Trends Biomedical Science
04c Finding Epigenetics Changes –
stimulus questions
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We can treat DNA with sodium bisulfite.
Unmethylated cytosine is changed to uracil,
while methylated cytosine is unaffected.
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How can we use this to find
methylated DNA?
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What will happen to the these DNA sequences
when we treat them with bisulfite?
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What would happen if you PCR amplified the
DNA?
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What would happen if you PCR amplified the DNA?
Cytosine residues at originally methylated
positions, and thymine residues at originally
unmethylated position (that were converted to
uracil).
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What would we have to think about
when we chose the primers for the
PCR?
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What would we have to think about when
we chose the primers for the PCR?
Primers used during this step do not
contain CpG sites (the common target of
cytosine methylation), so the
amplification works for both methylated
and unmethylated DNA.
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How could we use restriction enzymes to tell us
about the methylation?
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How could we use restriction enzymes to tell us
about the methylation?
PCR products are treated with a restriction
enzyme (e.g. BstUI), which will only cut sites
that were originally methylated (CGCG), while
leaving sites that were originally
unmethylated (TGTG).
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https://en.wikipedia.org/wiki/Combined_bisulfite_restriction_analysis#/media/File:Cobra_workflow.svg
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Quantification
The digested fragments are then separated
by polyacrylamide gel electrophoresis
with the expected appearance of bands
corresponding to a single large
undigested fragment, and many smaller
bands corresponding to digested
fragments.
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Quantitative amount of DNA in these
bands can be measured.
The methylation percentage of the original
sample can be calculated by:
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This technique is called
Combined Bisulfite Restriction
Analysis (or COBRA)
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Uses
COBRA has been in many applications such as:
• screening for DNA methylation changes
at gene promoters in cancer studies,
• detecting altered methylation patterns at
imprinted genes, and
• characterizing methylation patterns in the
genome during development in mammals.
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In medicine, COBRA canhelp diagnose
human disease involving incorrect DNA
methylation. COBRA is used in the
diagnosis of the genetic imprinting
disorder Russell-Silver syndrome where
hypomethylation of the imprinted gene
H19 is responsible for the disorder in up
to 50% of patients.
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What other ways could we use the bisulfite
technique to find methylated sites?
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Non-methylation-specific PCR based methods
• Direct sequencing
• Pyrosequencing
• Methylation-sensitive single-strand
conformation analysis (MS-SSCA)
• High resolution melting analysis (HRM)
• Methylation-sensitive single-nucleotide
primer extension (MS-SnuPE)
• Base-specific cleavage/MALDI-TOF
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Methylation-specific PCR (MSP)
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Immunological techniques.
Especially immunoprecipitation
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Methylated DNA immunoprecipitation (MeDIP
or mDIP) is a large-scale (chromosomeor genome-wide) purification technique in
molecular biology that is used to enrich
for methylated DNA sequences.
An antibody raised against 5-methylcytosine
(5mC) is used to enrich methylated DNA
fragments .
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Can you explain
what happens
here?
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Applications of MeDIP
Inactive X-chromosome in females is
hypermethylated on a chromosome wide level
(MeDIP coupled with microarray).
Colon and prostate cancer cells using MeDIP-chip.
Gives genome-wide analysis of genes lying in
hypermethylated regions – there is de novo
methylation in cancer cells.
High resolution methylome mapping in Arabidopsis
using MeDIP-chip.
MeDIP-chip to investigate human breast cancer for
methylation associated silencing. Found the
inactivation of the HOXA gene cluster.
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Restriction Landmark Genomic Scanning
(or RLGS)
Lets us quickly see thousands of restriction sites.
Using a combination of restriction enzymes
some of which are specific to DNA
modifications, the technique can be used to
see differences in methylation levels across
the genome of a given organism.
RLGS can be used to find the methylation of
thousands of CpG islands—simultaneously.
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RLGS becomes very useful when doing wholegenome scans, and can do the work of
thousands of polymerase chain reactions at
once.
It easily finds changes from normal, so is very
effective in finding hyper/hypomethylation
in tumors, deletions or amplifications of
genes, or changes in gene expression
throughout the development of an organism.
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Chromatin immunoprecipitation (ChIP)
• used to find DNA binding sites on
the genome for a particular protein of
interest.
• gives a picture of the protein–DNA
interactions that occur inside the nucleus of
living cells or tissues.
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https://en.wikipedia.org/wiki/Chromatin_immunoprecipitation#/media/File:Chromatin_immunoprecipitation_
sequencing.svg
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DNA-binding proteins (including transcription
factors and histones) in living cells can be crosslinked to the DNA that they are binding.
By using an antibody that is specific to a DNA binding
protein, we can immunoprecipitate the protein–DNA
complex out of cellular lysates.
The crosslinking is often done by
applying formaldehyde to the cells (or tissue).
Cells are then lysed and the DNA is broken into pieces
0.2–1.0 kb in length by sonication.
Immunoprecipitation purifies protein–DNA complexes.
The purified protein–DNA complexes are then heated to
reverse the formaldehyde cross-linking , allowing the
DNA to be separated from the proteins.
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The identity and quantity of the DNA can then be found
by PCR.
We must have an idea which genomic region is being
targeted in order to generate the correct PCR primers.
We could get around this by cloning the isolated genomic
DNA into a plasmid vector and then using primers that
are specific to the cloning region of that vector.
To find where the protein binds on a genome-wide scale,
a DNA microarray can be used (ChIP-on-chip or ChIPchip).
ChIP-Sequencing can localize protein binding sites in a
high-throughput, cost-effective fashion.
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