SpringerProtocols: Abstract: Methyl DNA Immunoprecipitation

15. Methyl DNA Immunoprecipitation

By: Juana Magdalena¹, Jean-Jacques Goval¹

Abstract

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Epigenetics is the study of heritable changes in gene expression. Chromatin immunoprecipitation (ChIP) and methylation status analysis of genes have been applied to the study of epigenetic modifications, often perturbed in human cancer. ChIP is a technique allowing the analysis of the protein association with specific genomic regions in the context of intact cells. ChIP and immunoprecipitation (IP) of methylated DNA, both rely on the use of well-characterized specific antibodies. The first is described in Chapter 2 and the second is shown here. At Diagenode, a novel METHYL kit has been designed to immunoprecipitate methylated DNA (Methyl DNA IP). This kit allows you to perform DNA methylation analysis of your sample together with optimized internal IP controls, all in one tube. This brand new Methyl DNA IP method provides methylated DNA (meDNA) and unmethylated DNA (unDNA) controls to be used together with your DNA sample, allowing direct correlation between immunoprecipitated material and methylation status. Such methylation analysis is highly specific and each IP is quality controlled, two essential keys for reliable results. In addition, the kit protocol is fast and user-friendly.

Affiliation(s): (1) Diagenode sa, Sart-Tilman, Liège, Belgium

Book Title: Chromatin Immunoprecipitation Assays: Methods and Protocols

Series: Methods in Molecular Biology | Volume: 567 | Pub. Date: Aug-01-2009 | Page Range: 237-247 | DOI: 10.1007/978-1-60327-414-2_15

Subject: Genetics/Genomics

Key Words: Immunoprecipitation - methylated DNA - MeDIP - internal controls

References

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1.	Ushijima, T., Morimura, K., Hosoya, Y., Okonogi, H., Tatematsu, M., Sugimura, T. and Nagao, M. (1997) Establishment of methylation-sensitive-representational difference analysis and isolation of hypo- and hypermethylated genomic fragments in mouse liver tumors. Proc. Natl. Acad. Sci. U.S.A. 94, 2284–2289.

2.	Clark, S. J., Harrison, J., Paul, C. L. and Frommer, M. (1994) High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 22, 2990–2997.

3.	Fraga, M. F. and Esteller, M. (2002) DNA methylation: a profile of methods and applications. Biotechniques 33, 636–649.

4.	Yang, H. J., Liu, V. W., Wang, Y., Chan, K. Y., Tsang, P. C., Khoo, U. S., Cheung, A. N. and Ngan, H. Y. (2004) Detection of hypermethylated genes in tumor and plasma of cervical cancer patients. Gynecol. Oncol. 93, 435–440.

5.	Cross, S. H., Charlton, J. A., Nan, X. and Bird, A. P. (1994) Purification of CpG islands using a methylated DNA binding column. Nat. Genet. 6, 236–244.

6.	Yegnasubramanian, S., Lin, X., Haffner, M. C., DeMarzo, A. M. and Nelson, W. G. (2006) Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation.Nucleic Acids Res. 34, e19.

7.	Gebhard, C., Schwarzfischer, L., Pham, T. H., Andreesen, R., Mackensen, A. and Rehli, M. (2006) Rapid and sensitive detection of CpG-methylation using methyl-binding (MB)-PCR. Nucleic Acids Res. 34, e82.

8.	Rauch, T., Li, H., Wu, X. and Pfeifer, G. P. (2006) MIRA-assisted microarray analysis, a new technology for the determination of DNA methylation patterns, identifies frequent methylation of homeodomain-containing genes in lung cancer cells. Cancer Res. 66, 7939–7947.

9.	Weber, M., Davies, J. J., Wittig, D., Oakeley, E. J., Haase, M., Lam, W. L. and Schübeler, D. (2005) Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat. Genet. 37, 853–862.

10.	Klose, R. J., Sarraf, S. A., Schmiedeberg, L., McDermott, S. M., Stancheva, I. and Bird, A. P. (2005) DNA binding selectivity of MeCP2 due to a requirement for A/T sequences adjacent to methyl-CpG. Mol. Cell 19, 667–678.

11.	Zhang, X., Yazaki, J., Sundaresan, A., Cokus, S., Chan, S. W., Chen, H., Henderson, I. R., Shinn, P., Pellegrini, M., Jacobsen, S. E. and Ecker, J. R. (2006) Genome-wide high-resolution mapping and functional analysis of DNA methylation in arabidopsis. Cell 126, 1189–1201.

12.	Rauch, T. A., Zhong, X., Wu, X., Wang, M., Kernstine, K. H., Wang, Z., Riggs, A. D. and Pfeifer, G. P. (2008) High-resolution mapping of DNA hypermethylation and hypomethylation in lung cancer. Proc. Natl. Acad. Sci. U.S.A. 105, 252–257.

13.	Weber, M. and Schübeler, D. (2007) Genomic patterns of DNA methylation: targets and function of an epigenetic mark. Curr. Opin. Cell Biol. 19, 273–280.

14.	Liu, C. L., Schreiber, S. L. and Bernstein, B. E. (2003) Development and validation of a T7 based linear amplification for genomic DNA. BMC Genomics 4, 19.

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