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          Institute: MPI für molekulare Genetik     Collection: Sequencing Group     Display Documents



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ID: 469006.0, MPI für molekulare Genetik / Sequencing Group
DNA methylation analysis by bisulfite conversion, cloning, and sequencing of individual clones.
Authors:Zhang, Yingying; Rohde, Christian; Tierling, Sascha; Stamerjohanns, Heinrich; Reinhardt, Richard; Walter, Jörn; Jeltsch, Albert
Language:English
Place of Publication:New York [et al]
Publisher:Humana Press
Date of Publication (YYYY-MM-DD):2009-01-01
Title of Book:DNA Methylation, 2nd edition
Start Page:177
End Page:187
Physical Description:391
Full Name of Book-Editor(s):Tost, Jörg
Title of Series:Methods in Molecular Biology
Volume:507
Full Name(s) of Series Editor(s):Walker, John M.
Corporate Body (Series):Humana Press
Copyright:Springer. Part of Springer Science+Business Media
Review Status:not specified
Audience:Experts Only
Abstract / Description:DNA methylation is an essential epigenetic modification in the human genome. For the investigation of DNA methylation patterns, bisulfite conversion and DNA sequencing is a method of choice, because it provides detailed information on the methylation pattern of individual DNA molecules at single CG site resolution. The method is based on the deamination of cytosine residues to uracils in the presence of NaOH and sodium bisulfite. Since methylcytosine is not converted under these conditions, the original methylation state of the DNA can be analyzed by sequencing of the converted DNA. After the conversion reaction, the DNA sequence under investigation is amplified by polymerase chain reaction (PCR) with primers specific for one strand of the bisulfite-converted DNA. The PCR product is cloned and individual clones are sequenced. Here, we describe an advanced protocol for bisulfite conversion, protocols for cloning, and tools for primer design (Methprimer, Bisearch). In addition, we present tools for the web display of primary data and data analysis (BiQ Analyzer, BDPC) and describe the setup of a sequencing and analysis pipeline for medium to high throughput.
Free Keywords:DNA methylation,
bisulfite;
sequencing;
cloning;
External Publication Status:published
Document Type:InBook
Communicated by:Richard Reinhardt
Affiliations:MPI für molekulare Genetik
External Affiliations:1.School of Engineering and Science, Jacobs University Bremen, Bremen, Germany;
2.Institut für Genetik, FB Biowissenschaften, Universität des Saarlandes, Saarbrücken, Germany.
Identifiers:URL:http://www.springerlink.com/content/v438623q311843...
DOI:10.1007/978-1-59745-522-0
ISBN:978-1-934115-61-9
Full Text:
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