novel nucleoside in embryonic stem cells was confirmed using high-resolution mass
spectrometry [29].
HPLC-ESI-MS/MS analysis of 4mC is challenging because it is present in
relatively low amounts but also has the same molecular mass and similar biophysical
properties as 5mC (Fig. 1), making it difficult to detect the two isomers separately.
HPLC resolution of 4mC and 5mC was achieved by gradient elution of a
C18 column under acidic conditions, allowing for 4mC quantification in
C. kristjanssonii containing high concentrations of 4mC [33].
3 Indirect Methods to Map Epigenetic DNA Modifications
Along the Genome
Mapping epigenetics DNA marks along the genome is essential for our understanding of their biological roles, interrelationships, and dynamics. The majority of
techniques used currently employ bisulfite conversion, modified bisulfite conversion, or antibody capture to help locate epigenetic DNA modifications. Although the
readout from these techniques is indirect, they have yielded a wealth of important
information over the years. Novel methodologies such as nanopore sequencing and
single-molecule real-time (SMRT) sequencing allow for a direct sequencing of
modified DNA and are discussed in Sect. 4.
3.1 Bisulfite Conversion to Distinguish Between C and 5mC
Early studies of chemical reactivity of native and modified DNA nucleosides have
revealed an important difference in chemical reactivity of C and 5mC [63, 64]. Upon
treatment with bisulfite, cytosine is readily deaminated to uracil, while much slower
reaction is observed for 5mC. The bisulfite reaction with cytosine proceeds through a
C-6 sulfonate intermediate, which undergoes hydrolysis in the presence of base to
release uracil (Fig. 7a) [63]. Unlike C, 5mC is resistant to deamination under the
same conditions [65]. Therefore, bisulfite treatment of genomic DNA can be used to
distinguish between methylated and unmethylated cytosines in the genome. Following bisulfite treatment, cytosines are deaminated and subsequently read as T, while
5mC will resist deamination and will be read as C (Fig. 7a). This unique property has
been used by various sequencing technologies that employ bisulfite conversion to
determine the locations of 5mC bases in the genomes of various species including
mammals, plants, and bacteria [66, 67].
Experimental Methodologies for Detection and Mapping of Epigenetic DNA Marks
499
spectrometry [29].
HPLC-ESI-MS/MS analysis of 4mC is challenging because it is present in
relatively low amounts but also has the same molecular mass and similar biophysical
properties as 5mC (Fig. 1), making it difficult to detect the two isomers separately.
HPLC resolution of 4mC and 5mC was achieved by gradient elution of a
C18 column under acidic conditions, allowing for 4mC quantification in
C. kristjanssonii containing high concentrations of 4mC [33].
3 Indirect Methods to Map Epigenetic DNA Modifications
Along the Genome
Mapping epigenetics DNA marks along the genome is essential for our understanding of their biological roles, interrelationships, and dynamics. The majority of
techniques used currently employ bisulfite conversion, modified bisulfite conversion, or antibody capture to help locate epigenetic DNA modifications. Although the
readout from these techniques is indirect, they have yielded a wealth of important
information over the years. Novel methodologies such as nanopore sequencing and
single-molecule real-time (SMRT) sequencing allow for a direct sequencing of
modified DNA and are discussed in Sect. 4.
3.1 Bisulfite Conversion to Distinguish Between C and 5mC
Early studies of chemical reactivity of native and modified DNA nucleosides have
revealed an important difference in chemical reactivity of C and 5mC [63, 64]. Upon
treatment with bisulfite, cytosine is readily deaminated to uracil, while much slower
reaction is observed for 5mC. The bisulfite reaction with cytosine proceeds through a
C-6 sulfonate intermediate, which undergoes hydrolysis in the presence of base to
release uracil (Fig. 7a) [63]. Unlike C, 5mC is resistant to deamination under the
same conditions [65]. Therefore, bisulfite treatment of genomic DNA can be used to
distinguish between methylated and unmethylated cytosines in the genome. Following bisulfite treatment, cytosines are deaminated and subsequently read as T, while
5mC will resist deamination and will be read as C (Fig. 7a). This unique property has
been used by various sequencing technologies that employ bisulfite conversion to
determine the locations of 5mC bases in the genomes of various species including
mammals, plants, and bacteria [66, 67].
Experimental Methodologies for Detection and Mapping of Epigenetic DNA Marks
499