Abstract
The accurate and quantitative detection of 5-methylcytosine is of great importance in the field of epigenetics. The method of choice is usually bisulfite sequencing because of the high resolution and the possibility to combine it with next generation sequencing. Nevertheless, also this method has its limitations. Following the bisulfite treatment DNA strands are no longer complementary such that in a subsequent PCR amplification the DNA methylation patterns information of only one of the two DNA strand is preserved. Several years ago Hairpin Bisulfite sequencing was developed as a method to obtain the pattern information on complementary DNA strands. The method requires fragmentation (usually by enzymatic cleavage) of genomic DNA followed by a covalent linking of both DNA strands through ligation of a short DNA hairpin oligonucleotide to both strands. The ligated covalently linked dsDNA products are then subjected to a conventional bisulfite treatment during which all unmodified cytosines are converted to uracils. During the treatment the DNA is denatured forming noncomplementary ssDNA circles. These circles serve as a template for a locus specific PCR to amplify chromosomal patterns of the region of interest. As a result one ends up with a linearized product, which contains the methylation information of both complementary DNA strands.
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References
Laird CD, Pleasant ND, Clark AD et al (2004) Hairpin-bisulfite PCR: assessing epigenetic methylation patterns on complementary strands of individual DNA molecules. Proc Natl Acad Sci U S A 101:204–209
Arand J, Spieler D, Karius T et al (2012) In vivo control of CpG and non-CpG DNA methylation by DNA methyltransferases. PLoS Genet 8:e1002750
Ficz G, Hore TA, Santos F et al (2013) FGF signaling inhibition in ESCs drives rapid genome-wide demethylation to the epigenetic ground state of pluripotency. Cell Stem Cell 13:351–359
Seisenberger S, Andrews S, Krueger F et al (2012) The dynamics of genome-wide DNA methylation reprogramming in mouse primordial germ cells. Mol Cell 48:849–862
Lutsik P, Feuerbach L, Arand J et al (2011) BiQ Analyzer HT: locus-specific analysis of DNA methylation by high-throughput bisulfite sequencing. Nucleic Acids Res 39:W551–W556
Giehr P, Kyriakopoulos C et al (2016) The influence of hydroxylation on maintaining CpG methylation patterns: a hidden Markov model approach. PLoS Comput Biol 12(5):e1004905
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Giehr, P., Walter, J. (2018). Hairpin Bisulfite Sequencing: Synchronous Methylation Analysis on Complementary DNA Strands of Individual Chromosomes. In: Tost, J. (eds) DNA Methylation Protocols. Methods in Molecular Biology, vol 1708. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7481-8_29
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DOI: https://doi.org/10.1007/978-1-4939-7481-8_29
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7479-5
Online ISBN: 978-1-4939-7481-8
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