Name: chip methods for dna-protein binding
Uploaded: 2021-04-07T12:12:00.000Z
Duration: 2 min 36 s
Channel: JoVE

K12 Standards

Tennessee Academic Standards

High School

Biology

links-among-engineering-technology-science-and-society

Links Among Engineering, Technology, Science, and Society

BIO1.ETS2.1

Chromatin Immunoprecipitation- ChIP

Chromatin immunoprecipitation, abbreviated as ChIP, is a technique for studying the protein-DNA interactions that regulate gene expression.
In eukaryotes, DNA is wrapped around histone proteins and forms a complex known as a nucleosome, which further groups together into a structure known as chromatin to aid in the tight packaging of DNA in the cells.
Gene expression is regulated through histone modifications that uncoil or tighten&#160; these structures as well as proteins that associate with cis-regulatory regions on the DNA.
In ChIP, the chromatin is broken down and antibodies that bind to histone modifications or regulatory proteins, are used to isolate the target molecules with the associated&#160; DNA.
The first step in this process is to crosslink the protein to the DNA with the help of a crosslinking agent, such as formaldehyde.&#160; This immobilizes the protein on the DNA marking the binding site of the protein. After crosslinking, the chromatin is mechanically sheared into short fragments ranging from 100 to 200 base pairs. This process is known as X-ChIP.
An alternative method is N-ChIP, in which nucleases directly digest DNA from the chromatin into short fragments, without any prior cross-linking.
Immunoprecipitation is carried out by introducing antibodies that target the regulatory proteins in the solution containing sheared or digested DNA. These antibodies are linked to agents that help in selective isolation.
One common method is linking the antibodies to magnetic beads. A magnet is used to isolate the antibodies along with any bound molecules.
The complex is rinsed to wash off any loosely associated contaminants.&#160; The target molecules are detached with the help of a detergent, such as SDS.
In the case of X-ChiP, crosslinking is reversed with the help of elevated temperatures, and the associated regulators or histones are degraded with the help of proteases, leaving behind the&#160; DNA.&#160;
Sequencing of the associated DNA can help identify the cis-regulatory sequence that the protein of interest was bound to or the gene whose expression is modulated by a particular histone modification.

chromatine-immunoprecipitatie - chip

Chromatine-immunoprecipitatie, of ChIP, is een op antilichamen gebaseerde techniek die wordt gebruikt om plaatsen op DNA te identificeren die binden aan interessante transcriptiefactoren of histoneiwitten. Het helpt ook bij het bepalen van het type histonmodificaties zoals acetylering, fosforylering of methylering.

Soorten ChIP

ChIP kan worden onderverdeeld in twee typen: X-ChIP en N-ChIP. X-ChIP omvat in vivo verknoping van histonen en regulerende eiwitten aan DNA, het fragmenteren van het DNA door sonicatie en het isoleren van de eiwit-DNA-complexen door ze te immunoprecipiteren met specifieke antilichamen. Aan de andere kant omvat N-ChIP geen verknoping van DNA aan eiwitten, en wordt de vertering uitgevoerd met behulp van nucleasen.

Stroomafwaartse analyse van DNA-eiwitinteracties

Na het isoleren van het DNA van interesse kunnen technieken zoals PCR, microarrays of Southern blot worden gebruikt voor analyse. Als alternatief kan dit DNA worden gebruikt voor diepe sequencing, bekend als ChIP-Seq. ChIP kan worden aangepast met behulp van andere methodologieën voor verschillende analyses, zoals ChIA-PET, een techniek die de principes van ChIP combineert met het vastleggen van chromosoomconformatie om chromatine-interacties over lange afstand te detecteren die worden gemedieerd via een eiwit van belang; enChIP, een techniek die gebruik maakt van het CRISPR/Cas9-systeem om specifieke genomische regio's te targeten, en RIP-ChIP/RIP-Seq, een modificatie van ChIP die wordt gebruikt om eiwit-RNA-interacties te analyseren.

Vergelijking tussen N-ChIP en X-ChIP

N-ChIP en X-ChIP hebben hun eigen voor- en nadelen. N-ChIP resulteert in sterkere antilichaambinding en efficiënte en zeer specifieke immunoprecipitatie. N-ChIP is echter alleen geschikt in het geval van strak gebonden eiwitten zoals histonen, omdat transcriptiefactoren tijdens de verwerking los kunnen raken. Bovendien wordt niet al het door nuclease gedigereerde chromatine oplosbaar gemaakt, wat resulteert in het missen van bepaalde fracties van het monster. X-ChIP is een uitstekende methodologie voor het bestuderen van transcriptiefactoren die vanwege de verknopingsstap niet stevig aan het DNA zijn gebonden. De X-ChIP-test is ook gevoeliger dan N-ChIP en vereist kleinere hoeveelheden monsters en antilichamen. Nadelen van X-chip zijn onder meer mogelijke problemen bij fragmentatie als gevolg van overmatige verknoping en valse positieven als gevolg van de verknoping van voorbijgaande DNA-eiwitinteracties.

Chromatine-immunoprecipitatie - ChIP

Chromatin immunoprecipitation, or ChIP, is an antibody-based technique used to identify sites on DNA that bind to transcription factors of interest or ...

Education

JoVE Core

Molecular Biology

Moleculaire biologie

Unit 2

Onderdeel 2

Analyzing Gene Expression and Function

Analyse van genexpressie en functie

KEY TERMS AND CONCEPTS

BELANGRIJKE TERMEN EN CONCEPTEN

chip methods for dna-protein binding

Chromatin immunoprecipitation, or ChIP, is an antibody-based method for finding where proteins bind to DNA. It is used to identify DNA sites bound by transcription factors or histone proteins. ChIP also helps show which histone changes are present, including acetylation, phosphorylation, and methylation.

ChIP is commonly divided into two forms: X-ChIP and N-ChIP. X-ChIP first cross-links proteins to DNA inside the cell, then breaks the DNA into pieces by sonication. The protein-DNA complexes are then isolated by immunoprecipitation with specific antibodies. N-ChIP does not use cross-linking. Instead, it uses nucleases to digest the chromatin.

After the DNA of interest is isolated, it can be analyzed in several ways. PCR, microarrays, and Southern blot can be used to study the captured DNA. The sample can also be sent for deep sequencing, which is called ChIP-Seq.

ChIP can also be adapted for other kinds of analysis. ChIA-PET combines ChIP with chromosome conformation capture to detect long-range chromatin interactions that are mediated by a protein of interest. enChIP uses the CRISPR/Cas9 system to target specific genomic regions. RIP-ChIP and RIP-Seq are used to study protein-RNA interactions.

N-ChIP and X-ChIP each have strengths and limits. N-ChIP gives stronger antibody binding and efficient, highly specific immunoprecipitation. It works best with tightly bound proteins such as histones, but transcription factors may be lost during processing. Some nuclease-digested chromatin may also remain insoluble, so parts of the sample can be missed.

X-ChIP is useful for studying transcription factors that do not bind tightly to DNA because it includes a cross-linking step. It is also more sensitive than N-ChIP and uses less sample and fewer antibodies. Its drawbacks include possible problems with fragmentation if cross-linking is too strong and false positives from transient DNA-protein interactions.

ChIP Methods for DNA-Protein Binding

in vitro mutagenesis

In Vitro Mutagenesis for Gene Analysis

In vitro mutagenesis is a lab method used to change DNA and study gene function. It helps scientists test how specific DNA changes affect a gene’s activity and the traits it controls. A common approach is to make a known change in a DNA sequence and then examine the result. This can be done by deleting, inserting, or altering selected bases in a gene. The method is useful for linking a DNA change to a biological effect. Researchers use in vitro mutagenesis in molecular biology, genetics, and...

genetic screens

Using Genetic Screens to Find Disease Genes

Genetic screens are tools used to find genes and mutations linked to specific traits. They are also used to identify people or groups at risk for genetic diseases. That can support early intervention, targeted therapy, and reproductive choices. Forward genetic screens, also called classical screens, start with random DNA changes in an organism. Researchers may use radiation, mutagens, or added bases to create mutations that lead to visible phenotype changes. The mutant is then inbred so...

test cross

Test Cross in Genetic Inheritance

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complementation tests

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A complementation test uses a simple cross to find out whether two mutations are in the same gene or in different genes. It helps show how mutations are arranged on chromosomes. Edward Lewis first performed this test in the 1940s while studying fruit flies. To do the test, organisms heterozygous for different mutations are crossed pairwise in all combinations. If the mutations are in different genes, they can complement each other. In that case, each mutation provides the missing genetic...

single nucleotide polymorphisms-snps

SNPs as Genetic Markers and Variants

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bacterial transformation

Bacterial Transformation in the Lab

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transgenic organisms

Making Transgenic Organisms

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reproductive cloning

Cloning an Organism by Reproduction

Reproductive cloning creates a new organism that is genetically the same as the donor. It is different from cloning genes or cells because the goal is to make a whole living individual. This process usually relies on somatic cell nuclear transfer. In this method, the nucleus from a body cell is placed into an egg cell that has had its own nucleus removed. The egg can then develop into an embryo with DNA from the donor cell. Reproductive cloning is used to produce a copy of an organism, such...

crispr

CRISPR Gene Editing Basics

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experimental rnai

RNAi for Gene Silencing and Discovery

RNA interference (RNAi) is a gene-silencing process used to reduce gene expression. It can block transcription or trigger RNA degradation. Scientists first discovered this mechanism in plants in 1998 through Andrew Fire and Craig Mello. RNAi has since been found in many eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. Researchers use it as a practical tool to study gene function. It can show what a gene does without permanently changing the organism’s DNA. <br...

reporter genes

Reporter Genes in Cells and Plasmids

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in-situ hybridization

ISH Probes for DNA and RNA Detection

In situ hybridization (ISH) uses labeled probes to detect and localize specific DNA or RNA molecules in cells, tissues, or tissue sections. The probe is a complementary strand of DNA or RNA that binds to a matching nucleotide sequence in the sample. Since its first use in 1969 for nucleic acid studies, ISH has become an important tool in research and in clinical diagnosis. Several probe types can be used in ISH. These include single-stranded DNA probes, double-stranded DNA probes, antisense...

chromatin immunoprecipitation- chip

Chromatin immunoprecipitation, or ChIP, is an antibody-based method for finding where proteins bind to DNA. It is used to identify DNA sites bound by transcription factors or histone proteins. ChIP also helps show which histone changes are present, including acetylation, phosphorylation, and methylation. ChIP is commonly divided into two forms: X-ChIP and N-ChIP. X-ChIP first cross-links proteins to DNA inside the cell, then breaks the DNA into pieces by sonication. The protein-DNA complexes...

synthetic biology

Synthetic Biology in Crops and Cells

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ribosome profiling

Ribosome Profiling for Translation Snapshot

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transgenic plants

Transgenic Plants for Better Crops

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Gene Interaction Maps in Epistasis

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genome-wide association studies-gwas

Using SNP Patterns to Link Disease Risk

Genome-wide association studies, or GWAS, look for common SNPs, or single nucleotide polymorphisms, that may be linked to disease. Researchers compare how often a SNP appears in people with a disease and in people without it. When a SNP is seen more often in the disease group, it is considered associated with that disease. GWAS uses chi-square analysis to test the chance that an allele is linked to the disease. This approach does not require scientists to first find the target gene behind the...

key terms and concepts

Explore how ChIP detects DNA-protein binding, compares X-ChIP and N-ChIP, and shows how captured DNA can be analyzed by PCR, sequencing, and more.

ChIP Methods for DNA-Protein Binding | JoVE K12

molecular biology

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Obtain, evaluate, and communicate information on how molecular biotechnology may be used in a variety of fields.
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BIO1.ETS2.2

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BIO1.ETS2.3

ChIP Methods for DNA-Protein Binding

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