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

Bu çalışma, Ulusal Sağlık Enstitüleri (CH ve NS051630/MH076090/MH078972 için PJ için GM071440) tarafından kısmen desteklenmiştir.

1. Genomik DNA Parçalanma İstediğiniz bir boyut aralığına sonication kullanarak Fragment genomik DNA genom dizileme platformu için uygundur. (Genellikle ~ 300 bp ile sonikasyon.)% 1 agaroz jel (Şekil 1) üzerinde dağınık genomik DNA&#39;nın boyutu dağılımı kontrol edin. 2. DNA Preparation Genomik DNA 5-HMC bolluğu dayalı başlayarak DNA miktarları belirleyin. 5-HMC düzeyleri farklı doku tiplerinin anl...

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Hydroxymethylcytosine 5-(5-HMC) belirli memeli hücre türleri içinde önemli miktarda yeni tanımlanmış bir epigenetik modifikasyon mevcuttur. Burada sunulan yöntem, 5-HMC arasında genom dağılımı belirlemek içindir. Biz 5-HMC bir hidroksil grubu üzerine bir azid grubu ihtiva eden bir mühendislik glikoz yarımı aktarmak için β-glukoziltransferaz T4 bakteriyofaj kullanır. Azid grubu kimyasal memeli genomları 5-HMC içeren DNA parçalarının tespiti, afinite zenginleştirme ve sıralama için biotin ile ...

<table border="1"><tbody><tr><td> Isim </td><td> Şirket </td><td> # Katalog </td><td> Yorumlamak </td></tr><tr><td></td><td></td><td></td><td> Reaktifler </td></tr><tr><td> 5M Sodyum klorür (NaCl) </td><td> Promega </td><td> V4221 </td><td></td></tr><tr><td> 0.5 M pH 8.0 Ethylenediaminetetraacetic asit (EDTA) </td><td> Promega </td><td> V4231 </td><td></td></tr><tr><td> 1M Trizma tabanı (Tris) pH7.5 </td><td> Invitrogen </td><td> 15567-027) </td><td></td></tr><tr><td> HEPES 1M pH7.4 </td><td> Invitrogen </td><td> 15630 </td><td></td></tr><tr><td> Magnezyum klorür (MgCl2) 1M </td><td> Ambion </td><td> AM9530g </td><td></td></tr><tr><td> Dimetil sülfoksit (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> Kimya Araçlar&#39;ı tıklayın </td><td> A112P3 </td><td></td></tr><tr><td> 1,4-ditiyotretol, son derece saf (DTT) Superpure </td><td> Invitrogen </td><td> 15508-013 </td><td></td></tr><tr><td> QIAquick Nükleotid Kazıma Seti </td><td> Qiagen </td><td> 28304 </td><td></td></tr><tr><td> Mikro Bio-Spin 6 Sütun </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 Arıtma Kiti </td><td> Qiagen </td><td> 28004 </td><td></td></tr><tr><td> Ultrapure Agaroz </td><td> Invitrogen </td><td> 16500500 </td><td></td></tr><tr><td> UDP-6-N 3-glikoz </td><td> Aktif Motif </td><td> 55013 </td><td></td></tr><tr><td></td><td></td><td></td><td> Enzim </td></tr><tr><td> β-glukoziltransferaz (β-GT) </td><td> New England Biolab </td><td> M0357 </td><td></td></tr><tr><td></td><td></td><td></td><td> Ekipman </td></tr><tr><td> Sonikasyon cihazı </td><td> Covaris </td><td></td><td></td></tr><tr><td> Masaüstü santrifüj </td><td></td><td></td><td></td></tr><tr><td> Su banyosu </td><td> Fisher Scientific </td><td></td><td></td></tr><tr><td> Jel çalışan cihazlar </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 Tüp Çalkalayıcı </td><td> Barnstead </td><td></td><td></td></tr><tr><td> Labquake Tüp Çalkalayıcı </td><td> Thermolyne </td><td></td><td></td></tr><tr><td> Manyetik Ayırma Standı </td><td> Promega </td><td> Z5342 </td><td></td></tr><tr><td> Qubit 2.0 Fluorometre </td><td> Invitrogen </td><td></td&gt; <td></td></tr><tr><td></td><td></td><td></td><td> Reaktif kurulum 10 X β-GT Reaksiyon Tamponu (500 mM HEPES pH 7.9, 250 mM MgCl2) 2 X bağlayarak ve (10 mM Tris, pH 7.5, 1 mM EDTA, 2 M NaCl,% 0.02 Tween 20) (B) tampon yıkama . </td></tr></tbody></table>

selective capture of 5-hydroxymethylcytosine from genomic dna

5-metilsitozin (5-mC) insan genomik DNA&#39;daki toplam sitozinler arasında ~% 2-8 oluşturan ve gen ekspresyonu, genom bütünlüğünün korunması, ebeveyn baskılama, X kromozomu inaktivasyonu, düzenlenmesi de dahil olmak üzere biyolojik fonksiyonları, geniş bir yelpazede etkiler gelişme, yaşlanma ve kanser 1. Son zamanlarda, bir okside 5-mC, 5-hydroxymethylcytosine (5-HMC), varlığında embriyonik sap (ES) hücreleri ve nöronal hücreler 2-4, özellikle de memeli hücrelerinde tespit edilmiştir. 5-HMC TET aile demir (II) / α-ketoglutarat-bağımlı dioxygenases 2, 3 ile 5-mC katalizli oksitlenmesi ile elde edilir. 5-HMC embriyonik kök (MES) hücre normal hemopoeziste ve maligniteler ve zigot gelişimi 2, 5-10 bakımı dahil olmak üzere önerilmiştir. Iyi 5-HMC fonksiyonunu anlamak için, güvenilir ve kolay sıralama sistemi esastır. Geleneksel bisülfit dizileme 5-mC 11 5-HMC ayırt edemez </sup&gt;. 5-HMC biyolojisini aydınlatma için, özellikle 5-HMC 12 glikoz parçası ekler bir bakteriyofaj enzim yararlanarak, etiketlemek ve 5-HMC yakalamak için oldukça etkili ve seçici kimyasal bir yaklaşım geliştirdik. Burada 5-HMC seçici kimyasal etiketleme için basit bir iki aşamalı bir prosedür açıklanmaktadır. Birinci adımda etiketleme, genomik DNA 5-HMC 5-HMC için 6-azit-glikoz aktaran bir şekilde, β-GT, T4 bakteriyofaj bir glukoziltransferaz ile bir 6-azit-glikoz katalize ile etiketlenir Modifiye kofaktör, UDP-6-N3-GLC (6-N3UDPG). İkinci adımda, biyotinilasyon, bir disülfid biyotin linker klik kimyası ile azid grubu eklenir. Iki adım genomik bölgelerde 5-HMC bolluğu bakılmaksızın etiketlenmesi ve son derece düşük arka plan vererek tamamlamak için lider, son derece spesifik ve etkili. 5-HMC takibi biyotinilasyon, 5-HMC içeren DNA parçaları daha sonra seçici yakalanırbir yoğunluk-bağımsız bir şekilde streptavidin boncuklar kullanarak. Sonuçta ortaya çıkan 5-HMC-zenginleştirilmiş DNA fragmanları, yeni nesil dizi analizi de dahil olmak üzere aşağı akım analizleri için kullanılabilir. Bizim seçici etiketleme ve yakalama protokol çeşitli / değişken 5-HMC bolluğu ile genomik DNA herhangi bir kaynak için geçerli yüksek hassasiyet, bahşeder. Bu protokolün amacı ve mansabındaki uygulaması (yani., Yeni nesil dizileme genomunda 5-HMC dağıtım haritaya) olsa da, tek-molekül, bir gerçek zamanlı SMRT (DNA) dizi ile uyumlu 5-HMC tek-baz çözünürlük sıralama sunma yeteneğine.

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

Açıklanan kolay ve yoğunluk bağımsız zenginleştirme için bir biyotin bağlayıcının aktarmak tıkırtı kimya ve ardından, 5-HMC için bir azit-glikoz aktarmak için β-glukoziltransferaz (β-GT) ile iki aşamalı bir etiketleme bir işlemdir. Bu etkili ve spesifik bir etiketleme yöntemi nesil dizileme ile son derece düşük arka plan ve yüksek verimlilik epigenomic haritalama ile 5-HMC zenginleşmesine olanak sağlar.

Genomik DNA 5-hydroxymethylcytosine seçici Yakalama

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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