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

iş CPRIT Araştırma Eğitim Ödülü RP140105 tarafından desteklenen yanı sıra kısmen ABD Ulusal Sağlık Enstitüleri (NIH) R01 GM114142 verir tarafından ve William &amp; Ella Owens Tıbbi Araştırma Vakfı tarafından desteklenmektedir.

Bütün dokular Kurumsal Değerlendirme Kurulu komitesinin onayını takip eden ve elde edilen tüm katılımcılar moleküler analizler ve takip çalışmaları hem rıza zaman. protokoller San Antonio Texas Sağlık Bilimleri Merkezi Üniversitesi&#39;nde İnsan Araştırmaları Komitesi tarafından onaylanmıştır. 1. Metil-DNA bağlama Yakalama (MBDCap) <ol><li> Örnek toplama ve DNA izolasyonu <ol><li> Hasta parafin doku örneklerinden toplu tümör veya normal doku örnekleri toplamak. </li><li> üreticinin protokolüne uygun olarak parafine gömülü doku örneklerinden genomik DNA izole etmek için ticari bir DNA mini kiti kullanarak. </li><li> üreticinin protokolüne göre burada açıklanan prosedüre sahip metillenmiş DNA zenginleştirme kiti kullanın. </li></ol></li><li> İlk boncuk yıkama Not: boncuk genom DNA metilasyonu tespit MBD-Biyotin protein ile çift için kullanılır. Boncuklar genelde askıya alınırstok çözelti içinde. Bu sıvıları yıkayın için aşağıdaki adımları kullanın. <ol><li> Homojen bir süspansiyon elde etmek için yavaşça yukarı pipetleme streptavidin boncuk (Malzeme Tablo bakınız) stoğunu tekrar süspansiyon ve. vorteks boncuk karışmaz. </li><li> Giriş DNA&#39;nın bir ug için, 1 x Cilt / Yıkama Tamponu 90 uL olan bir tüpe tanelerin add10 uL. 1 dakika boyunca çevirin. vorteks karıştırmayınız. </li><li> 1 dakika boyunca manyetik rafa (ler) yerleştirin. </li><li> Bir pipet ile sıvı çıkarın ve sıvıyı atın. </li><li> boncuk 1x Bind / Yıkama Tamponu 250 uL ekleyin ve 1 dakika boyunca döndürün. </li><li> iki kere 1.2.5 - Tekrar 1.2.3 adımları. </li></ol></li><li> Boncuklar MBD-Biotin proteini Kavrama <ol><li> giriş DNA 1.2 ug için, her bir tüpe MBD-Biyotin proteininin 7 uL (3.5 ug) ilave edilir. </li><li> 100 ul&#39;lik nihai bir hacimde protein 1x bağlama / Yıkama Tamponu 93 uL ekleyin. </li><li> Boncuklar-protein karışımı1 saat boyunca oda sıcaklığında bir döner karıştırıcıda karışımı ile gerçekleştirilmektedir. </li></ol></li><li> Parçalanmış DNA (giriş) <ol><li> TE tamponu 96 uL ve 24 uL 5x Bind / yıkama tamponu toplam genomik DNA 1.2 ug ekleyerek sonikasyon DNA 120 uL çözelti yapmak için. 25 kez - 30 s güç AÇIK ve sonication sırasında 30 saniyelik güç KAPALI, 20 kullanın. </li><li> üreticinin protokolüne uygun olarak 350 bp - 150 aralığında olması fragmanlarının boyutunu kontrol etmek için ticari bir yüksek hassasiyetli bir DNA kiti ile, bir Bioanalyzer sistemi kullanılarak sonikasyona tabi tutulmuş, DNA solüsyonuna 1 ul çalıştırın. </li></ol></li><li> MBD-boncuk yıkayın <ol><li> 1 dakika için bir manyetik raf MBD-boncuk içeren tüp yerleştirin. </li><li> Çıkarın ve boncuk dokunmadan bir pipet ile sıvı atın. </li><li> 1x Bind / Yıkama Tamponu 250 mcL boncuk süspanse. </li><li> 5 dakika boyunca oda sıcaklığında bir döner karıştırıcıda boncuk karıştırın. </li><li> iki kere 1.5.4 - Tekrar 1.5.1 adımları. </li&gt; </ol></li><li> Parçalanmış DNA yakalama reaksiyonu (1 ug giriş DNA) <ol><li> MBD-boncuk içeren tüp DNA / Tampon karışımı aktarın. </li><li> Oda sıcaklığında 1 saat için bir döner karıştırıcıda DNA MBD-boncuk karıştırın. Seçenek olarak ise, 4 O ° C&#39;de O / N karıştırın </li></ol></li><li> Boncuk olmayan yakalanan DNA Çıkarma <ol><li> 1 dakika borunun iç duvarı üzerinde boncuklar her konsantre için DNA ve MBD-boncuk karıştırdıktan sonra, manyetik rafa yerleştirilir. </li><li> Bir pipet yardımıyla süpernatant sıvı çıkarın ve nonmethylated DNA gibi temiz bir DNaz ücretsiz mikrosantrifüj tüp kaydedin. Buz üzerinde bu örnek saklayın. </li><li> boncuk yıkamak için boncuk 1x Bind / Yıkama Tamponu 200 uL ekleyin. </li><li> 3 dakika için bir döner karıştırıcıda boncuk karıştırın. </li><li> 1 dakika boyunca manyetik rafa yerleştirilir. </li><li> Bir pipet ile sıvı çıkarın ve bir mikrosantrifüj tüp kaydedin. </li><li> kapağı içinToplamda 2 daha yıkama fraksiyonları için 1.7.6 - Giriş DNA ≤1 mikrogram tepkilerini Ture, tekrar 1.7.3 yineleyin. </li></ol></li><li> Tek fraksiyon yıkama <ol><li> yüksek tuzlu tampon düşük tuz tamponu karıştırın (1: 1) elüsyon tamponu (1.000 mM NaCI) alır. </li><li> elüsyon tamponu (1000 mM NaCI) 200 uL boncuk tekrar. </li><li> 3 dakika için bir döner karıştırıcıda boncuk inkübe edin. </li><li> 1 dakika boyunca manyetik rafa yerleştirilir. </li><li> Manyetik rafa yerinde tüp ile, pipet ile boncuk dokunmadan bir pipet ile sıvı kaldırmak ve temiz bir DNaz-özgür 1.5 mL mikrosantrifüj tüpe kaydedin. Buz üzerinde bu örnek saklayın. </li><li> Tekrarlayın 1.8.1 adımları - aynı tüpte ikinci örnek toplama, bir zamanlar 1.8.4 (toplam hacim 400 uL olacaktır). Buz üzerinde toplanmış numune saklayın. </li></ol></li><li> Etanol yağış (DNA temizleme) <ol><li> Her noncaptured, yıkama ve elüsyon fraksiyonu fr içinÖnceki adım OM, 1 pL glikojen ilave (20 ug / kit içerisinde bulunan uL), 3M sodyum asetatın 1/10 örnek hacmi, pH 5.2 (örneğin, 400 başına 40 uL numune uL) ve% 100 2 numune hacimleri etanol (örneğin, 400 800 ul örnek uL). toplam hacmi 1.241 uL olduğunu. </li><li> İyice karıştırın ve en azından 2 saat boyunca o C -80 ° C&#39;de inkübe edin. </li><li> 4 O ° C&#39;de 11,363 x g&#39;de 15 dakika boyunca santrifüj tüpü </li><li> Dikkatle pelet bozmadan süpernatant atın. </li><li> Soğuk% 70 etanol 500 ul ekle. </li><li> 4 O ° C&#39;de 11,363 x g&#39;de 5 dakika boyunca tüp santrifüj </li><li> Dikkatle pelet bozmadan süpernatant atın. </li><li> 1.9.7 kez ve kalan kalıntı süpernatant kaldırmak - Tekrar 1.9.6 adımları. </li><li> ~ 5 dakika boyunca pelet hava-kurutun. tamamen pelet kurutmayın. </li><li> DNaz-serbest su ya da diğer 37.5 uL DNA pelettampon uygun hacmi. </li><li> Buz üzerinde DNA yerleştirin veya -20 o C&#39;de ya da altında daha sonra kullanmak kadar DNA saklayın. </li><li> DNA toplam tutarı 20 ng üzerinde olup olmadığını kontrol edin (örneğin, florometrik kantitatif sistemi kullanmak, Malzeme Tablo). </li></ol></li></ol> 2. Sıralama <ol><li> dizileme için MBDCap prosedür elde DNA fragmanları kullanın. Her bir numune, sekanslanacak için, 20 toplam miktarı - 40 ng DNA kütüphanesi hazırlamak için gereklidir. </li><li> DNA seq kütüphane hazırlığı tam otomatik kütüphane inşaat sistemi kullanmak için (Malzeme Tablo bakınız) ve üretici tarafından sağlanan standart protokol izleyin. Kütüphane hazırlanması için 400 bp - boyutu 200 Select DNA fragmanları. otomasyon kütüphane hazırlama sistemi kütüphane hazırlık prosedürü standartlaştırılması ve bağlı manuel hazırlanması örneklerin arasında varyasyonu minimize sağlar. </li><li> Kullanım adaptör asalrs ve 100x konsantrasyonu sulandırmak. Bu nedenle, her örnek için 10 uL kullanımı. </li><li> Adım 2.2 takiben, reaksiyonu almak ve bir florometrik kantitatif sistemi (Malzeme Tablosu) kullanılarak DNA-seq kütüphane ölçmek. <ol><li> PCR işlemi ayarlayın. PCR Master Mix seçimi genomunun GC zenginleştirilmiş bölgenin PCR etkinliğini arttırmaktır. Bir PCR tüpünde, 15 uL DNA seq kütüphane eklemek, 25 ul PCR master mix (Malzeme Tablosu), 2 uL PCR primer karışımı (Malzeme Tablo bakınız), ve 8 uL H 2 O </li></ol></li><li> Başlangıç maddeleri farklılaşan bisiklet süreleri ve sıcaklıkları değiştirerek PCR Master Mix üreticiden PCR tavsiyesini izleyin: 98 o C 45 sn, 15 sn için 98 ° C&#39;nin, 30 sn için 65 ° C, 30 72 o C&#39;de X döngüleri s, 1 dakika süre ile daha sonra 72 o C. 4 o C&#39;de tutun <ol><li> perf(- 10 döngü 8) kütüphane DNA 10 nM - 2 ile bitirmek için yeterli döngüleri kapatýnýz. </li></ol></li><li> 1 ile PCR reaksiyonunu temizleyin: Üreticinin protokolüne göre PCR saflaştırma boncuk 1 oranında (Malzeme Tablosu bakınız). </li><li> 30 uL yıkama tamponu - 20 yıkayın. </li><li> Bir akış hücresinin bir şeride 4 numune birlikte her bir örnek ve havuz barkod. Sıralama için standart 50 bp tek okuma protokolü (Malzeme Tablosu bakınız) kullanın. </li></ol> 3. Biyoinformatik Analizi Not: sıralama elde edilen ayrıntılı süreç ham fastq dosyaları kalite kontrolünü yapmak ve genomuna kısa DNA dizileri (okur) haritalama. <ol><li> Kullanım Papyon kısa okumak hizalama, her örnek fastq dosya üzerinde genom haritalama aracı referans genomuna okur eşleştirmek için. Birçok okuma haritalama araçları bu adımı gerçekleştirmek için kullanılabilir ve bireysel tercihinize göre kullanılabilir. <ol><li> whol 2 uyumsuzlukları kadar izinReferans genomuna eşleme sırasında e okur. Okuma için en iyi 20 saflaşma kadar bildirin. Örneğin, fiyonkunu v 2 --best k 20 --chunkmbs 200 &lt;ebwt&gt; &lt;fastqFile&gt; &lt;alignFile&gt; kullanımı. </li></ol></li><li> Çarpma eşlenen zenginleştirilmiş bölgelerin tespiti geliştirmek için okuma kurtarmak için LONUT 9 kullanın. Her okuma, en fazla bir uyum azından yukarıda adı verilen tepelerin herhangi yeterince yakınken kurtarıldı olacaktır. Örneğin, kullanın: perl lonut.pl -g hg19 -w 250 -p 99 &lt;alignFile&gt; &lt;OutputPath&gt;. LONUT aşağıdaki adımları gerçekleştirir. <ol><li> Böl hizalamalar benzersiz okur eşlenen ve çarpma okur haritası çizilmiştir. Benzersiz eşlenen Çağrı pik tepe arayan KEMER 11 kullanılarak okur. örnek komutu 250 ve -p 99 -w seçeneği KEMER içindir. </li><li> benzersiz eşlenen Kombine LONUT elde okur geri ile okur ve birleştirilen YATAK biçiminde, daha fazla analiz kullanılacak okur. combin sayısını hesaplayıned gelecek normalleşmesi için okur. </li></ol></li><li> Bin okur. Özü Perl script kullanarak ilgi bölgede okur: perl methyPipeline.pl &lt;sampleInfoFile&gt; &lt;refGeneFile&gt;-up &lt;yukarı uzanan uzunluk&gt; dn &lt;aşağı uzanan uzunluk&gt;. sampleInfoFile listelenen her yatak dosyası için perl komut aşağıda görevleri gerçekleştirir. Not: Perl komut dosyası için ek kodlama dosyasına bakın. <ol><li> Bin 24 kromozom için, sabit bin boyutu, örneğin, 100 bp okur. </li><li> refGeneFile belirtilen her bir gen için, opsiyon-up ve dn ile belirtilen uzanan uzunluğa kadar, transkripsiyon başlama site çevresinde bidonları (TSS) ayıklayın. Örneğin, yukarı ve aşağı TSS 4 kb bölgedeki veri ayıklamak. Bin boyutu 100 bp, bu veriler 81 bidonları var çıkarılan ve merkez bin TSS karşılık gelir. </li><li> Soldan sağa doğru yukan depo her zaman için antisens zinciri üzerindeki geni için PLA ekstre bölge çevirmekPerl script ile soldaki ced. </li><li> 2 kb akıntıya karşı Sol, yukarı 4 kb;: Diğer üç alt bölgeye TSS ± ​​4 kb bölgesini bölmek Orta, yukarı ve aşağı 2 kb; Sağ, 4 kb 2 kb aşağı. </li><li> 3.3.3 hesaplanan okur eşlenen kombine toplam sayısına göre her bin okur her bir numune için, sayım bölün. Örnek özgü ortadan kaldıracak normalleşme farklılıkları okumak ve farklı örnekleri arasında okur karşılaştırmanızı sağlar. </li></ol></li><li> sol bölgeden elde okur normalize istatistiki testi gerçekleştirmek için (komut dosyasında belirtildiği gibi) yan bölge olarak tanımlanan bölgelerde normal ve olgu grubu arasında farklı metilasyon tespit etmek, bash komut dosyasını çalıştırın. Not: Bash komut dosyası için ek kodlama dosyasına bakın. diferansiyel metillenmiş bölgelerde dayanarak, yedi kombinasyonları vardır: Tüm bölge: AKM ± 4 kb bölge genelinde diferansiyel metilasyon middleLeft: Her iki orta ve sol bölgede ayırıcı metilasyon MiddleRight: Her iki orta ve sağ bölgede diferansiyel metilasyon Kanat: Sağ ve sol bölgede diferansiyel metilasyon Sol: Sol bölgedeki diferansiyel metilasyon sadece Sağ: Sağ bölgede diferansiyel metilasyon sadece Orta: orta bölgesinde diferansiyel metilasyon sadece </li><li> bir kasırga arsa diferansiyel metilasyonu görselleştirmek için aynı bash komut dosyasını kullanın. Ayrı bir panelde hiper ve hipo metile genleri arsa, sırasıyla, 3.4 anlatıldığı gibi bölge kombinasyonunun sırasına göre sıralayın. </li></ol>

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Bu protokol San Antonio Texas Sağlık Bilimleri Merkezi Üniversitesi&#39;nde Dr. Tim Huang ve Dr. Victor Jin laboratuarlarında geliştirilmiştir.

MBDCap seq tekniği hastalar 15 çok sayıda kohortları araştırılırken maliyetli bir alternatif olarak kabul bir afinite zenginleştirme yaklaşımı 3 vardır. Burada sunulan boru hattı veri analizi ve yorumlanması örnek temininden kapsamlı bir yaklaşım anlatılmaktadır. En önemli adımlardan biri DNA metilasyonu meydana geldiği bu gibi genomunda GC zenginleştirilmiş bölgelerinin PCR verimliliğini artırmak için bir PCR prosedürü kuruyor. Ayrıca, dizi analizi sonrasında, her bir örnek benzersiz...

DNA metilasyonu yoluyla gen epigenetik düzenlemesi gövde 1 &#39;de, farklı doku tiplerinin stabil farklılaşması, hücre akıbetini belirlemek için gerekli olan temel mekanizmalardan birisidir. Bu işlemin bozulması kanseri 2 de dahil olmak üzere, çeşitli hastalıklara sebep olduğu bilinmektedir. Bu işlem, esas olarak DNA 3 CpG dinükleotidleri sitozin tortu üzerindeki metil gruplarının eklenmesini içerir. Şu anda birçok çalışmada 2-8 belirtildiği gibi her biri kendi avantajları olan, bu mekanizmayı araştırmak için kullanılan birkaç farklı teknikler vardır. İşte biz DNA&#39;nın metillenmiş bölgeleri tanımlamak için bir afinite zenginleştirme tekniği kullanmak (MBDCap seq) Metil-Bağlama DNA Yakalama dizi olarak adlandırılan bu tekniklerin birini tartışacağız. Bu teknik, metile CpG ihtiva eden genomik DNA parçaları için ettirecek MBD2 proteininin metil-bağlanma yeteneği üzerine inşa edilmiştir. Bu, ticari bir metillenmiş DNA zenginleştirme kiti kullanmaktadırBu metile bölgelerin izole edilmesi için. Laboratuvarımız bu tekniği kullanarak hasta numunelerinin yüzlerce ekranlı ve burada büyük hasta kohortlarında araştırmak için kullanılabilecek kapsamlı bir optimize protokol sağlamak. Herhangi bir yeni nesil dizileme teknolojisi ile belirgin olarak, MBDCap seq de doğru örneklerin arasında metilasyon seviyelerini ölçmek amacıyla belirli bir biyoinformatik yaklaşım gerektirir. Sıralama verilerine 9, 10 normalleşmesi ve analiz sürecini optimize etmek amacıyla birçok yeni çalışmalar olmuştur. LONUT - - hasta örneklerinin çok sayıda genelinde tarafsız karşılaştırmalar etkinleştirmek için her numunenin doğrusal normalleşme ve ardından bu protokolde, benzersiz bir okuma kurtarma yaklaşımı uygulayan aşağıdaki yöntemlerden birini göstermektedir.

<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

Metilasyon, farklı doku tiplerinin istikrarlı gelişimi ve farklılaşması için gereken genlerin kesin düzenlenmesinden sorumlu olan DNA, için gerekli epigenetik değişiklikler biridir. Bu sürecin düzensizliği sık sık kanser gibi çeşitli hastalıkların özelliğidir. Burada, son sıralama tekniklerden birini anahat, Metil-Bağlama geniş hasta grupları için çeşitli normal ve hastalık dokularda metilasyonu ölçmek için kullanılan DNA Yakalama sıralama (MBDCap seq). Biz en uygun kantifikasyon ulaşmak için biyoinformatik boru hattı ile birlikte bu afinite zenginleştirme yaklaşımı ayrıntılı bir protokol açıklar. Bu teknik, 1000 methylome projesi (Kanser Methylome sistemi) bir parçası olarak çeşitli kanser türlerinde hastanın yüz sıralamak için kullanılmaktadır.

Bu, diğerleri arasında meme 12, endometriyal 13, prostat 14 ve karaciğer kanseri dahil olmak üzere çeşitli kanser türlerinden hasta çok sayıda DNA metilasyonu değişiklikleri çalışma MBDCap seq kullandık. Burada biz son zamanlarda 12 yayınlanmış meme kanseri çalışmasında bazı bilgileri göstermektedir. Bu durumda, farklı şekilde farklı bir genomik bölgedeki normale göre tümör metile CpG adaları tespit etmek tüm genom sekanslama yaklaşım kullanılır. Araştırma, t...

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

Burada bir protokol Metil-Bağlama DNA Yakalama sıralama (MBDCap seq veya MBD-seq) teknolojisi ve sonraki biyoinformatik analiz boru hattı kullanılarak büyük ölçekli klinik hasta tarama çalışmalarında genom DNA metilasyonu araştırmak için sunuyoruz.

Hasta Dokularda metil-bağlayıcı DNA yakalama Dizi

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 Görüntüleme.  University of Texas Health Science Center at San Antonio.  Burada bir protokol Metil-Bağlama DNA Yakalama sıralama (MBDCap seq veya MBD-seq) teknolojisi ve sonraki biyoinformatik analiz boru hattı kullanılarak büyük ölçekli klinik hasta tarama çalışmalarında genom DNA metilasyonu araştırmak için sunuyoruz. 

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.

Kronik Lenfositik Lösemi Hastalarında Metil-CpG-Bağlayıcı Alan Yakalamaya Dayalı Bir Yöntem Kullanarak Kapsamlı DNA Metilasyon Analizi

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

Genetik

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.

Mitokondrial DNA metilasyonu doğru algılanması için metodoloji

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).

Metillenmiş 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 ...

Biyoloji

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.

Beyin Küçük, Anatomik tanımlı alanları DNA metilasyon ve Gen İfade Optimize Analizi

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 ...

Nörobilim

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.

Gastrointestinal Kanserde DNA Metilasyonunun Genom Çapında Analizi

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

Kanser Araştırmaları

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.

DNA Metilasyonu: bisülfit modifikasyonu ve Analiz

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.

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 ...

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 Hedeflenen Epigenetik Susturma için Tasarlanmış Transkripsiyonel Baskılayıcıların Seçimi

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

Biyomühendislik

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.

Baz Çifti Çözünürlük DNA metilasyon Değerlendirme Temsil bisülfit sıralamayı İndirimli pekiştirilmiş

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.

DNA Metilasyon Analizi nesil Dizilimine tarafından hedef

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 ...