उच्च संवेदनशीलता / Balb सी मस्तिष्क के ऊतकों में जांच 5-hydroxymethylcytosine
Summary
5 HMC EpiMark और 5-MC विश्लेषण किट का विश्लेषण और एक विशेष cific बिन्दुपथ के भीतर 5 मिथाइलसिटोसाइन और 5-hydroxymethylcytosine quantitate करने के लिए इस्तेमाल किया जा सकता है. किट 5 – HMC से ग्लूकोज की एक enzymatic β-glucosyltransferase (टी -4 BGT) का उपयोग प्रतिक्रिया के माध्यम से 5-HMC के हाइड्रॉक्सिल समूह के अलावा द्वारा 5 MC अलग. जब 5-HMC CCGG के संदर्भ में होता है, इस संशोधन के एक गैर cleavable साइट के लिए एक cleavable MspI साइट धर्मान्तरित.
Abstract
DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research. Mammalian DNA is enzymatically modified at the 5th carbon position of cytosine (C) residues to 5-mC, predominately in the context of CpG dinucleotides. 5-mC is amenable to enzymatic oxidation to 5-hmC by the Tet family of enzymes, which are believed to be involved in development and disease. Currently, the biological role of 5-hmC is not fully understood, but is generating a lot of interest due to its potential as a biomarker. This is due to several groundbreaking studies identifying 5-hydroxymethylcytosine in mouse embryonic stem (ES) and neuronal cells.
Research techniques, including bisulfite sequencing methods, are unable to easily distinguish between 5-mC and 5-hmC . A few protocols exist that can measure global amounts of 5-hydroxymethylcytosine in the genome, including liquid chromatography coupled with mass spectrometry analysis or thin layer chromatography of single nucleosides digested from genomic DNA. Antibodies that target 5-hydroxymethylcytosine also exist, which can be used for dot blot analysis, immunofluorescence, or precipitation of hydroxymethylated DNA, but these antibodies do not have single base resolution.In addition, resolution depends on the size of the immunoprecipitated DNA and for microarray experiments, depends on probe design. Since it is unknown exactly where 5-hydroxymethylcytosine exists in the genome or its role in epigenetic regulation, new techniques are required that can identify locus specific hydroxymethylation. The EpiMark 5-hmC and 5-mC Analysis Kit provides a solution for distinguishing between these two modifications at specific loci.
The EpiMark 5-hmC and 5-mC Analysis Kit is a simple and robust method for the identification and quantitation of 5-methylcytosine and 5-hydroxymethylcytosine within a specific DNA locus. This enzymatic approach utilizes the differential methylation sensitivity of the isoschizomers MspI and HpaII in a simple 3-step protocol.
Genomic DNA of interest is treated with T4-BGT, adding a glucose moeity to 5-hydroxymethylcytosine. This reaction is sequence-independent, therefore all 5-hmC will be glucosylated; unmodified or 5-mC containing DNA will not be affected.
This glucosylation is then followed by restriction endonuclease digestion. MspI and HpaII recognize the same sequence (CCGG) but are sensitive to different methylation states. HpaII cleaves only a completely unmodified site: any modification (5-mC, 5-hmC or 5-ghmC) at either cytosine blocks cleavage. MspI recognizes and cleaves 5-mC and 5-hmC, but not 5-ghmC.
The third part of the protocol is interrogation of the locus by PCR. As little as 20 ng of input DNA can be used. Amplification of the experimental (glucosylated and digested) and control (mock glucosylated and digested) target DNA with primers flanking a CCGG site of interest (100-200 bp) is performed. If the CpG site contains 5-hydroxymethylcytosine, a band is detected after glucosylation and digestion, but not in the non-glucosylated control reaction. Real time PCR will give an approximation of how much hydroxymethylcytosine is in this particular site.
In this experiment, we will analyze the 5-hydroxymethylcytosine amount in a mouse Babl/C brain sample by end point PCR.
Protocol
Discussion
वहाँ कई महत्वपूर्ण बातें जब तक इस प्रयोग की स्थापना पर विचार करने के लिए कर रहे हैं. सबसे पहले, यह महत्वपूर्ण है कि जीनोमिक डीएनए के glucosylation पूरा करने के लिए आय. टी -4 – BGT के अनुक्रम विशिष्टता में जाना जाता है, नहीं है और यह सब्सट्रेट के अनुक्रम के लिए कोई विकल्प नहीं है प्रकट होता है है. इसलिए, कुछ मामलों में, अब ऊष्मायन बार आवश्यक हो सकता है. दूसरा MspI, और HpaII पाचन पूरा हो ताकि पृष्ठभूमि संकेत से बचने के लिए करना चाहिए. इन प्रतिबंध एंजाइमों के लिए, हम 4 घंटे की एक ऊष्मायन समय सलाह देते हैं, लेकिन अब incubations जब अधूरा दरार मनाया जाता है निष्पादित किया जा सकता है. तीसरा, इनपुट डीएनए राशि उपलब्धता पर निर्भर करता है, समायोजित किया जा सकता है के बाद डीएनए की 20 ngs पीसीआर अंत बिंदु के लिए इस्तेमाल किया जा सकता है. अंत में, हम आपूर्ति नियंत्रण डीएनए जो 5 – एम सी और 5 – HMC मात्रा का ठहराव के लिए जीनोमिक डीएनए के साथ समानांतर में चलाया जा सकता है है के उपयोग की सलाह देते हैं.
Disclosures
The authors have nothing to disclose.
Acknowledgements
Sriharsa प्रधान, शान्नोन मोरे Kinney, रुको Gyeong चिन, Jurate Bitinaite, यू झेंग, पियरे Olivier Esteve, Romualdas Vaisvila, स्टीवन ई. Jacobsen प्रयोगशाला, UCLA. इस काम आंशिक रूप से 1R44GM095209 01 NIH द्वारा समर्थित किया गया था.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| EpiMark™ 5-hmC and 5-mC Analysis Kit | New England Biolabs | E3317 | ||
| Locus specific primers, flanking a CCGG site of interest | Custom per experiment; provided by user | |||
| LongAmp® Taq DNA Polymerase | New England Biolabs | M0323 | ||
| dNTPs | New England Biolabs | N0447 | ||
| molecular biology grade water |
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