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  DNA methylation analysis by bisulfite conversion, cloning, and sequencing of individual clones.

Zhang, Y., Rohde, C., Tierling, S., Stamerjohanns, H., Reinhardt, R., Walter, J., et al. (2009). DNA methylation analysis by bisulfite conversion, cloning, and sequencing of individual clones. In J. Tost (Ed.), DNA Methylation, 2nd edition (pp. 177-187). New York [et al]: Humana Press.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0010-7E44-2 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0010-7E45-F
Genre: Book Chapter

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 Creators:
Zhang, Yingying, Author
Rohde, Christian, Author
Tierling, Sascha, Author
Stamerjohanns, Heinrich, Author
Reinhardt, Richard1, Author              
Walter, Jörn2, Author              
Jeltsch, Albert, Author
Affiliations:
1High Throughput Technologies, Max Planck Institute for Molecular Genetics, Max Planck Society, ou_1433552              
2Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society, ou_1433550              

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Free keywords: DNA methylation, bisulfite; sequencing; cloning;
 Abstract: 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.

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Language(s): eng - English
 Dates: 2009-01-01
 Publication Status: Published in print
 Pages: -
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 Table of Contents: -
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Title: DNA Methylation, 2nd edition
Source Genre: Book
 Creator(s):
Tost, Jörg, Editor
Affiliations:
-
Publ. Info: New York [et al] : Humana Press
Pages: 391 Volume / Issue: - Sequence Number: - Start / End Page: 177 - 187 Identifier: ISBN: 978-1-934115-61-9

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Title: Methods in Molecular Biology
Source Genre: Series
 Creator(s):
Walker, John M., Editor
Humana Press, Editor  
Affiliations:
-
Publ. Info: -
Pages: - Volume / Issue: 507 Sequence Number: - Start / End Page: - Identifier: -