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

ChIP olarak kısaltılan kromatin immünopresipitasyonu, gen ekspresyonunu düzenleyen protein-DNA etkileşimlerini incelemek için kullanılan bir tekniktir.
Ökaryotlarda DNA, histon proteinlerinin etrafına sarılır ve nükleozom olarak bilinen bir kompleks oluşturur, bu da hücrelerde DNA'nın sıkı bir şekilde paketlenmesine yardımcı olmak için kromatin olarak bilinen bir yapı halinde gruplanır.
Gen ekspresyonu, bu yapıları çözen veya sıkılaştıran histon modifikasyonlarının yanı sıra DNA üzerindeki cis-düzenleyici bölgelerle ilişkili proteinler yoluyla düzenlenir.
ChIP'de, kromatin parçalanır ve histon modifikasyonlarına veya düzenleyici proteinlere bağlanan antikorlar, hedef molekülleri ilişkili DNA ile izole etmek için kullanılır.
Bu işlemdeki ilk adım, formaldehit gibi bir çapraz bağlama maddesi yardımıyla proteini DNA'ya çapraz bağlamaktır. Bu, proteinin bağlanma bölgesini işaretleyerek proteini DNA üzerinde hareketsiz hale getirir. Çapraz bağlamadan sonra, kromatin mekanik olarak 100 ila 200 baz çifti arasında değişen kısa parçalara kesilir. Bu işlem X-ChIP olarak bilinir.
Alternatif bir yöntem, nükleazların önceden herhangi bir çapraz bağlanma olmaksızın kromatinden DNA'yı doğrudan kısa parçalara sindirdiği N-ChIP'dir.
İmmünopresipitasyon, kesilmiş veya sindirilmiş DNA içeren çözeltideki düzenleyici proteinleri hedef alan antikorların eklenmesiyle gerçekleştirilir. Bu antikorlar, seçici izolasyona yardımcı olan ajanlarla bağlantılıdır.
Yaygın bir yöntem, antikorları manyetik boncuklara bağlamaktır. Antikorları herhangi bir bağlı molekülle birlikte izole etmek için bir mıknatıs kullanılır.
Kompleks, gevşek bir şekilde ilişkili kirleticileri yıkamak için durulanır. Hedef moleküller, SDS gibi bir deterjan yardımıyla ayrılır.
X-ChiP durumunda, çapraz bağlama, yüksek sıcaklıkların yardımıyla tersine çevrilir ve ilişkili düzenleyiciler veya histonlar, proteazların yardımıyla parçalanır ve DNA'yı geride bırakır. 
İlişkili DNA'nın dizilimi, ilgilenilen proteinin bağlandığı cis-düzenleyici dizinin veya ekspresyonu belirli bir histon modifikasyonu ile modüle edilen genin tanımlanmasına yardımcı olabilir.

<ol>
	<li>Gade, P. ve Kalvakolanu, D. V. (2012). Transkripsiyon faktörü aktivitesini analiz etmek için bir araç olarak kromatin immünopresipitasyon testi. Moleküler biyolojide yöntemler (Clifton, N.J.), 809, 85–104. https://doi.org/10.1007/978-1-61779-376-9_6</li>
	<li>Nelson, J. D., Denisenko, O. ve Bomsztyk, K. (2006). Hızlı kromatin immünopresipitasyon (ChIP) yöntemi için protokol. Doğa Protokolleri, 1(1), 179–185. doi:10.1038/nprot.2006.27</li>
	<li>Das, P. M., Ramachandran, K., VanWert, J., & Singal, R. (2004). Kromatin immünopresipitasyon testi. Biyoteknik, 37(6), 961-969.</li>
</ol>

kromatin İmmünopresipitasyonu - chip

Kromatin immünopresipitasyonu veya ChIP, DNA üzerinde ilgilenilen transkripsiyon faktörlerine veya histon proteinlerine bağlanan bölgeleri tanımlamak için kullanılan antikor bazlı bir tekniktir. Ayrıca asetilasyon, fosforilasyon veya metilasyon gibi histon modifikasyonu türünün belirlenmesine de yardımcı olur. <h4>ChIP Türleri</h4> ChIP iki türe ayrılabilir: X-ChIP ve N-ChIP. X-ChIP, histonların ve düzenleyici proteinlerin DNA'ya in vivo çapraz bağlanmasını, DNA'nın sonikasyonla parçalanmasını ve protein-DNA komplekslerinin spesifik antikorlarla immünopresipitasyon yoluyla izole edilmesini içerir. Öte yandan N-ChIP, DNA'nın proteinlere çapraz bağlanmasını içermez ve sindirim, nükleazlar kullanılarak gerçekleştirilir. <h4>DNA - protein etkileşimlerinin aşağı akış analizi</h4> İlgili DNA'nın izole edilmesinden sonra analiz için PCR, mikrodiziler veya Southern blot gibi teknikler kullanılabilir. Alternatif olarak bu DNA, ChIP-Seq olarak bilinen derin dizileme için kullanılabilir. ChIP, ilgilenilen bir proteinin aracılık ettiği uzun menzilli kromatin etkileşimlerini tespit etmek için ChIP ilkelerini kromozom konformasyon yakalamasıyla birleştiren bir teknik olan ChIA-PET gibi farklı analizler için başka metodolojiler kullanılarak değiştirilebilir; spesifik genomik bölgeleri hedeflemek için CRISPR/Cas9 sistemini kullanan bir teknik olan enChIP ve protein-RNA etkileşimlerini analiz etmek için kullanılan ChIP'in bir modifikasyonu olan RIP-ChIP/RIP-Seq. <h4>N-ChIP ve X-ChIP’in karşılaştırılması</h4> N-ChIP ve X-ChIP'in kendi avantajları ve dezavantajları vardır. N-ChIP, daha güçlü antikor bağlanması ve etkili ve son derece spesifik immünopresipitasyonla sonuçlanır. Bununla birlikte, N-ChIP yalnızca histonlar gibi sıkı bağlı proteinler durumunda uygundur çünkü transkripsiyon faktörleri işlem sırasında ayrılabilir. Ek olarak, nükleazla sindirilen kromatinin tümü çözünmez, bu da numunenin belirli parçalarının kaybolmasına neden olur. X-ChIP, çapraz bağlanma adımı nedeniyle DNA'ya sıkı bir şekilde bağlanmayan transkripsiyon faktörlerini incelemek için mükemmel bir metodolojidir. X-ChIP testi ayrıca N-ChIP'den daha hassastır ve daha az antikor ve daha az miktarda numune gerektirir. X çipinin dezavantajları arasında aşırı çapraz bağlanma nedeniyle parçalanmadaki olası zorluklar ve geçici DNA protein etkileşimlerinin çapraz bağlanması nedeniyle yanlış pozitifler yer alır.

Kromatin İmmünopresipitasyonu - ChIP

Kromatin immünopresipitasyon veya ChIP, DNA üzerinde ilgilenilen transkripsiyon faktörlerine bağlanan bölgeleri tanımlamak için kullanılan antikor bazlı bir tekniktir.

Education

JoVE Core

Molecular Biology

Moleküler Biyoloji

Unit 2

Ünite 2

Analyzing Gene Expression and Function

Gen İfadesinin ve İşlevinin Analizi

KEY TERMS AND CONCEPTS

TEMEL TERİMLER VE KAVRAMLAR

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

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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...

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transgenic plants

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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 Moleküler Biyoloji, hücrenin moleküler süreçlerini açık ve özlü animasyonlu video dersleriyle sergiler. Buna ek olarak, bilim insanı-eylem videolarında, bu kavramların uygulaması modern laboratuvarlarda gerçek deneylerle gösterilmektedir.

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BIO1.ETS2.3

ChIP Methods for DNA-Protein Binding

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