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

Denne studien ble støttet i en del av National Institutes of Health (GM071440 til CH og NS051630/MH076090/MH078972 til PJ).

1. Genomisk DNA fragmentering Fragment genomisk DNA ved hjelp av sonikering i et ønsket størrelsesområde egnet for genom-wide sekvensering plattform. (Vi vanligvis sonicate til ~ 300 bp). Verifiser størrelsesfordelingen av den fragmenterte genomisk DNA på 1% agarosegel (figur 1). 2. DNA Forberedelse Bestem utgangsmaterialene DNA beløp basert på overflod av 5-HMC i genomisk DNA. Siden 5-HMC-nivåene varierer betydel...

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5-hydroxymethylcytosine (5-HMC) er et nylig identifisert epigenetic modifikasjon tilstede i betydelige mengder i visse pattedyr celletyper. Metoden som presenteres her er for å bestemme genom-wide distribusjon av 5-HMC. Vi bruker T4 bakteriofag β-glucosyltransferase å overføre en konstruert glukose moiety inneholdende en azide gruppe på hydroksylgruppen av 5-HMC. Azidet gruppen kan være kjemisk modifisert med biotin for deteksjon, affinitet berikelse, og sekvensering av 5-HMC-holdige DNA-fragmenter i pattedyr geno...

<table border="1"><tbody><tr><td> Navn </td><td> Selskapet </td><td> Katalogisere # </td><td> Kommentar </td></tr><tr><td></td><td></td><td></td><td> Reagenser </td></tr><tr><td> 5M Natriumklorid (NaCl) </td><td> Promega </td><td> V4221 </td><td></td></tr><tr><td> 0.5M pH8.0 etylendiamintetraeddiksyre (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> 1M HEPES, pH 7,4 </td><td> Invitrogen </td><td> 15630 </td><td></td></tr><tr><td> Magnesiumklorid (MgCl 2) 1M </td><td> Ambion </td><td> AM9530g </td><td></td></tr><tr><td> Dimetylsulfoksyd (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-Biotin konjugat </td><td> Klikk kjemi Verktøy </td><td> A112P3 </td><td></td></tr><tr><td> 1,4-Dithiothreitol, ultrarent (DTT) superrene </td><td> Invitrogen </td><td> 15508-013 </td><td></td></tr><tr><td> QIAquick nukleotid fjerning Kit </td><td> Qiagen </td><td> 28304 </td><td></td></tr><tr><td> Micro Bio-Spin 6 Column </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> Streptavidin C1 </td><td></td><td></td><td></td></tr><tr><td> Qiagen MinElute PCR Rensing Kit </td><td> Qiagen </td><td> 28004 </td><td></td></tr><tr><td> Ultrapure Agarose </td><td> Invitrogen </td><td> 16500500 </td><td></td></tr><tr><td> UDP-6-N 3-glukose </td><td> Aktiv Motif </td><td> 55013 </td><td></td></tr><tr><td></td><td></td><td></td><td> Enzym </td></tr><tr><td> β-glucosyltransferase (β-GT) </td><td> New England Biolab </td><td> M0357 </td><td></td></tr><tr><td></td><td></td><td></td><td> Utstyr </td></tr><tr><td> Sonikering enhet </td><td> Covaris </td><td></td><td></td></tr><tr><td> Desktop sentrifuge </td><td></td><td></td><td></td></tr><tr><td> Vannbad </td><td> Fisher Scientific </td><td></td><td></td></tr><tr><td> Gel kjører apparater </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 Tube Shaker </td><td> Barnstead </td><td></td><td></td></tr><tr><td> Labquake Tube Shaker </td><td> Thermolyne </td><td></td><td></td></tr><tr><td> Magnetisk separasjon Stand </td><td> Promega </td><td> Z5342 </td><td></td></tr><tr><td> Qubit 2,0 fluorometer </td><td> Invitrogen </td><td></td&gt; <td></td></tr><tr><td></td><td></td><td></td><td> Reagens oppsett 10 X β-GT reaksjonsbuffer (500 mM HEPES pH 7,9, 250 mM MgCl 2) 2 X innbinding og vasking (svart-hvitt) 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-methylcytosine (5-mC) utgjør ~ 2-8% av de totale cytosines i humant genomisk DNA og påvirker et bredt spekter av biologiske funksjoner, inkludert genekspresjon, vedlikehold av genom integritet, foreldrenes imprinting, X-kromosom inaktivering, regulering av utvikling, aldring og kreft en. Nylig, ble tilstedeværelsen av et oksydert 5-MC, 5-hydroxymethylcytosine (5-HMC), oppdaget i pattedyrceller, spesielt i embryonale stamceller (ES-celler) og neuronale celler 2-4. 5-HMC genereres ved oksidasjon av 5-mC katalysert av TET family jern (II) / α-ketoglutarat-avhengige 2 dioxygenases, 3. 5-HMC er foreslått å være involvert i vedlikehold av embryonale stamceller (MES) celle, normal hematopoiesis og maligniteter, og zygote utvikling 2, 5-10. For bedre å forstå funksjonen av 5-HMC, er en pålitelig og grei sekvensering system viktig. Tradisjonell bisulfite sekvensering kan ikke skille 5-HMC fra 5-mC 11 </sup&gt;. Å løse biologi 5-HMC, har vi utviklet en svært effektiv og selektiv kjemiske tilnærming til å merke og ta 5-HMC, dra nytte av en bakteriofag enzym som legger en glukose moiety til 5-HMC spesielt 12. Her beskriver vi en grei totrinns prosedyre for selektiv kjemisk merking av 5-HMC. I det første trinnet merking, er 5-HMC i genomisk DNA merket med en 6-azide-glukose katalysert av β-GT, en glucosyltransferase fra T4 bakteriofag, på en måte som overfører 6-azide-glukose til 5-HMC fra modifisert cofaktor, UDP-6-N3-Glc (6-N3UDPG). I det andre trinn, biotinylation, en disulfid biotin linkerarm festet til azid-gruppen ved klikk kjemi. Begge trinnene er meget spesifikke og effektive, fører til fullstendig merking uansett overflod av 5-HMC i genomisk regioner og gi ekstremt lav bakgrunn. Etter biotinylation av 5-HMC, de 5-HMC-holdige DNA-fragmenter blir deretter selektivt fangetbruker streptavidin perler i en tetthet-uavhengig måte. De resulterende 5-HMC-beriket DNA fragmenter kan bli brukt for nedstrøms analyser, inkludert neste generasjons sekvensering. Vår selektiv merking og fangst protokollen confers høy følsomhet, gjelder enhver kilde til genomisk DNA med variable / diverse 5-HMC abundances. Selv om hovedformålet med denne protokollen er dens nedstrøms søknaden (ie., Neste generasjons sekvensering å kartlegge 5-HMC distribusjon i genomet), er den kompatibel med single-molekyl, real-time SMRT (DNA) sekvensering, som er stand til å levere ett-grunnoppløsningen sekvensering av 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

Beskrevet er en to-stegs etiketteringsprosessen hjelp β-glucosyltransferase (β-GT) for å overføre en azid-glukose til 5-HMC, etterfulgt av klikk kjemi å overføre en biotin linker for enkel og tetthet uavhengig berikelse. Denne effektive og spesifikke merking metoden muliggjør berikelse av 5-HMC med ekstremt lav bakgrunn og høy gjennomstrømning epigenomic kartlegging via neste generasjons sekvensering.

Selektiv Capture av 5-hydroxymethylcytosine fra genomisk 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 ...

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