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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