Jayanth Krishnan
Mt. Sinai
ChIP-on-chip
7/13/09
Summary
What is ChIP-on-chip?
ChIP-on-chip, also known as genome-wide location analysis, is a technique that is used by scientists in
order to investigate Protein-DNA interactions. This technique combines elements from chromatin
immunoprecipitation (ChIP) with microarray technology (chip) hence giving it the name ChIP-on-chip.
ChIP-on chip is most widely used in order to identify where the binding sites are for a given protein.
Once these binding sites are found, scientists may have the opportunity to gain great knowledge on the
functions of various transcriptional regulators and perhaps identify many different functional elements
which include promoters, enhancers, sequences that control DNA replication etc. DNA replication and
recombination are also uses of ChIP-on chip, however the technique is most well known to identify
binding sites for given transcription factors. The discovery of the binding sites of the model organism
budding yeast or Saccharomyces cerevisiae was when the ChIP-on-chip technique was first successfully
used.
DNA microarrays – The technological platform for ChIP-on-chip
ChIP-on chip experiments are conducted on DNA microarrays. The only difference between a standard
microarray and a DNA microarray is DNA is being measured or somehow being used in the detection
system. A microarray is basically a tool that draws connections based on expression. It consists of a vast
number of microscopic spots of DNA oligonucleotides which have been bound at high density whose
purpose is to hybridize the cDNA. Basically using hybridization probes and fluorescent tags expression
of various genes can be observed. Regarding ChIP-on-chip experiments DNA sequences for a given
protein are isolated by immunoprecipitation, then parts of the sequences are hybridized by the
microarray and the protein binding sites can be identified.
A DNA Microarray Experiment
1. Prepare your DNA
chip using your
chosen target DNAs.
2. Generate a
hybridization solution
containing a mixture
of fluorescently
labeled cDNAs.
3. Incubate your
hybridization mixture
containing fluorescently
labeled cDNAs with your
DNA chip.
4. Detect bound cDNA
using laser technology
and store data in a
computer.
5. Analyze data
using computational
methods.
http://www.ncbi.nlm.nih.gov/About/primer/microarrays.html
Pros and Cons of ChIP-on-chip
Although ChIP-on-chip can identify binding sites and chromatin modifications it can be quite expensive.
DNA microarrays are often very expensive for a laboratory to afford. Another disadvantage in the
technique is in order to obtain meaningful results experiments must be run several times. Also the size
of the DNA fragments is a constraint (usually the minimum size is around 200bp). Also this technique has
been limited only to the mammalian genomes. Antibodies are also a limiting factor, only certain
antibodies are recognized by this technique.
The ChIP-on-chip experiment
Wet Lab
Dry Lab
Part 1: Wet Lab
1) Choose a protein and crosslink it to DNA site (formaldehyde fixation usually used)
2) Cells must be Lysed and DNA is sonicated and a Protein-DNA complex is formed
3) Complex is filtered using antibody complementary to the chosen protein
(immunoprecipitation)
4) The complex is reverse cross linked; purifying the DNA
5) Fragments are labeled with a fluorescent tag
6) Fragments poured into the microarray where they essentially go and find their
complementary single stranded DNA once found they hybridize and form a double stranded
DNA fragment.
7) Lastly base pairs attach at the snip sites
Part 2: Dry Lab
1) Areas of successful hybridization will emit fluorescence which is captured by a camera.
2) Data is then converted into numbers
3) Algorithms that normalize the data will provide proper statistical evidence to the
experimenter
4) They using a peak software such as mpeak one can identify where the protein binding sites
are located.
Extra Links
PubMed Home
PubMed Central Homepage
DNA microarray - Wikipedia, the free encyclopedia
ChIP-on-chip - Wikipedia, the free encyclopedia
Immunoprecipitation - Wikipedia, the free encyclopedia
Oligonucleotide - Wikipedia, the free encyclopedia
Saccharomyces cerevisiae - Wikipedia, the free encyclopedia
Proteome - Wikipedia, the free encyclopedia
Northern blot - Wikipedia, the free encyclopedia
Telomerase - Wikipedia, the free encyclopedia
CHIP_zmdl_1036.pdf (application/pdf Object)
Mining ChIP-chip data for transcription factor and cofactor binding sites -- Smith et al.
21 (Supplement 1): i403 -- Bioinformatics
Mapping of transcription factor binding regions in mammalian cells by ChIP:
Comparison of array- and sequencing-based technologies
Transcriptional Regulatory Network
Microarrays Factsheet
ChIP on Chip
Ming's Homepage
CHIP Bioinformatics | Mapper
lecture13.pdf (application/pdf Object)
Michael J. Buck
cmb.2006.13.579 (application/pdf Object)