डीएनए methylated immunoprecipitation

Summary

इस वीडियो डीएनए methylated immunoprecipitation (MeDIP) के लिए प्रोटोकॉल को दर्शाता है. MeDIP एक दो दिन की प्रक्रिया है कि चुनिंदा एक जीनोमिक डीएनए नमूने से डीएनए methylated टुकड़े अर्क विशिष्टता के साथ 5 मिथाइलसिटोसाइन के लिए एंटीबॉडी का उपयोग (MC विरोधी 5) है.

Abstract

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}.

Protocol

डीएनए निष्कर्षण और नमूना तैयारी MeDIP के विभिन्न नमूनों की एक किस्म (संवर्धित कोशिकाओं, के रूप में formalin निर्धारित पैराफिन एम्बेडेड ऊतकों के रूप में अच्छी तरह से ताजा जमे हुए) से डीएनए के लिए इस्ते…

Discussion

रोग में महत्वपूर्ण भूमिका डीएनए मेथिलिकरण नाटकों का एक बढ़ती जागरूकता है, इसलिए assays के विकास के लिए इस संशोधन को मापने के तेजी से महत्वपूर्ण होते जा ^{रहे हैं 3, 12,} 13. MeDIP तकनीक दोनों जीनोम और पूरे ठिकाना व?…

Materials

Material Name	Tipo	Company	Catalogue Number	Comment
Biorupter sonicator	Tool	Diagenode	UCD-200 TM
1.7ml SafeSeal Microcentrifuge Tubes	Outro	Sorenson BioScience	11510
ND 3300 Spectrophotometer	Tool	NanoDrop
Primary Antibody: Anti-5′-methylcytosine mouse mAb	Reagent	CalBiochem	162 33 D3
Secondary Antibody: Dynabeads M-280 Sheep anti-mouse IgG	Reagent	Invitrogen	112-01D
Magnetic Tube Rack	Tool	Invitrogen	CS15000
Mini LabRoller	Tool	Labnet International	H5500
IP Buffer				10 mM NaPO4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100. Stored at room temperature
Digestion Buffer				10 mM Tris pH8.0, 100 mM EDTA, 0.5% SDS, 50 mM NaCl