuro </td><td> Invitrogen </td><td> 15508-013 </td><td></td></tr><tr><td> QIAquick Kit Rimozione Nucleotide </td><td> Qiagen </td><td> 28304 </td><td></td></tr><tr><td> Micro Bio-Spin 6 Colonna </td><td> Bio-Rad </td><td> 732-6222 </td><td></td></tr><tr><td> Dynabeads MyOne </td><td> Invitrogen </td><td> 650-01 </td><td></td></tr><tr><td> Streptavidina C1 </td><td></td><td></td><td></td></tr><tr><td> Qiagen MinElute PCR Purification Kit </td><td> Qiagen </td><td> 28004 </td><td></td></tr><tr><td> Ultrapura agarosio </td><td> Invitrogen </td><td> 16500500 </td><td></td></tr><tr><td> UDP-6-N 3-glucosio </td><td> Motif attivo </td><td> 55013 </td><td></td></tr><tr><td></td><td></td><td></td><td> Enzima </td></tr><tr><td> β-glucosil (β-GT) </td><td> New England Biolab </td><td> M0357 </td><td></td></tr><tr><td></td><td></td><td></td><td> Attrezzatura </td></tr><tr><td> Sonicazione dispositivo </td><td> Covaris </td><td></td><td></td></tr><tr><td> Desktop centrifuga </td><td></td><td></td><td></td></tr><tr><td> Bagnomaria </td><td> Fisher Scientific </td><td></td><td></td></tr><tr><td> Gel apparecchi in esecuzione </td><td> Bio-Rad </td><td></td><td></td></tr><tr><td> NanoDrop1000 </td><td> Thermo Scientific </td><td></td><td></td></tr><tr><td> Labquake Tubo Shaker </td><td> Barnstead </td><td></td><td></td></tr><tr><td> Labquake Tubo Shaker </td><td> Thermolyne </td><td></td><td></td></tr><tr><td> Separazione supporto magnetico </td><td> Promega </td><td> Z5342 </td><td></td></tr><tr><td> Qubit 2,0 fluorimetro </td><td> Invitrogen </td><td></td&gt; <td></td></tr><tr><td></td><td></td><td></td><td> Reagente di installazione 10 X β-GT Reaction Buffer (500 mM HEPES pH 7,9, 250 mM MgCl2) 2 X Legatura e lavaggio (B &amp; W) buffer (10 mM Tris pH 7.5, 1 mM EDTA, 2 M NaCl, 0,02% Tween 20) . </td></tr></tbody></table>

selective capture of 5-hydroxymethylcytosine from genomic dna

5-metilcitosina (5-mC) costituisce ~ 2-8% dei cytosines totali in DNA genomico umano e influisce un&#39;ampia gamma di funzioni biologiche, tra l&#39;espressione genica, il mantenimento dell&#39;integrità genomica, imprinting parentale, inattivazione del cromosoma X, regolazione sviluppo, invecchiamento e cancro 1. Recentemente, la presenza di un ossidato 5-mC, 5-hydroxymethylcytosine (5-HMC), è stato scoperto in cellule di mammifero, in particolare nel staminali embrionali (ES) cellule e cellule neuronali 2-4. 5-HMC è generato per ossidazione di 5-mC catalizzata da TET famiglia ferro (II) / α-chetoglutarato-dipendente diossigenasi 2, 3. 5-HMC si propone di essere coinvolto nel mantenimento di staminali embrionali (MES) delle cellule, emopoiesi normale e le neoplasie, e zigote sviluppo 2, 5-10. Per capire meglio la funzione di 5-HMC, un sistema di sequenziamento affidabile e semplice è essenziale. Sequenziamento bisolfito tradizionale non è possibile distinguere 5-HMC da 5-MC 11 </sup&gt;. Per svelare la biologia di 5-HMC, abbiamo sviluppato un approccio chimico altamente efficiente e selettivo di etichettare e catturare 5-HMC, approfittando di un enzima batteriofago che aggiunge una frazione di glucosio al 5-HMC specifico 12. Qui si descrive una semplice procedura in due fasi per l&#39;etichettatura chimica selettiva di 5-HMC. Nella prima fase di etichettatura, 5-HMC nel DNA genomico viene etichettato con un 6-catalizzata azide-glucosio da β-GT, un glucosiltransferasi dal batteriofago T4, in modo che trasferisce il 6-azide-glucosio al 5-HMC dal modificato cofattore, UDP-6-N3-Glc (6-N3UDPG). Nella seconda fase, biotinilazione, un linker biotina disolfuro è attaccato al gruppo azide dalla chimica click. Entrambe le fasi sono altamente specifici ed efficienti, portando a completare etichettatura indipendentemente l&#39;abbondanza di 5-HMC in regioni genomiche e dando fondo estremamente bassa. Biotinilazione seguito di 5-HMC, i 5-HMC contenenti frammenti di DNA vengono selettivamente catturatiutilizzando perline streptavidina in una densità modo indipendente. La risultante 5-HMC arricchiti frammenti di DNA potrebbe essere utilizzato per analisi a valle, sequenziamento di prossima generazione. La nostra etichettatura selettiva e il protocollo di acquisizione conferisce elevata sensibilità, applicabile a qualsiasi fonte di DNA genomico con variabili / 5-HMC diverse abbondanze. Anche se lo scopo principale di questo protocollo è la sua applicazione a valle (ad es., Sequenziamento di prossima generazione per tracciare il 5-HMC distribuzione nel genoma), è compatibile con singola molecola, in tempo reale SMRT (DNA) sequenziamento, che è in grado di erogare singola base sequenziamento risoluzione del 5-HMC.

Watch this Scientific Journal Video about Selective Capture of 5-hydroxymethylcytosine from Genomic DNA at JoVE.com

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

Descritto è un processo a due fasi usando etichettatura β-glucosil (β-GT) per trasferire una azide-glucosio al 5-HMC, seguita dalla chimica click per trasferire un linker biotina per l&#39;arricchimento facile e densità-indipendenti. Questo metodo efficace di etichettatura e specifica consente l&#39;arricchimento di 5-HMC con fondo estremamente basso e high-throughput mappatura epigenomic tramite sequenziamento di prossima generazione.

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 ...

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