Abstract
Quantitative polymerase chain reaction (Q-PCR) allows for the accurate and reproducible determination of the amount of target DNA in a sample through the measurement of PCR product accumulation in “real time.” This method determines starting target DNA quantity over a large assay dynamic range and requires no post-PCR sample manipulation. When used in combination with the method of chromatin immunoprecipitation (ChIP), the amount of protein binding to a specific region of DNA can be accurately and rapidly determined. A method for quantifying the presence of acetylated histones H3 and H4 on different regions of a target locus using Q-PCR after ChIP is described.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Irvine, R. A. and Hsieh, C.-L. (2002) DNA methylation has a local effect on transcription and histone acetylation. Mol. Cell. Biol. 22, 6689–6696.
Orlando, V. (2000) Mapping chromosomal proteins in vivo by formaldehyde-crosslinked-chromatin immunoprecipitation. Trends Biochem. Sci. 25, 99–104.
Ginzinger, D. G. (2002) Gene quantification using real-time quantitative PCR: An emerging technology hits the mainstream. Exp. Hematology 30, 503–512.
Stevens, S. J., Verschuuren, E. A., Pronk, I., van Der Bij, W., Harmsen, M. C, The, T. H., Meijer, C. J., van Den Brule, A. J., and Middeldorp, J., M. (2001) Frequent monitoring of Epstein-Barr virus DNA load in unfractionated whole blood is essential for early detection of posttransplant lymphoproliferative disease in high-risk patients. Blood 97, 1165–1171.
Heid, C. A., Stevens, J., Livak, K. J., and Williams, P. M. (1996) Real Time Quantitative PCR. Genome Res. 6, 986–994.
Solomon, M. J., Larsen, P. L., and Varshavsky, A. (1988) Mapping protein-DNA interactions in vivo with formaldehyde: evidence that histone H4 is retained on a highly transcribed gene. Cell 53, 937–947.
Braunstein, M., Rose, A. B., Holmes, S. G., Allis, C. D., and Broach, J. R. (1993) Transcriptional silencing in yeast is associated with reduced nucleosome acetylation. Genes Dev. 7, 592–604.
Hsieh, C.-L. (1994) Dependence of transcriptional repression on CpG methylation density. Mol. Cell. Biol. 14, 5487–5494.
Chen, H., Lin, R. J., Xie, W., Wilpitz, D., and Evans, R. M. (1999) Regulation of hormone-induced histone hyperacetylation and gene activation via acetylation of an acetylase. Cell 98, 675–686.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Irvine, R.A., Hsieh, CL. (2004). Q-PCR in Combination With ChIP Assays to Detect Changes in Chromatin Acetylation. In: Tollefsbol, T.O. (eds) Epigenetics Protocols. Methods in Molecular Biology™, vol 287. Humana Press. https://doi.org/10.1385/1-59259-828-5:045
Download citation
DOI: https://doi.org/10.1385/1-59259-828-5:045
Publisher Name: Humana Press
Print ISBN: 978-1-58829-336-7
Online ISBN: 978-1-59259-828-1
eBook Packages: Springer Protocols