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

इस अध्ययन के हिस्से में स्वास्थ्य के राष्ट्रीय संस्थान (GM071440 से CH और NS051630/MH076090/MH078972 तक पी.जे. करने के लिए) द्वारा समर्थित किया गया था.

1. जीनोमिक डीएनए विखंडन Fragment जीनोमिक इच्छित आकार सीमा sonication का उपयोग डीएनए अनुक्रमण जीनोम चौड़ा मंच के लिए अनुकूल है. (हम आमतौर पर ~ 300 बीपी sonicate.) 1% agarose जेल (1 चित्रा) पर खंडित जीनोमिक डीएनए के आका?...

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+Tet3+DNA+dioxygenase+in+epigenetic+reprogramming+by+oocytes.">The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes.</a> Nature. 477, 606-610 (2011).</li><li>Dawlaty, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tet1+is+dispensable+for+maintaining+pluripotency+and+its+loss+is+compatible+with+embryonic+and+postnatal+development.">Tet1 is dispensable for maintaining pluripotency and its loss is compatible with embryonic and postnatal development.</a> Cell Stem Cell. 9, 166-175 (2011).</li><li>Huang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+behaviour+of+5-hydroxymethylcytosine+in+bisulfite+sequencing.">The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing.</a> PLoS One. 5, e8888 (2010).</li><li>Song, C. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+chemical+labeling+reveals+the+genome-wide+distribution+of+5-hydroxymethylcytosine.">Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine.</a> Nat. Biotechnol. 29, 68-72 (2011).</li><li>Pastor, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+mapping+of+5-hydroxymethylcytosine+in+embryonic+stem+cells.">Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells.</a> Nature. 473, 394-397 (2011).</li><li>Matarese, F., Pau, C. a. r. r. i. l. l. o. -. d. e. S. a. n. t. a., E, ., Stunnenberg, H. 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S., Spada, F., Leonhardt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+enzymatic+quantification+of+5-hydroxymethylcytosine+in+genomic+DNA.">Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA.</a> Nucleic Acids Res. 38, 181 (2010).</li><li>Terragni, J., Bitinaite, J., Zheng, Y., Pradhan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+characterization+of+recombinant+&#946;-glucosyltransferase+and+analysis+of+global+5-hydroxymethylcytosine+in+unique+genomes.">Biochemical characterization of recombinant &#946;-glucosyltransferase and analysis of global 5-hydroxymethylcytosine in unique genomes.</a> Biochemistry. , (2012).</li><li>Rusmintratip, V., Sowers, L. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+5-hydroxymethylcytosine+into+the+epigenomic+landscape+of+human+embryonic+stem+cells.">Integrating 5-hydroxymethylcytosine into the epigenomic landscape of human embryonic stem cells.</a> PLoS Genet. 7, e1002154 (2011).</li><li>Szulwach, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-hmC-mediated+epigenetic+dynamics+during+postnatal+neurodevelopment+and+aging.">5-hmC-mediated epigenetic dynamics during postnatal neurodevelopment and aging.</a> Nat. Neurosci. 14, 1607-1616 (2011).</li></ol>

5 hydroxymethylcytosine (5 HMC) एक हाल ही में पहचाना कुछ स्तनधारी प्रकार की कोशिकाओं में पर्याप्त मात्रा में मौजूद epigenetic संशोधन है. यहाँ प्रस्तुत विधि 5-HMC जीनोम चौड़ा वितरण का निर्धारण करने के लिए है. हम टी -4 जीवाणुभोजी β glucosyltr...

<table border="1"><tbody><tr><td> नाम </td><td> कंपनी </td><td> # सूची </td><td> टिप्पणी </td></tr><tr><td></td><td></td><td></td><td> अभिकर्मकों </td></tr><tr><td> 5M सोडियम क्लोराइड (NaCl) </td><td> PROMEGA </td><td> V4221 </td><td></td></tr><tr><td> 0.5M pH8.0 ethylenediaminetetraacetic एसिड (EDTA) </td><td> PROMEGA </td><td> V4231 </td><td></td></tr><tr><td> 1M Trizma (Tris) आधार pH7.5 </td><td> Invitrogen </td><td> 15567-027) </td><td></td></tr><tr><td> 1M HEPES, pH7.4 </td><td> Invitrogen </td><td> 15630 </td><td></td></tr><tr><td> मैग्नेशियम क्लोराइड (2 MgCl) 1M </td><td> Ambion </td><td> AM9530G </td><td></td></tr><tr><td> डाइमिथाइल sulfoxide (DMSO) </td><td> सिग्मा </td><td> D8418 </td><td></td></tr><tr><td> 20 Tween </td><td> फिशर BioReagents </td><td> BP337-100 </td><td></td></tr><tr><td> DBCO एसएस PEG3 बायोटिन संयुग्म </td><td> रसायन विज्ञान उपकरण क्लिक करें </td><td> A112P3 </td><td></td></tr><tr><td> 1,4-Dithiothreitol, ultrapure Superpure (डीटीटी) </td><td> Invitrogen </td><td> 15508-013 </td><td></td></tr><tr><td> QIAquick Nucleotide हटाने किट </td><td> क्विएज़न </td><td> 28304 </td><td></td></tr><tr><td> जैव स्पिन 6 कॉलम माइक्रो </td><td> जैव रेड </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 पीसीआर शोधन किट </td><td> क्विएज़न </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-ग्लूकोज </td><td> सक्रिय आकृति </td><td> 55013 </td><td></td></tr><tr><td></td><td></td><td></td><td> किण्वक </td></tr><tr><td> β glucosyltransferase (β जी.टी.) </td><td> न्यू इंग्लैंड BIOLAB </td><td> M0357 </td><td></td></tr><tr><td></td><td></td><td></td><td> उपकरण </td></tr><tr><td> Sonication डिवाइस </td><td> Covaris </td><td></td><td></td></tr><tr><td> डेस्कटॉप अपकेंद्रित्र </td><td></td><td></td><td></td></tr><tr><td> जल स्नान </td><td> फिशर साइंटिफिक </td><td></td><td></td></tr><tr><td> जेल चल रहा तंत्र </td><td> जैव रेड </td><td></td><td></td></tr><tr><td> NanoDrop1000 </td><td> थर्मो वैज्ञानिक </td><td></td><td></td></tr><tr><td> Labquake ट्यूब शेखर </td><td> Barnstead </td><td></td><td></td></tr><tr><td> Labquake ट्यूब शेखर </td><td> Thermolyne </td><td></td><td></td></tr><tr><td> चुंबकीय जुदाई स्टैंड </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> अभिकर्मक 10 सेटअप एक्स बफर β-जी.टी. रिएक्शन (500 मिमी 7.9 पीएच HEPES, 250 मिमी 2 MgCl) 2 एक्स बाध्यकारी और (B &amp; W) बफर धोने (10 मिमी Tris 7.5 पीएच, 1 मिमी EDTA, 2 एम NaCl, 0.02% 20 Tween) . </td></tr></tbody></table>

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.

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

वर्णित एक दो कदम लेबलिंग β glucosyltransferase (β-GT) प्रक्रिया का उपयोग कर 5-HMC एक azide ग्लूकोज हस्तांतरण, रसायन शास्त्र क्लिक द्वारा पीछा करने के लिए आसान और घनत्व स्वतंत्र संवर्धन के लिए एक बायोटिन linker हस्तांतरण. यह कुशल और विशिष्ट लेबलिंग विधि अत्यंत कम पृष्ठभूमि और उच्च throughput epigenomic मानचित्रण के साथ अगली पीढ़ी के अनुक्रमण के माध्यम से 5 HMC के संवर्धन के लिए सक्षम बनाता है.

जीनोमिक डीएनए से 5-hydroxymethylcytosine के चुनिंदा कब्जा

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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