DNA Technology,

Functional
Genomics &
Proteomics
Lecture 2
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By:
Nahla Osama, PhD
Investigating Histones
modification; Practical
approach
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ChIP-on-chip
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ChIP-on-chip

§ ChIP-on-chip, also known as ChIP-chip, is a technology that

combines chromatin immunoprecipitation with DNA
microarray.

§ Like regular ChIP, ChIP-on-chip is used to investigate

interactions between proteins and DNA in vivo and to
study many of the epigenomic phenomena.
Crosslinking and lysis

• In vivo covalent crosslinking stabilizes protein-DNA interactions for analysis at a specific

point in time.

• Formaldehyde is commonly used for cross-linking, while glycine is added to quench the
reaction.

• The crosslinkers permeate into the intact cells and lock the protein-DNA complexes together,
enabling analysis.

• Next, the cell membrane is permeabilized with lysis solutions, the cellular components are
liberated, and the protein-DNA complexes become soluble.

• Cytosolic proteins are removed to reduce background signal and increase sensitivity.

Note: The ChIP assay can be stopped at this point. After crosslinking, quenching, and
washing the cell pellet, the lysate can be stored at -80°C.
Chromatin fragmentation

The nuclear material must be fragmented, and this fragmentation is key for good ChIP
resolution, ideally resulting in fragment sizes between 200 and 800 base pairs.

Shearing is one of the most difficult steps to control. It can be achieved by sonication and/or
nuclease/enzymatic digestion, each with limitations and benefits.

Sonication requires significant hands-on time, but is ideal for hard-to-lyse cells, while
enzymatic digestion is relatively hands-off, accommodates multiple sample processing, but is
not random.
Micrococcal nuclease (MNase) digestion of chromatin cuts linker DNA between
neighboring nucleosomes and in this way generates mononucleosomes.
This enzyme requires Ca2+ as a cofactor for its activity.
Immunoprecipitation
• Antibodies are used to capture the protein of interest in the protein-DNA complex, and they
are a crucial factor in a successful experiment.
• Antibodies immunoprecipitate and isolate the protein from the nuclear components,
eliminating the unrelated cellular material.
• Purification of the resulting antibody-protein-DNA complex is accomplished using antibody-
binding beads. The bead-antibody-protein-DNA complex is washed extensively after
incubation, purified, and the protein-DNA complex eluted.

•Not all antibodies work for ChIP. Choose antibodies already shown to work for ChIP or IP, or
validate their use in this application. IHC and IF are reasonably good indicators the antibody
could work in ChIP
•Titer the antibody concentration for the best signal-to-noise ratio
•The wash buffer concentration can affect protein binding, signal-to-noise-ratio, and background
•Use a ‘no antibody’ control to distinguish ChIP signal from background
Preparation of DNA
Now that chromatin fractions containing your protein of interest have been isolated, the
crosslinks need to be reversed and the DNA purified.

The reversal of crosslinking is typically done through extensive heat incubation and/or
digestion of the protein component.

In order to obtain a purer DNA sample, treatment with RNase A is recommended.

To purify the DNA from the remaining proteins, phenol:chloroform extraction or spin columns
for DNA purification should be used.

Note: ChIP can be stopped at this point. After crosslink reversal and/or DNA
purification, samples can be stored at -20°C.
Quantitation of DNA

At this step, the purified DNA products are quantitated by qPCR, which can be achieved
using the NanoDrop spectrophotometer, Qubit Fluorometer, or other spectrophotometric
method.

The NanoDrop Spectrophotometer provides a very simple way to quantitate DNA as well as
assess for any solvent contaminants using the 230/260 nm absorbance ratio.

The Qubit Fluorometer can accurately quantitate low levels of purified DNA and be used to
normalize samples prior to qPCR or for use in sequencing applications.
Briefly:
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§ Modified chromatin is first purified by immunoprecipitating cross-linking
chromatin with an antibody that is specific to a particular histone
modification.

§ Then DNA is amplified to obtain sufficient DNA (labelled with one color).

§ The color-labelled ChIP DNA is hybridized to a DNA microarray with the

control DNA prepared from input chromatin and labelled with a different
color.

§ Subsequently, the microarray probes can be mapped to the genome to

yield genomic coordinates.

§ However, most ChIP on chip experiments still use polyclonal antibodies,

which are different in specificity between batches. The generation and use
of monoclonal antibodies for ChIP on chip is therefore an important goal.
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ChIP-SAGE
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ChIP-Seq
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§ ChIP-seq, also known as ChIP-sequencing, is one of the

most exciting recent advances in technologies for
studying epigenetic phenomena at a genomic scale
which relies on the combination of ChIP experiments
with high-throughput sequencing.

§ ChIP-seq combines ChIP with massively parallel DNA

sequencing to identify the binding sites of DNA-
associated proteins.