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ChIP-seq Methodology Overview

ChIP-seq is a method used to identify DNA regions bound by specific proteins, crucial for understanding transcriptional regulation and chromatin modifications. The methodology involves crosslinking DNA-binding proteins, lysing cells, and purifying DNA fragments, with careful selection of antibodies and controls to ensure data quality. The document also outlines a stepwise guide for utilizing genome data to identify diagnostic markers in major crops and pathogens.

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27 views7 pages

ChIP-seq Methodology Overview

ChIP-seq is a method used to identify DNA regions bound by specific proteins, crucial for understanding transcriptional regulation and chromatin modifications. The methodology involves crosslinking DNA-binding proteins, lysing cells, and purifying DNA fragments, with careful selection of antibodies and controls to ensure data quality. The document also outlines a stepwise guide for utilizing genome data to identify diagnostic markers in major crops and pathogens.

Uploaded by

Vijaya laxmi
Copyright
© All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPTX, PDF, TXT or read online on Scribd

CHIP seq

INTRODUCTION
 Chromatin immunoprecipitation (ChIP) followed by highthroughput DNA sequencing
(ChIP-seq) is a widely used approach to identify the DNA regions bound by a specific
protein in vivo.
 Genome-wide mapping of protein–DNA interactions and epigenetic markers is essential
for full understanding of transcriptional regulation. Chromatin immunoprecipitation
(ChIP) is an effective method for isolating the DNA fragments binding by a specific
protein in vivo.
 ChIP-seq has become a popular method for profiling chromatin modifications and
transcription factor (TF)-binding sites in eukaryote genomes.

 Chromatin immunoprecipitation (ChIP) is an effective method for isolating the DNA


fragments binding by a specific protein in vivo.
Methodology
DNA-binding protein is crosslinked to DNA in vivo by treating cells with formaldehyde.
the cells are lysed and chromatin is sheared by sonication into small fragments followed by
immunoprecipitation of protein–DNA complexes using the specific protein antibodies.
NOTE- To ensure high-quality data, the choices of antibody and negative control are critical. Many
antibodies are commercially available against the epitope tags, such as GFP, MYC or FLAG, which
are usually fused to interest proteins in transgenic plants. and non-transgenic plants or plants
only expressing the tags are the perfect negative controls.
last step is reversing the crosslinks to release the DNA fragments from proteins followed by
purification of the released DNA fragments.
NOTE- The quality of purified DNA can be tested by quantitative real-time PCR (ChIPqPCR) to
determine the enrichment of genomic regions of interest.
ENCODE encodes ChIP-seq overview information covering experimental
design to data analysis and contains some published standards to achieve
each step in ChIP-seq analysis.
• For example, Haring et al. (2007) used 3% formaldehyde to crosslinkprotein to DNA
region in Maize (Zea mays) while subsequent publications used 1% formaldehyde in
Arabidopsis thaliana.
• crosslinking protein–DNA with formaldehyde and quenching with glycine
• Reversal of protein–DNA crosslinking is typically achieved by heating (usually 65 C) in
the presence of high salt concentrations (example 5 M NaCl and 20% SDS) or treating
with proteinase K at 37 C
Stepwise guide
1. Identify the major diseases in major crops for which well defined genome data should be
available on genome datasets.
2. Download the genome of-
• Target crops
• Pathogen
3. Identify Unique Pathogen Sequences
• Align Pathogen and Host Genomes and compare the pathogen genome against: The host
genome and other closely related pathogens.
4. Select Regions for Diagnostic Markers
Select sequences with high specificity and minimal variation within
the target pathogen.

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