Top Med Chem (2020) 33: 487–522
DOI: 10.1007/7355_2019_77
© Springer Nature Switzerland AG 2019
Published online: 26 October 2019
Experimental Methodologies for Detection
and Mapping of Epigenetic DNA Marks
Christopher L. Seiler, Jenna Fernandez, Qiyuan Han,
and Natalia Y. Tretyakova
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489
2 Methods for Detection and Quantification of Epigenetic Modifications Within
the Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492
2.1 Initial Detection of 5mC and Its Oxidized Forms via Paper Chromatography
and Thin-Layer Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492
2.2 Antibody-Based Detection of Epigenetic Marks in DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494
2.3 High Performance Liquid Chromatography: Mass Spectrometry . . . . . . . . . . . . . . . . . . . . 496
3 Indirect Methods to Map Epigenetic DNA Modifications Along the Genome . . . . . . . . . . . . 499
3.1 Bisulfite Conversion to Distinguish Between C and 5mC . . . . . . . . . . . . . . . . . . . . . . . . . . . 499
3.2 Microarray Technology for Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501
3.3 Next-Generation Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503
3.4 Antibody/Enrichment-Based Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503
3.5 Whole-Genome Bisulfite Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505
3.6 Reduced Representation Bisulfite Sequencing (RRBS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505
3.7 Modified Bisulfite Sequencing Methods for Detection of 5hmC, 5fC, and 5caC . . . 506
4 Direct Detection of Epigenetic Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
4.1 Single-Molecule Real-Time (SMRT) Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
4.2 Nanopore Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
Abstract Epigenetic marks of DNA such as 5-methylcytosine control the levels of
gene expression in cells and tissues by altering DNA structure, inhibiting transcription factor binding, and recruiting chromatin-modifying enzymes. DNA methylation
marks are introduced in both strands of DNA by DNA methyltransferases, allowing
for heritable gene silencing. In addition to 5-methylcytosine, cells contain its oxidized
forms 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine, as well
as recently discovered DNA marks, 4-methylcytosine and 6-methyladenosine.
C. L. Seiler, J. Fernandez, Q. Han, and N. Y. Tretyakova (*)
Department of Medicinal Chemistry and the Masonic Cancer Center, University of Minnesota,
Minneapolis, MN, USA
e-mail: trety001@umn.edu
DOI: 10.1007/7355_2019_77
© Springer Nature Switzerland AG 2019
Published online: 26 October 2019
Experimental Methodologies for Detection
and Mapping of Epigenetic DNA Marks
Christopher L. Seiler, Jenna Fernandez, Qiyuan Han,
and Natalia Y. Tretyakova
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489
2 Methods for Detection and Quantification of Epigenetic Modifications Within
the Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492
2.1 Initial Detection of 5mC and Its Oxidized Forms via Paper Chromatography
and Thin-Layer Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492
2.2 Antibody-Based Detection of Epigenetic Marks in DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494
2.3 High Performance Liquid Chromatography: Mass Spectrometry . . . . . . . . . . . . . . . . . . . . 496
3 Indirect Methods to Map Epigenetic DNA Modifications Along the Genome . . . . . . . . . . . . 499
3.1 Bisulfite Conversion to Distinguish Between C and 5mC . . . . . . . . . . . . . . . . . . . . . . . . . . . 499
3.2 Microarray Technology for Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501
3.3 Next-Generation Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503
3.4 Antibody/Enrichment-Based Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503
3.5 Whole-Genome Bisulfite Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505
3.6 Reduced Representation Bisulfite Sequencing (RRBS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505
3.7 Modified Bisulfite Sequencing Methods for Detection of 5hmC, 5fC, and 5caC . . . 506
4 Direct Detection of Epigenetic Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
4.1 Single-Molecule Real-Time (SMRT) Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
4.2 Nanopore Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
Abstract Epigenetic marks of DNA such as 5-methylcytosine control the levels of
gene expression in cells and tissues by altering DNA structure, inhibiting transcription factor binding, and recruiting chromatin-modifying enzymes. DNA methylation
marks are introduced in both strands of DNA by DNA methyltransferases, allowing
for heritable gene silencing. In addition to 5-methylcytosine, cells contain its oxidized
forms 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine, as well
as recently discovered DNA marks, 4-methylcytosine and 6-methyladenosine.
C. L. Seiler, J. Fernandez, Q. Han, and N. Y. Tretyakova (*)
Department of Medicinal Chemistry and the Masonic Cancer Center, University of Minnesota,
Minneapolis, MN, USA
e-mail: trety001@umn.edu