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

Иммунопреципитация хроматина, сокращенно ChIP, является методом изучения белково-ДНК-взаимодействий, которые регулируют экспрессию генов.
У эукариот ДНК оборачивается вокруг белков гистонов и образует комплекс, известный как нуклеосома, который в дальнейшем группируется в структуру, известную как хроматин, чтобы помочь плотной упаковке ДНК в клетках.
Экспрессия генов регулируется с помощью модификаций гистонов, которые разматывают или ужесточают эти структуры, а также белков, которые связываются с цис-регуляторными областями ДНК.
В ChIP хроматин расщепляется, и антитела, которые связываются с модификациями гистонов или регуляторными белками, используются для выделения молекул-мишеней с ассоциированной ДНК.
Первым шагом в этом процессе является сшивание белка с ДНК с помощью сшивающего агента, такого как формальдегид. Это обездвиживает белок на ДНК, обозначая место связывания белка. После сшивки хроматин механически разрезается на короткие фрагменты в диапазоне от 100 до 200 пар оснований. Этот процесс известен как X-ChIP.
Альтернативным методом является N-ChIP, при котором нуклеазы непосредственно переваривают ДНК из хроматина в короткие фрагменты, без какой-либо предварительной сшивки.
Иммунопреципитация осуществляется путем введения антител, нацеленных на регуляторные белки в растворе, содержащем срезанную или переваренную ДНК. Эти антитела связаны с агентами, которые помогают в селективной изоляции.
Одним из распространенных методов является связывание антител с магнитными шариками. Магнит используется для выделения антител вместе с любыми связанными молекулами.
Комплекс промывается, чтобы смыть любые слабо связанные загрязнения. Целевые молекулы отделяются с помощью моющего средства, такого как SDS.
В случае X-ChiP сшивание обращается вспять с помощью повышенных температур, а связанные с ним регуляторы или гистоны разрушаются с помощью протеаз, оставляя позади ДНК. 
Секвенирование ассоциированной ДНК может помочь идентифицировать цис-регуляторную последовательность, с которой был связан интересующий белок, или ген, экспрессия которого модулируется определенной модификацией гистона.

<ol>
	<li>Гейд,., и Калваколану, Д. В. (2012). Иммунопреципитация хроматина как инструмент для анализа активности транскрипционного фактора. Методы молекулярной биологии (Клифтон, Нью-Джерси), 809, 85–104. https://doi.org/10.1007/978-1-61779-376-9_6</li>
	<li>Нельсон, Д. Д., Денисенко, О., и Бомштык, К. (2006). Протокол для метода быстрой иммунопреципитации хроматина (ChIP). Протоколы природы, 1(1), 179–185. DOI:10.1038/nProt.2006.27</li>
	<li>Дас,. М., Рамачандран, К., ВанВерт, Д., и Сингал, Р. (2004). Иммунопреципитация хроматина. Биотехнологии, 37(6), 961-969.</li>
</ol>

Иммунопреципитация хроматина - chip

Иммунопреципитация хроматина, или ChIP, представляет собой метод на основе антител, используемый для идентификации участков на ДНК, которые связываются с интересующими транскрипционными факторами или гистоновыми белками. Это также помогает определить тип модификаций гистонов, таких как ацетилирование, фосфорилирование или метилирование. <h4>Виды ChIP</h4> ChIP можно разделить на два типа – X-ChIP и N-ChIP. X-ChIP включает в себя перекрестное связывание гистонов и регуляторных белков с ДНК in vivo, фрагментацию ДНК путем обработки ультразвуком и выделение комплексов белок-ДНК путем иммунопреципитации их специфическими антителами. С другой стороны, N-ChIP не предполагает сшивания ДНК с белками, а расщепление осуществляется с помощью нуклеаз. <h4>Последующий анализ взаимодействия ДНК и белка</h4> После выделения интересующей ДНК для анализа можно использовать такие методы, как ПЦР, микрочипы или Саузерн-блоттинг. Альтернативно, эту ДНК можно использовать для глубокого секвенирования, известного как ChIP-Seq. ChIP можно модифицировать с использованием других методологий для различных анализов, таких как ChIA-PET, метод, который сочетает в себе принципы ChIP с захватом конформации хромосом для обнаружения дальних взаимодействий хроматина, опосредованных интересующим белком; enChIP, метод, который использует систему CRISPR/Cas9 для нацеливания на определенные области генома и RIP-ChIP/RIP-Seq, модификацию ChIP, используемую для анализа взаимодействий белок-РНК. <h4>Сравнение N-ChIP и X-ChIP</h4> N-ChIP и X-ChIP имеют свои преимущества и недостатки. N-ChIP приводит к более сильному связыванию антител и эффективной и высокоспецифичной иммунопреципитации. Однако N-ChIP подходит только в случае прочно связанных белков, таких как гистоны, поскольку факторы транскрипции могут отсоединяться во время процессинга. Кроме того, не весь расщепленный нуклеазой хроматин растворяется, что приводит к потере определенных фракций образца. X-ChIP — отличный метод для изучения факторов транскрипции, которые не связаны прочно с ДНК из-за стадии перекрестного сшивания. Анализ X-ChIP также более чувствителен, чем N-ChIP, и требует меньшего количества образцов, а также антител. Недостатки X-ChIP включают возможную сложность фрагментации из-за избыточного сшивания и ложноположительных результатов из-за перекрестного сшивания взаимодействий временных белков ДНК.

Иммунопреципитация хроматина - ChIP

Иммунопреципитация хроматина, или ChIP, — это метод на основе антител, используемый для идентификации участков ДНК, которые связываются с факторами транскрипции, представляющими интерес или ...

Education

JoVE Core

Molecular Biology

Молекулярная биология

Unit 2

Раздел 2

Analyzing Gene Expression and Function

Анализ экспрессии и функций генов

KEY TERMS AND CONCEPTS

ОСНОВНЫЕ ТЕРМИНЫ И ПОНЯТИЯ

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

A test cross is a genetics tool used to determine the genotype of an organism with a dominant trait. It helps reveal whether the dominant individual is homozygous or heterozygous by crossing it with a recessive individual. The outcome of the cross shows the hidden genotype. If any offspring show the recessive trait, the dominant parent must carry one recessive allele. If all offspring show the dominant trait, the dominant parent is likely homozygous dominant. Test crosses are common in basic...

complementation tests

Using Crosses to Group Mutations

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

Single nucleotide polymorphisms, or SNPs, are small DNA changes at one specific genomic position. They are the most common type of sequence variation in the human genome. A point mutation counts as an SNP when it appears in more than 1% of the population.

bacterial transformation

Bacterial Transformation in the Lab

Bacterial transformation is a lab method used to move new DNA into bacteria. Scientists use it to change how bacteria behave or to help them carry a gene of interest. In high school biology, this process is often shown with plasmids, which are small circular DNA molecules. During transformation, bacteria take up DNA from their surroundings. The new DNA can then be used by the cell, depending on the experiment. This technique is an important part of genetic engineering and is widely used in...

transgenic organisms

Making Transgenic Organisms

Transgenic organisms are living things with DNA added from another organism. A transgene is the inserted gene. Scientists use this method to change an organism’s traits and to study how genes work. To make a transgenic organism, researchers first identify the gene they want to add. They then place that gene into the target organism’s DNA. Once the gene is integrated, the organism can pass it to new cells as it grows. This approach is useful in biology because it helps scientists analyze gene...

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

CRISPR is a gene editing tool that helps scientists make targeted changes to DNA. It uses a guide system to find a chosen sequence and a cutting protein to act at that spot. This method is based on a natural defense system found in bacteria. In high school biology, it is often studied as a way to see how genes can be edited with precision. CRISPR is widely used in molecular biology research. It helps scientists explore how genes function and how DNA changes can affect living things.

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

Reporter genes are protein-coding genes used to track gene activity in cells. They are often tagged to a gene of interest so scientists can see whether that gene is present or active. When the tagged genes are expressed together, reporter genes can produce visible signals such as fluorescence or luminescence. These signals help show the presence or absence of a gene of interest in an organism. They can also reveal the pattern of gene expression or the location of a DNA segment or protein...

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

Synthetic biology combines engineering, molecular biology, cell biology, and systems biology to redesign living systems. Scientists use it to remodel organisms from nature or to build new synthetic organisms. These tools can support protein or enzyme production, bioremediation, the production of useful macromolecules, and the addition of helpful traits to crops. One well-known example is golden rice. This genetically modified rice plant makes grains rich in β-carotene, which is a precursor of...

ribosome profiling

Ribosome Profiling for Translation Snapshot

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transgenic plants

Transgenic Plants for Better Crops

Transgenic plants are crops that have been changed with recombinant DNA technology, also called transgenesis. This method adds a foreign gene or removes a harmful one from an organism. The result is a genetically modified organism. The first transgenic plant was a tobacco plant made in 1983, and it was resistant to tobacco mosaic virus. Since then, transgenic plants have been developed for many uses in agriculture, horticulture, and ornamental plants. Some are designed to resist pests or...

gene therapy

Gene Therapy for Disease Treatment

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epistasis analysis

Gene Interaction Maps in Epistasis

Gene interaction maps in epistasis show how different genes work together to shape a phenotype. In epistasis, the interaction of genes or alleles at different locations affects the final trait. This often happens when one gene masks or interferes with the expression of another gene. Epistasis is common when genes are part of the same biochemical pathway. In that case, the expression of one gene can depend on a gene product made earlier in the pathway. Genes that mask or inhibit other genes in...

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

JoVE Core Molecular Biology showcases the molecular processes of the cell through clear and concise animated video lessons. Additionally, in scientist-in-action videos, these concepts' application is demonstrated through real experiments in modern laboratories.

Molecular Cloning

Molecular cloning is a set of methods, which are used to insert recombinant DNA into a vector - a carrier of DNA molecules that will replicate recombinant ...

An Overview of Genetic Engineering

Genetic engineering &#8211; the process of purposefully altering an organism&#8217;s DNA &#8211; has been used to create powerful research tools and model ...

Recombineering and Gene Targeting

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Genome Editing

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Recombinant DNA

DNA Ligation Reactions

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Restriction Enzyme Digests

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DNA Isolation

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Restriction Enzymes

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DNA Agarose Gel Electrophoresis

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DNA Gel Electrophoresis

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Separating Protein with SDS-PAGE

Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis, or SDS-PAGE, is a widely-used technique for separating mixtures of proteins based on their ...

Plasmid Purification

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Gel Purification

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PCR - Polymerase Chain Reaction

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target ...

RACE - Rapid Amplification of cDNA Ends

Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known ...

PCR: The Polymerase Chain Reaction

The polymerase chain reaction, or PCR, is a technique used to amplify DNA through thermocycling &#8211; cyles of temperature changes at fixed time ...

bacterial-transformation-the-heat-shock-method

Bacterial Transformation: The Heat Shock Method

Transformation is the process that occurs when a cell ingests foreign DNA from its surroundings. Transformation can occur in nature in certain types of ...

Bacterial Transformation: Electroporation

The term &#x201C;transformation&#x201D; refers cellular ingestion of foreign DNA.  In nature, transformation can occur in certain types of bacteria.  In ...

An Introduction to Transfection

Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. The insertion of DNA into a cell ...

Labeling DNA Probes

DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize ...

Southern Blot

Agarose gel electrophoresis is very useful in separating DNA fragments by size. Running a DNA ladder containing fragments of the known length alongside ...

DNA Microarrays

Microarrays are high-throughput and relatively inexpensive assays that can be automated to analyze large quantities of data at a time. They are used in ...

An Overview of Gene Expression

Gene expression is the complex process where a cell uses its genetic information to make functional products. This process is regulated at multiple ...

Expression Profiling with Microarrays

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Complementary DNA

FISH - Fluorescent In-situ Hybridization

Fluorescence in situ hybridization, or FISH, was developed in the early 1980s and has quickly become one of the most widely used techniques in ...

Sanger Sequencing

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Maxam-Gilbert Sequencing

In the same year as the discovery of the Sanger sequencing method, another group of scientists, Allan Maxam and Walter Gilbert, demonstrated their ...

Next-generation Sequencing

The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and ...

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

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the ...

Synthetic Biology

Synthetic biology is an interdisciplinary science that involves using principles from disciplines such as engineering, molecular biology, cell biology, ...

Genome Annotation and Assembly

The genome refers to all of the genetic material in an organism. It can range from a few million base pairs in microbial cells to several billion base ...

Ribosome Profiling

Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences ...

The ELISA Method

An enzyme-linked immunosorbent assay (ELISA) is typically performed to detect the presence and/or amount of a target protein of interest within an ...

Transgenic Plants

Recombinant DNA technology called transgenesis is often used to add a foreign gene or remove a detrimental gene from an organism. Such genetically ...

Transgenic Organisms

Gene Therapy

Reproductive Cloning

CRISPR

Experimental RNAi

RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. ...

Reporter Genes

Reporter genes are a type of&#160;protein-coding gene&#160;that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually ...

In-situ Hybridization

In situ hybridization (ISH) is a technique used to detect and localize specific DNA or RNA molecules in cells, tissue, or tissue sections using a labeled ...

Obtain, evaluate, and communicate information on how molecular biotechnology may be used in a variety of fields.
EXPLANATION: The goal is to help students appreciate that the basic science knowledge being studied can be manipulated and used to design useful tools for further scientific investigations, medical treatments, agricultural yields, etc. A detailed understanding of any technical procedures is not expected, but rather a "big picture" view of concepts the technology utilizes and the applications the technology is being used for. These technologies may be best investigated along with the standards from LS1 From Molecules to Organisms: Structure and Functions, as the students are learning about DNA and protein. Comparative DNA and protein sequence analysis is also a large part of current phylogenic research and could accompany LS4 Biological Change: Unity and Diversity. Students may also consider the pros and cons of biotechnical applications. Investigation of techniques utilized could come from field trips to witness the use of these techniques in practice, lab exercises, virtual labs, simulation activities, interviews with professionals, etc. Molecular techniques could include: PCR, electrophoresis, restriction enzyme digestion of DNA, DNA sequencing, plasmid-based transformation, transfection, etc. These techniques are used in fields of medicine, agriculture, biomedical engineering, forensic science, etc.

Karyotyping

Lampbrush Chromosomes

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Polytene Chromosomes

Polytene chromosomes are giant interphase chromosomes with several DNA strands placed side by side. They were discovered in the year 1881 by Balbiani in ...

Euchromatin

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Heterochromatin

X and Y Chromosomes

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The Y Chromosome Determines Maleness

The Y chromosome is a sex chromosome&#160;found in several vertebrates and mammals, including humans. In addition to 22 pairs of autosomes, the human ...

The Ratio of X Chromosome to Autosomes

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Investigate means by which karyotypes are utilized in diagnostic medicine.
EXPLANATION: Engineers incorporate growing scientific knowledge in order to increase benefits to humans and decrease cost and risks. Karyotyping technology and its use in medical diagnosis can complement the standards from LS3: Hereditary: Inheritance, and Variation of Traits, as the students are learning about chromosomal organization of genomic information that determines one's phenotype. Analyze a large set of karyotypes to identify common patterns. Identify variations from the most commonly observed patterns and correlate them with patient phenotypes to propose a cause and effect relationship, discussing limitations of correlative data interpretation.

BIO1.ETS2.2

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Methods of Nuclear Reprogramming

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The first successfully cloned mammal was Dolly, a sheep, born on 5th July 1996 at Roslin Institute, Scotland. The cloned sheep was named after the ...

Analyze scientific and ethical arguments to support the pros and cons of applications of a specific biotechnology technique such as stem cell usage, in vitro fertilization, or genetically modified organisms.
EXPLANATION: The utilization of new technologies in any field of science is dependent on both economic and social factors. In addition to evaluating these factors, scientists must also consider long-term consequences that may not be initially apparent. The emphasis should be on the construction of a rationale argument that supports a position on the use of an application with ethical and social impact. Students should begin to appreciate the differences in ethical values that exist, and recognize that discussion of these values is imperative as knowledge and technology continue to advance, even when resolutions of differences can be rare. After investigation of a specific biotechnology application, students can write a position paper and/or participate in a classroom debate.

BIO1.ETS2.3

ChIP Methods for DNA-Protein Binding

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