The strength of TLC in studies aiming to identify new epigenetic modifications of
DNA lies in its high sensitivity following
32 P end labeling of native and modified
nucleosides. A key weakness of TLC is the necessity for a secondary confirmation of
the identified spot, which is most often done through mass spectrometry. Additionally, TLC is not scalable to a high-throughput format and thus is not well suited for
large studies.
2.2 Antibody-Based Detection of Epigenetic Marks in DNA
Antibodies that specifically recognize epigenetic marks of DNA are commonly used
to quantify their global levels. In 1980, Sano et al. developed an antibody against
5mC which could recognize methylated sites in purified DNA [48]. In brief, DNA
fragments were separated by agarose gel electrophoresis and transferred to nitrocellulose paper. The membrane was incubated in the presence of purified antibody
against 5mC, raised in rabbits, followed by incubation with
125 I-labeled goat antirabbit IgG. The 5mC was then visualized by autoradiography. This methodology
was capable of detecting 20 fmol of 5mC in immobilized restriction fragments of
DNA [48]. A more sensitive 5mC antibody, as well as an antibody specific for
N6MedA, was developed a few years later and utilized for their detection in human
and Drosophila DNA [27]. This more sensitive 5mC antibody enabled the detection
of a minimum of 5 fmol amounts in nanogram quantities of DNA [27].
Today, antibody-based detection of DNA epigenetic marks is a commonly used
technique, and antibodies for many epigenetic DNA modifications are commercially
available. These antibodies have been used in an enzyme-linked immunosorbent
assay (ELISA) [49, 50] (Fig. 5). Commercially available ELISA-based kits for
global DNA methylation profiling are available from a number of companies
including Zymo and EpiGentek. Generally, these kits capture DNA on an ELISA
plate. 5mC is detected through sequential incubations with a primary antibody
against 5mC, followed by a secondary antibody linked to luciferase or a similar
enzyme, and finally using colorimetric or fluorometric detection reagents [49]. The
development of antibodies specific for 5hmC and 5fC allowed for their quantitation
using similar commercially available kits (e.g., EpiGentek MethylFlash Global DNA
Hydroxymethylation (5-hmC) ELISA Easy Kit; Active Motif Global 5-hmC DNA
Quantification Kit; EpiGentek MethylFlash 5-Formylcytosine (5-fC) DNA Quantification Kit). These ELISA-based assays are easy to perform, employ simple equipment that is readily available at most research laboratories, and work well for
monitoring relative changes in global DNA methylation. However, it should be
noted that ELISA-based methods are prone to high variability, do not allow for
absolute quantitation, and are best suited for detecting large changes in global levels
of 5hmC or 5fC.
Dot blotting is another simple and rapid antibody-based method to quantify
global levels of epigenetic modifications of DNA. In this method, genomic DNA
is spotted on a membrane. The membrane is sequentially incubated in blocking
494
C. L. Seiler et al.
DNA lies in its high sensitivity following
32 P end labeling of native and modified
nucleosides. A key weakness of TLC is the necessity for a secondary confirmation of
the identified spot, which is most often done through mass spectrometry. Additionally, TLC is not scalable to a high-throughput format and thus is not well suited for
large studies.
2.2 Antibody-Based Detection of Epigenetic Marks in DNA
Antibodies that specifically recognize epigenetic marks of DNA are commonly used
to quantify their global levels. In 1980, Sano et al. developed an antibody against
5mC which could recognize methylated sites in purified DNA [48]. In brief, DNA
fragments were separated by agarose gel electrophoresis and transferred to nitrocellulose paper. The membrane was incubated in the presence of purified antibody
against 5mC, raised in rabbits, followed by incubation with
125 I-labeled goat antirabbit IgG. The 5mC was then visualized by autoradiography. This methodology
was capable of detecting 20 fmol of 5mC in immobilized restriction fragments of
DNA [48]. A more sensitive 5mC antibody, as well as an antibody specific for
N6MedA, was developed a few years later and utilized for their detection in human
and Drosophila DNA [27]. This more sensitive 5mC antibody enabled the detection
of a minimum of 5 fmol amounts in nanogram quantities of DNA [27].
Today, antibody-based detection of DNA epigenetic marks is a commonly used
technique, and antibodies for many epigenetic DNA modifications are commercially
available. These antibodies have been used in an enzyme-linked immunosorbent
assay (ELISA) [49, 50] (Fig. 5). Commercially available ELISA-based kits for
global DNA methylation profiling are available from a number of companies
including Zymo and EpiGentek. Generally, these kits capture DNA on an ELISA
plate. 5mC is detected through sequential incubations with a primary antibody
against 5mC, followed by a secondary antibody linked to luciferase or a similar
enzyme, and finally using colorimetric or fluorometric detection reagents [49]. The
development of antibodies specific for 5hmC and 5fC allowed for their quantitation
using similar commercially available kits (e.g., EpiGentek MethylFlash Global DNA
Hydroxymethylation (5-hmC) ELISA Easy Kit; Active Motif Global 5-hmC DNA
Quantification Kit; EpiGentek MethylFlash 5-Formylcytosine (5-fC) DNA Quantification Kit). These ELISA-based assays are easy to perform, employ simple equipment that is readily available at most research laboratories, and work well for
monitoring relative changes in global DNA methylation. However, it should be
noted that ELISA-based methods are prone to high variability, do not allow for
absolute quantitation, and are best suited for detecting large changes in global levels
of 5hmC or 5fC.
Dot blotting is another simple and rapid antibody-based method to quantify
global levels of epigenetic modifications of DNA. In this method, genomic DNA
is spotted on a membrane. The membrane is sequentially incubated in blocking
494
C. L. Seiler et al.