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DNA methylation: Bisulphite modification and analysis
Abstract
DNA methylation is an important epigenetic modification of DNA in mammalian genomes. DNA methylation patterns are established early in development, modulated during tissue-specific differentiation and disrupted in many disease states, including cancer. To understand further the biological functions of these changes, accurate and reproducible methods are required to fully analyze the DNA methylation sequence. Here, we describe the 'gold-standard' bisulphite conversion protocol that can be used to re-sequence DNA from mammalian cells in order to determine and quantify the methylation state of a gene or genomic region at single-nucleotide resolution. The process of bisulphite treatment exploits the different sensitivities of cytosine and 5-methylcytosine (5-MeC) to deamination by bisulphite under acidic conditions—in which cytosine undergoes conversion to uracil, whereas 5-MeC remains unreactive. Bisulphite conversion of DNA, in either single tubes or in a 96-well format, can be performed in a minimum of 8 h and a maximum of 18 h, depending on the amount and quality of starting DNA.
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Cited by
Pidsley, R., Zotenko, E., Peters, T. J., Lawrence, M. G., Risbridger, G. P., Molloy, P., Van Djik, S., Muhlhausler, B., Stirzaker, C., & Clark, S. J. (2016) · Genome biology · doi:10.1186/s13059-016-1066-1 · Cited by 1,480
Self-citationDirect detection of DNA methylation during single-molecule, real-time sequencing
Flusberg, B. A., Webster, D. R., Lee, J. H., Travers, K. J., Olivares, E. C., Clark, T. A., Korlach, J., & Turner, S. W. (2010) · Nature Methods · doi:10.1038/nmeth.1459 · Cited by 1,460
Plagemann, A., Harder, T., Brunn, M., Harder, A., Roepke, K., Wittrock‐Staar, M., Ziska, T., Schellong, K., Rodekamp, E., Melchior, K., et al. (2009) · The Journal of Physiology · doi:10.1113/jphysiol.2009.176156 · Cited by 439
Jin, S.-G., Kadam, S., & Pfeifer, G. P. (2010) · Nucleic Acids Research · doi:10.1093/nar/gkq223 · Cited by 437
Pullen, T. J., da Silva Xavier, G., Kelsey, G., & Rutter, G. A. (2011) · Molecular and Cellular Biology · doi:10.1128/mcb.01433-10 · Cited by 258
Metadata
- Authors
- Clark, S. J., Statham, A., Stirzaker, C., Molloy, P. L., & Frommer, M.
- Venue
- Nature Protocols
- Published
- 2006-12
- Volume
- 1
- Issue
- 5
- Pages
- 2353-2364
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- OpenAlex
- W2076234267
- Cited by
- 362
- References
- 0
- FWCI
- 4.67
- Type
- journal article
- Language
- en
- Open access
- closed
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- Clark, S. J., Statham, A., Stirzaker, C., Molloy, P. L., & Frommer, M. (2006). DNA methylation: Bisulphite modification and analysis. Nature Protocols. https://doi.org/10.1038/nprot.2006.324
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