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ChIP-on-chip
Chromatin immunoprecipitation (ChIP)With
Microarray technology (chip)
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ChIPChIPis used to determine whether a given protein binds to a
specific DNA sequence in vivo.
This technique allows us to map minute-by-minute changes at
a single promoter, follow a single transcription factor over the
entire human genome.
Specific DNA sites in direct physical interaction with
transcription factors and other proteins can be isolated
by chromatin immunoprecipitation
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o Isolation of total chromatin.
o Fragmentation of the chromatin -
Fragmentation of the chromatin is required to make
interactions accessible to antibody reagents. To fragment
chromatin, you can either sonicate it or digest it with usingmicrococcal nuclease.
Sonication- Sonication is the act of applying sound
energy to agitate particles in a sample.Sonication is often used to disrupt cell membranes &
release cellular contents.
The ChIPprocedure consists of the following steps:
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o Immunoprecipitation of the resulting chromatin fragments.
Immunoprecipitation- Immunoprecipitation is the
technique of precipitating protein antigen out of
solution using an antibody that specifically binds to
that particular protein.
o Analysis of the immunoprecipitate fraction to determine the
amount of a target chromatin.
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Wet Lab Workflow - 1
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1- POI (protein of interest) is cross-linked with the DNA site it binds
to it in vivo environment. formaldehyde is used for fixation thatis reversible with heat.
2- The cells are lysed and the DNA is sheared by sonication or using
micrococcal nuclease . This results in double-stranded chunks ofDNA fragments, (200 - 1,000bp).
3 -Antibodies against the POI are used to immunoprecipitate the
protein-DNA complex.
Wet Lab Portion
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4- The immune complexes are isolated by the use of agarose or
magnetic beads.
5- The POI-DNA complexes are reverse cross-linked by high salt
and heat treatment and the DNA are purified.
6- The fragments are poured over the surface of the DNA microarraywhich is spotted with short, single-stranded sequences that cover
the genomic portion of interest. Whenever a labeled fragment
"finds" a complementary fragment on the array, they will
hybridize and form again a double-stranded DNA fragment
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Microarray Technique
A DNA microarray-based analysis is a complex multistep process
involving numerous specific equipments, and requiring a strongexpertise in various areas, including molecular biology, image
analysis, computing, and statistics.
Microarray is a powerful tool for genome analysis. It gives the
global view of the genome analysis in a single experiment. Dataanalysis in the Microarray is a vital part as this part influences the
final result.
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Wet Lab Workflow - 2
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Dry Lab Workflow
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Dry Lab Work
1- After a sufficiently large time frame to allowhybridization, the array is illuminated with fluorescencelight.
2- Those probes on the array that are hybridized to one of
the labeled fragments will emit a light signal which canbe captured by a camera. This image contains all raw datafor the remaining part of the workflow.
3- This raw data, encoded as false color image needs to be
converted to numerical values before the actual analysiscan be done.
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4- The analysis and information extraction of the raw
data often remains the most challenging part for ChIP-on-chip experiments.
5- Problems arise throughout this portion of the
workflow, ranging from the initial chip read -out, tosuitable methods to subtract background noise, and
finally to appropriate algorithms that normalize the
data and make it available for subsequent statistical
analysis, which then hopefully lead to a better
understanding of the biological question sought to
answer.
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Data Analysis The captured fluorescence signals from the array are
normalized, using control signals.
Numerical and statistical tests are applied to control dataand to identify POI-enriched regions along the genome.
The following three methods are used widely:
Median percentile rank, Single-array error and Sliding -window. These methods generally differ in a way howlow-intensity signals are handled, how much backgroundnoise is accepted, and which trait for the data isemphasized during the computation.
These regions are analyzed further. If, for example, thePOI was a transcription factor, such regions wouldrepresent its binding sites.
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Application Protein binding site localization . Transcription factor binding
sites and gene regulatory networks have been successfully
identified in mammalian and yeast genomes.
Promoter DNA methylation in Haman genome DNAmethylation is an epigenetic mechanism of gene transcriptionregulation in eukaryotic cells. It has been implicated in anumber of biological process (e.g. X-chromosome inactivation,
gene imprinting) and di seases like cancer (e.g. abe rrant DNAmethylation causes silencing of tumor suppressor genes andpromotes chromosomal instability in cancer cells).
Characterization of histone modification. Post translationalmodification of histones (methylation, acetylation and
phosphorylation) are essential epigenetic marks to determinechromatin structure and its role in gene regulation.
Whole transcriptomics including identification of novelexpressed transcripts.
To estimate the proportion of undiscovered enriched probes.
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Application
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Thank you
Anuja V. Nawarange
MBI-08015