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

ChIP로 약칭되는 염색질 면역침전법(chromatin immunoprecipitation)은 유전자 발현을 조절하는 단백질-DNA 상호작용을 연구하는 기술입니다.
진핵생물에서 DNA는 히스톤 단백질을 감싸고 뉴클레오솜으로 알려진 복합체를 형성하며, 뉴클레오솜은 세포에서 DNA의 단단한 포장을 돕기 위해 염색질로 알려진 구조로 함께 그룹화됩니다.
유전자 발현은 DNA의 시스 조절 영역과 관련된 단백질뿐만 아니라 이러한 구조를 풀거나 조이는 히스톤 변형을 통해 조절됩니다.
ChIP에서는 염색질이 분해되고 히스톤 변형 또는 조절 단백질에 결합하는 항체를 사용하여 관련 DNA를 가진 표적 분자를 분리합니다.
이 과정의 첫 번째 단계는 포름알데히드와 같은 가교제의 도움으로 단백질을 DNA에 가교결합시키는 것입니다.이것은 단백질의 결합 부위를 표시하는 DNA의 단백질을 고정시킵니다. 가교결합 후, 염색질은 100개에서 200개의 염기쌍에 이르는 짧은 단편으로 기계적으로 전단됩니다. 이 프로세스를 X-ChIP라고 합니다.
또 다른 방법은 N-ChIP로, 뉴클레아제는 사전 교차 결합 없이 염색질에서 DNA를 짧은 단편으로 직접 분해합니다.
면역침전은 전단 또는 분해된 DNA를 포함하는 용액에서 조절 단백질을 표적으로 하는 항체를 도입하여 수행됩니다. 이러한 항체는 선택적 분리에 도움이 되는 약제와 연결되어 있습니다.
한 가지 일반적인 방법은 항체를 자성 비드에 연결하는 것입니다. 자석은 결합된 분자와 함께 항체를 분리하는 데 사용됩니다.
복합체를 헹궈서 느슨하게 관련된 오염 물질을 씻어냅니다.표적 분자는 SDS와 같은 세제를 사용하여 분리됩니다.
X-ChiP의 경우, 가교결합은 고온의 도움으로 역전되고, 관련 조절자 또는 히스톤은 프로테아제의 도움으로 분해되어 DNA를 남깁니다. 
관련 DNA의 염기서열분석은 관심 단백질이 결합된 시스 조절 서열 또는 특정 히스톤 변형에 의해 발현이 조절되는 유전자를 식별하는 데 도움이 될 수 있습니다.

<ol>
	<li>Gade, P., & Kalvakolanu, D. V. (2012). 전사 인자 활성을 분석하기 위한 도구로서의 크로마틴 면역침전 분석법. 분자 생물학 방법 (Clifton, N.J.), 809, 85–104. https://doi.org/10.1007/978-1-61779-376-9_6</li>
	<li>Nelson, J. D., Denisenko, O., & Bomsztyk, K. (2006). 빠른 염색질 면역침전(ChIP) 방법을 위한 프로토콜. 자연 의정서, 1(1), 179–185. 도이 : 10.1038 / nprot.2006.27</li>
	<li>Das, P. M., Ramachandran, K., VanWert, J., & Singal, R. (2004). 염색질 면역침전 분석법. 생물 기술, 37 (6), 961-969.</li>
</ol>

염색질 면역침전 - chip

크로마틴 면역침전(ChIP)은 관심 있는 전사 인자나 히스톤 단백질에 결합하는 DNA 부위를 식별하는 데 사용되는 항체 기반 기술입니다. 또한 아세틸화, 인산화 또는 메틸화와 같은 히스톤 변형 유형을 결정하는 데 도움이 됩니다.

염색질 면역침전(ChIP)의 종류

ChIP은 X-ChIP과 N-ChIP의 두 가지 유형으로 나눌 수 있습니다. X-ChIP에는 생체 내에서 히스톤과 조절 단백질을 DNA에 교차 연결하고, 초음파 처리로 DNA를 단편화하고, 특정 항체로 면역침전시켜 단백질-DNA 복합체를 분리하는 과정이 포함됩니다. 반면, N-ChIP은 DNA와 단백질의 가교결합을 수반하지 않으며, 뉴클레아제를 사용하여 소화가 수행됩니다.

DNA의 다운스트림 분석 - 단백질 상호작용

관심 있는 DNA를 분리한 후 PCR, 마이크로어레이 또는 서던 블롯과 같은 기술을 사용하여 분석할 수 있습니다. 또는 이 DNA는 ChIP-Seq로 알려진 심층 시퀀싱에 사용될 수 있습니다. ChIP는 ChIA-PET와 같은 다양한 분석을 위한 다른 방법론을 사용하여 수정할 수 있습니다. 이 기술은 ChIP의 원리와 염색체 구조 캡처를 결합하여 관심 있는 단백질을 통해 매개되는 장거리 염색질 상호 작용을 감지합니다. enChIP은 특정 게놈 영역을 표적으로 삼기 위해 CRISPR/Cas9 시스템을 사용하는 기술과 단백질-RNA 상호 작용을 분석하는 데 사용되는 ChIP의 변형인 RIP-ChIP/RIP-Seq입니다.

N-ChIP과 X-ChIP의 비교

N-ChIP과 X-ChIP에는 고유한 장점과 단점이 있습니다. N-ChIP은 더 강한 항체 결합과 효율적이고 고도로 특이적인 면역 침전을 초래합니다. 그러나 N-ChIP은 처리 중에 전사 인자가 분리될 수 있으므로 히스톤과 같이 단단히 결합된 단백질의 경우에만 적합합니다. 또한, 뉴클레아제로 소화된 염색질 전체가 용해되지 않아 표본의 특정 부분이 누락될 수 있습니다. X-ChIP은 교차 연결 단계로 인해 DNA에 단단히 결합되지 않은 전사 인자를 연구하기 위한 탁월한 방법입니다. X-ChIP 분석은 N-ChIP보다 더 민감하며 더 적은 양의 표본과 항체가 필요합니다. X-칩의 단점은 과도한 가교로 인한 단편화의 어려움과 일시적인 DNA 단백질 상호작용의 가교로 인한 위양성(false positive)을 포함합니다.

염색질 면역침전 - ChIP

염색질 면역침전(ChIP)은 관심 전사 인자에 결합하는 DNA의 부위를 식별하는 데 사용되는 항체 기반 기술입니다.

Education

교육

JoVE Core

Molecular Biology

분자생물학

Unit 2

제2단원

Analyzing Gene Expression and Function

유전자 발현 및 기능 분석

KEY TERMS AND CONCEPTS

주요 용어 및 개념

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

Ribosome profiling is a deep sequencing method that gives a snapshot of active translation in a cell. It sequences the mRNAs protected by ribosomes. This shows which messages are being read to make proteins at a specific moment. The method has several uses in biology. It can track translation in a living organ or tissue type. It also helps measure new protein synthesis levels. Researchers use it to find translated open reading frames, which are stretches of RNA that can make protein. It can...

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

Gene therapy is a treatment approach that changes gene activity to help address disease. It focuses on the genetic cause of a problem rather than only easing symptoms. In this way, it is used in disease intervention. One common strategy is to replace a faulty gene with a normal copy. Another strategy is to inactivate a gene that is causing harm. Gene therapy can also introduce a new gene to help cells work more normally.

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 분자생물학은 명확하고 간결한 애니메이션을 통해 분자 단계에서 일어나는 세포 과정을 설명합니다.또한, 과학자가 직접 실험을 진행하는 실사 영상을 통해 분자생물학 개념의 응용 사례를 보여줍니다.

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Recombinant DNA

DNA Ligation Reactions

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Restriction Enzyme Digests

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PCR - Polymerase Chain Reaction

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bacterial-transformation-the-heat-shock-method

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An Introduction to Transfection

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Southern Blot

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Expression Profiling with Microarrays

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Complementary DNA

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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

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Ribosome Profiling

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Transgenic Plants

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Transgenic Organisms

Gene Therapy

Reproductive Cloning

CRISPR

Experimental RNAi

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Reporter Genes

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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

In 1882, Flemming observed lampbrush chromosomes (LBC) in salamander eggs. Later in 1892, R&#252;ckert observed LBCs in shark egg cells and coined the ...

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

The extent of chromatin compaction can be studied by staining chromatin using specific DNA binding dyes. Under the microscope, the dense-compacted regions ...

Heterochromatin

X and Y Chromosomes

Among mammals, the gender of an organism is determined by the sex chromosomes. Humans have two sex chromosomes, X and Y. Every human diploid cell has 22 ...

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

In most organisms, sex is determined by the ratio of X and Y chromosomes. However, in some organisms, such as Drosophila and C.elegans, sex is determined ...

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

Whole Body Regeneration

Regeneration is the process of restoring injured or lost tissues, organs, or&#160;body parts. While simpler organisms generally show greater ability to ...

Liver Regeneration

The liver is an important organ in vertebrates that plays an essential role in metabolism. It is also responsible for storing and redistributing nutrients ...

stem-cell-therapy-for-tissue-regeneration

Stem Cell Therapy for Tissue Regeneration

Stem cell therapy is a method used in regenerative medicine to repair and restore function to&#160;damaged tissues and organs. Stem cells have the ...

Introduction to Nuclear Reprogramming

Nuclear reprogramming is the process of switching gene expression of one cell type to that of another cell type, usually from a differentiated cell state ...

Methods of Nuclear Reprogramming

Nuclear reprogramming is a process of transforming one cell type into an unrelated cell type by epigenetic changes that alter the cell&#8217;s original ...

Cloning of Dolly the Sheep

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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