Abstract
noindent Sodium bisulfite modification-based fine mapping of methylated cytosines represents the gold standard technique for DNA methylation studies. A major problem with this approach, however, is that it results in considerable DNA degradation, and large quantities of genomic DNA material are needed if numerous genomic regions are to be profiled. This chapter describes a method for profiling DNA methylation from small amounts of genomic-DNA starting material utilizing an efficient sodium bisulfite conversion method followed by whole-genome amplification (WGA). WGA is a useful method to overcome the problem of low initial amount of DNA and/or severe DNA degradation during conventional sodium bisulfite treatment in studies investigating DNA methylation. WGA is a relatively inexpensive process that can be optimized for high-throughput application and enables the thorough investigation of methylation at numerous genomic locations on samples for which DNA availability is low. Data from our lab has demonstrated that bisulfite-treated DNA amplified using WGA can be used for a range of downstream DNA methylation mapping procedures, including bisulfite-primer optimization, the sequencing of cloned PCR products, MS-SNuPE, and Pyrosequencing.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Henikoff, S., Matzke, M. A. (1997) Exploring and explaining epigenetic effects. Trends Genet 13, 293–295.
El-Maarri, O. (2003) Methods: DNA methylation. Adv Exp Med Biol 544, 197–204.
Clark, S. J., Harrison, J., Paul, C. L., et al. (1994) High sensitivity mapping of methylated cytosines. Nucleic Acids Res 22, 2990–2997.
Schumacher, A., Kapranov, P., Kaminsky, Z., et al. (2006) Microarray-based DNA methylation profiling: technology and applications. Nucleic Acids Res 34, 528–542.
Tost, J., El Abdalaoui, H., Gut, I.G. (2006) Serial pyrosequencing for quantitative DNA methylation analysis. Biotechniques 40, 721–726.
Grunau, C., Clark, S.J., Rosenthal, A. (2001) Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res 29, e65
Olek, A., Oswald, J., Walter, J. (1996) A modified and improved method for bisulphite based cytosine methylation analysis. Nucleic Acids Res 24, 5064–5066.
Ballantyne, K.N., van Oorschot, R.A., Mitchell, R.J. (2007) Comparison of two whole genome amplification methods for STR genotyping of LCN and degraded DNA samples. Forensic Sci Int 166, 35–41.
Zhang, L., Cui, X., Schmitt, K., et al. (1992) Whole genome amplification from a single cell: implications for genetic analysis. Proc Natl Acad Sci USA 89, 5847–5851.
Dean, F.B., Hosono, S., Fang, L., et al. (2002) Comprehensive human genome amplification using multiple displacement amplification. Proc Natl Acad Sci USA 99, 5261–5266.
Kuivaniemi, H., Yoon, S., Shibamura, H., et al. (2002) Primer-extension preamplified DNA is a reliable template for genotyping. Clin Chem 48, 1601–1604.
Hosono, S., Faruqi, A.F., Dean, F.B., et al. (2003) Unbiased whole-genome amplification directly from clinical samples. Genome Res 13, 954–964.
Mill, J., Yazdanpanah, S., Guckel, E., et al. (2006) Whole genome amplification of sodium bisulfite-treated DNA allows the accurate estimate of methylated cytosine density in limited DNA resources. Biotechniques 41, 603–607.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Mill, J., Petronis, A. (2009). Profiling DNA Methylation from Small Amounts of Genomic DNA Starting Material: Efficient Sodium Bisulfite Conversion and Subsequent Whole-Genome Amplification. In: Tost, J. (eds) DNA Methylation. Methods in Molecular Biology, vol 507. Humana Press. https://doi.org/10.1007/978-1-59745-522-0_27
Download citation
DOI: https://doi.org/10.1007/978-1-59745-522-0_27
Publisher Name: Humana Press
Print ISBN: 978-1-934115-61-9
Online ISBN: 978-1-59745-522-0
eBook Packages: Springer Protocols