Name: linear amplification mediated pcr &#8211; localization of genetic elements and characterization of unknown flanking dna
Uploaded: 2014-06-25T14:00:00
Description: Watch this Scientific Journal Video about Linear Amplification Mediated PCR Ã¢â‚¬â€œ Localization of Genetic Elements and Characterization of Unknown Flanking DNA at JoVE.com

Funding was provided by the Deutsche Forschungsgemeinschaft (SPP1230, grant of the Tumor Center Heidelberg/Mannheim), by the Bundesministerium f&#252;r Bildung und Forschung (iGene), by the VIth + VIIth Framework Programs of the European Commission (CONSERT, CLINIGENE and PERSIST). We thank Ina Kutschera for demonstrating the protocol technique in the video.

Bağlayıcı Kaset 1. Hazırlama (LC) <ol><li> LC1 oligonükleotid (Tablo 1), (uygun kısıtlayıcı enzim çıkıntı ile Tablo 1) LC2 oligonükleotid, 110 ul Tris-HCl (100 mM, pH 7.5) ve 10 ul 250 mM MgCl2, 40 ul, 40 ul karıştırın. </li><li> 5 dakika boyunca 95 ° C&#39;de inkübe edildi ve reaksiyon yavaş yavaş oda sıcaklığına soğumaya bırakın. 300 ul H2O ilave edin ve bir santrifüj filtre üzerinde dsLinker-DNA konsantre edilir. 0.2 PCR tüpleri...

<ol>
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LAM-PCR tekniği, bilinen bir DNA bölgesine eşlik bilinmeyen DNA sekanslarının belirlenmesi izin verir. Çünkü bilinen DNA dizisinde spesifik primerler hibridize olan birleşme kısımlarının amplifikasyon kaynaklanan yüksek duyarlılığın, bu tek hücre seviyesine kadar da nadir kavşaklar yükseltmek ve tespit etmek mümkündür. Aksine, bir poliklonal durumda LAM-PCR tek bir reaksiyonda farklı birleşme binlerce amplifiye edebilir. Bununla birlikte, sınırlama kullanımı ned...

Doğrusal amplifikasyon, PCR (LAM-PCR) aracılık ettiği herhangi bir kökenli bilinen DNA komşu bilinmeyen DNA yan belirlenmesi ve karakterize sağlar. Daha özel olarak, LAM-PCR, konakçı genomu içinde 1,2 (IS) viral vektör entegrasyon alanlarının yerini belirlemek üzere geliştirilmiştir. Retrovirüslere veya transpozonlar gibi Genetik elemanlar, bir (yarı-) rastgele bir şekilde 3-6 konakçı genom içine entegre genom. Pek çok durumda, bu gibi vektörler entegre tam konumunu bilmek belirleyicidir. LAM-PCR ligasyon aracılığında PCR 7 ve onun varyantları ya da ters PCR 8 gibi alternatif teknikler üstün olduğu kanıtlanmıştır. Bu yöntemin duyarlılığı ve sağlamlığı vektör genom birleşme ve amplifiye PCR ürünleri manyetik seçim başlangıç ​​amplifikasyon doğar. Bahsedilen alternatif yöntemler gibi, LAM-PCR IS 9-11 alma kapasiteli bir önyargı sokulması, kısıtlama enzimlerinin kullanımına dayanır. Bu durumda,IS repertuar (integrome) yalnızca bir alt kümesi, bir reaksiyonda tespit edilebilir. Bu eğilim kısıtlama enzimleri 9 en uygun kombinasyonları kullanılarak belirli bir örnek paralel analizi ile en aza indirilmiştir. Son zamanlarda, teknoloji bir varyant kısıtlama enzimlerinin kullanımı önlediği ve tek bir reaksiyonda 9,12 içinde bir numunenin ilgili genom analizleri sağlayan LAM-PCR (nrLAM-PCR) geliştirilmiştir sınırlayıcı olmayan adlandırılır. Geçmişte, LAM-PCR neden olan retroviral gen terapisi klinik çalışmalarda 13-15 birkaç hastada lösemi sebebiyet veren IS tanımlamak için kullanılmıştır. O zamandan beri, LAM-PCR belirlemek için adapte edilmiş diğer entegre vektörleri (lentiviral vektörler, transpozonlar) ve pasif adeno-ilişkili vektörler (AAV) veya integraz-kusurlu lentiviral vektörleri (IDLV) gibi vektörleri entegre entegrasyonu biçimlerini belirlemek için her 16 -21. LAM-PCR Uygulamaları geniş yayılır: Gelenekselly, teknik yaygın gen tedavisi geçirmiş olan hastalarda gen modifiye edilmiş hücrelerin klonal çalışma bileşimi için ya da entegre davranışlarını 15,16,22-24 çözülmesine göre yeni vektör sistemleri Biogüvenlik değerlendirmek için kullanılır. Son zamanlarda, LAM-PCR bir IDLV yakalama deneyi 25 özgüllük ve tasarımcı nükleazların hedef dışı aktivitesinin belirlenmesinde etkin. Ayrıca, LAM-PCR kolay bir organizmada zamanla transduse edilmiş hücrenin kaderini takip sağlar. Bu proto-onkogenlerin yanı sıra tümör baskılayıcı genleri tespit etmek ve aynı zamanda hematopoiezis veya kanser kök hücre biyolojisi 26-28 çalışma sağlar. Son fakat en az değil, LAM-PCR insanlarda 29 (ve yayınlanmamış veriler) T-hücre reseptör çeşitliliğini incelemek için adapte edilmiştir. Teknolojinin iç güç tek nükleotid r ile komşu bilinmeyen DNA milyonlarca karakterize izin derin sıralama teknolojileri için yöntem bağlayarak güçlendirilirTüm genomların esolution. Aşağıdaki protokolde, biz adım adım amplifikasyon ve lentiviral vektör IS tanımlamak için örnekli bilinmeyen kuşatıcı DNA tanımlanması için. Protokolünde kullanılan oligonükleotidler,. Olan kaynak Ekstre edilen DNA, cDNA veya LAM-PCR ve nrLAM-PCR için DNA şablonu olarak kullanılabilir Tablo 1 &#39;de listelenmiştir.

<table border="0" cellpadding="0" cellspacing="0" width="1209">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Name of Material/ Equipment</td>
			<td style="width:203px;">Company</td>
			<td style="width:120px;">Catalog Number</td>
			<td style="width:527px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Taq DNA Polymerase</td>
			<td style="width:203px;">Genaxxon Bioscience GmbH</td>
			<td style="width:120px;">M3001.5000</td>
			<td>Alternative Taq Polymerases may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">PCR Buffer</td>
			<td style="width:203px;">Qiagen</td>
			<td style="width:120px;">201203</td>
			<td>Use of this buffer is recommended</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">dNTP-Mixture</td>
			<td style="width:203px;">Genaxxon Bioscience GmbH</td>
			<td style="width:120px;">M3015.4020</td>
			<td>or any other dNTPs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Oligonucleotides (Primers)</td>
			<td style="width:203px;">MWG Biotech</td>
			<td style="width:120px;"></td>
			<td>HPLC purified</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Dynabeads M-280 Streptavidin&#160;</td>
			<td style="width:203px;">Invitrogen</td>
			<td style="width:120px;">11206D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">PBS</td>
			<td style="width:203px;">Gibco</td>
			<td style="width:120px;">14190-086</td>
			<td>0.1 % wt/vol BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">6M LiCl</td>
			<td style="width:203px;">Roth</td>
			<td style="width:120px;">3739.1</td>
			<td>10 mM Tris-HCl&#160; (pH 7.5)/1 mM EDTA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Tris-HCl, pH 7.5</td>
			<td style="width:203px;">USB Corporation&#160;</td>
			<td style="width:120px;">22637</td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">EDTA</td>
			<td style="width:203px;">Applichem</td>
			<td style="width:120px;">A1103,0250</td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Klenow Polymerase</td>
			<td style="width:203px;">Roche Diagnostics</td>
			<td style="width:120px;">10104523001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Hexanucleotide mixture</td>
			<td style="width:203px;">Roche Diagnostics</td>
			<td style="width:120px;">11277081001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Restriction endonuclease</td>
			<td style="width:203px;">NEB</td>
			<td style="width:120px;"></td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Fast-Link DNA ligation kit</td>
			<td style="width:203px;">Epicentre Biotechnologies</td>
			<td style="width:120px;">LK11025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">CircLigase ssDNA Ligase Kit</td>
			<td style="width:203px;">Epicentre Biotechnologies</td>
			<td style="width:120px;">CL4111K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">NaOH</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:120px;">72068</td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Agarose LE</td>
			<td style="width:203px;">Roche Diagnostics</td>
			<td style="width:120px;">11685660001</td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">TBE buffer</td>
			<td style="width:203px;">Amresco</td>
			<td style="width:120px;">0658</td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Ethidium bromide</td>
			<td style="width:203px;">Applichem</td>
			<td style="width:120px;">A2273,0005</td>
			<td>Ethidium bromide is mutagenic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">100 bp DNA Ladder</td>
			<td style="width:203px;">Invitrogen</td>
			<td style="width:120px;">15628-050</td>
			<td>or any other DNA ladder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">20 mM NaCl</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:120px;">71393-1L</td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Magna-Sep Magnetic Particle Separator&#160;</td>
			<td style="width:203px;">Life Technologies</td>
			<td style="width:120px;">K158501</td>
			<td>for use with 1.5 ml Tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Magna-Sep Magnetic Particle Separator&#160;</td>
			<td style="width:203px;">Life Technologies</td>
			<td style="width:120px;">K158696</td>
			<td>for use with 96 well plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon Ultra-0.5, Ultracel-30 membrane</td>
			<td style="width:203px;">Millipore</td>
			<td>UFC503096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">PerfectBlue Gelsystem Midi S</td>
			<td style="width:203px;">PeqLab</td>
			<td style="width:120px;">40-1515</td>
			<td>or other electrophoresis system&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">TProfessional 96</td>
			<td style="width:203px;">Biometra</td>
			<td style="width:120px;">050-551</td>
			<td>or other Thermocycler for 96-well plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Orbital shaker KS 260 basic</td>
			<td style="width:203px;">IKA</td>
			<td style="width:120px;">2980200</td>
			<td>or other horizontal shaker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">PCR softtubes 0.2 ml</td>
			<td style="width:203px;">Biozym Scientific GmbH</td>
			<td style="width:120px;">711082</td>
			<td>or other 0.2 ml PCR tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">1.5 ml tubes</td>
			<td style="width:203px;">Eppendorf</td>
			<td style="width:120px;">12682</td>
			<td>or other 1.5 ml tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Gel documentation system</td>
			<td style="width:203px;">PeqLab</td>
			<td style="width:120px;"></td>
			<td>or any other gel documentation system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Nanodrop ND-1000 spectrophotometer</td>
			<td style="width:203px;">Thermo Scientific</td>
			<td style="width:120px;">ND-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Spreadex EL1200 precast gel</td>
			<td style="width:203px;">Elchrom Scientific</td>
			<td style="width:120px;">3497</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:360px;">Submerged gel electrophoresis apparatus SEA 2000&#160;</td>
			<td style="width:203px;">Elchrom Scientific</td>
			<td style="width:120px;">2001E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">2100 Electrophoresis Bioanalyzer</td>
			<td style="width:203px;">Agilent Technologies</td>
			<td>G2939AA</td>
			<td></td>
		</tr>
	</tbody>
</table>

linear amplification mediated pcr &#8211; localization of genetic elements and characterization of unknown flanking dna

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with integrating vector systems. Due to the stable integration of retroviral vectors, integration sites can be used to study the clonal fate of individual cells and their progeny. LAM- PCR for the first time provided evidence that leukemia in gene therapy treated patients originated from provirus induced overexpression of a neighboring proto-oncogene. The high sensitivity and specificity of LAM-PCR compared to existing methods like inverse PCR and ligation mediated (LM)-PCR is achieved by an initial preamplification step (linear PCR of 100 cycles) using biotinylated vector specific primers which allow subsequent reaction steps to be carried out on solid phase (magnetic beads). LAM-PCR is currently the most sensitive method available to identify&#160;unknown DNA which is located in the proximity of known DNA. Recently, a variant of LAM-PCR has been developed that circumvents restriction digest thus abrogating retrieval bias of integration sites and enables a comprehensive analysis of provirus locations in host genomes. The following protocol explains step-by-step the amplification of both 3&#8217;- and 5&#8217;- sequences adjacent to the integrated lentiviral vector.

LAM-PCR her bir birleşme için tanımlı bir parça boyutu ile birleşme vektör genom amplifikasyonu ile sonuçlanır. Tek tek PCR parçalarının büyüklüğü genomunda bilinen DNA&#39;nın konumu ve en yakın sınır enzimi tanıma sitesi arasındaki mesafeye bağlıdır. Bu jel elektroforezi ile analiz örneğin örneklerde amplifiye birleşme çeşitliliğini görselleştirme sağlar., Sadece tek bir (monoklonal), birkaç (oligoklonal) ya da birden fazla (poliklonal) bantlar jel üzerinde mevcut ise. LA...

Watch this Scientific Journal Video about Linear Amplification Mediated PCR Ã¢â‚¬â€œ Localization of Genetic Elements and Characterization of Unknown Flanking DNA at JoVE.com

Linear Amplification Mediated PCR ÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Å“ Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Doğrusal Amplifikasyon Dolayımlı PCR - Bilinmeyen Yollardan DNA Genetik Elemanları ve Karakterizasyonu lokalizasyonu

Doğrusal amplifikasyon aracılı (LAM)-PCR genomu içinde entegre viral vektörlerin tam konumlarını belirlemek için geliştirilen bir yöntemdir. Teknik gen tedavisi hastaların, vb roman vektör teknolojileri, T-hücresi çeşitlilik, kanser kök hücre modelleri, biyogüvenlik klonal dinamiklerini incelemek için üstün bir yöntem olarak gelişmiştir

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with ...

Research

JoVE Journal

Biology

Linear Amplification Mediated PCR &#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Method for Culture of Early Chick Embryos ex vivo (New Culture)

Method for Culture of Early Chick Embryos ex vivo New Culture

Erken Civciv Embriyolar ex vivo (Kültür) Kültür Yöntemi

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

Erken Civciv Embriyolar in situ Hibridizasyon Çift Tüm Dağı

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

İnsan DNA Onarım Proteinler Genetik Araştırmalar Maya Model Sistemi olarak kullanma

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

Kültür İlköğretim Fare Embriyonik Fibroblastlar Sinyal Molekülleri Adenovirüs aracılı Genetik Kaldırma

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Genetik ve epigenetik Analizleri Parafin Gömülü Malzeme DNA Ekstraksiyon

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Genetik İfade Profilleri Belirlenmesi C. elegans Mikroarray ve Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

Protein-DNA Etkileşiminin Çalışmaları Tek molekül Manipülasyon ve Görüntüleme birleştiren

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

DNA Electroporation, Isolation and Imaging of Myofibers

DNA Elektroporasyon, Myofibers İzolasyon ve Görüntüleme

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Sıçan Karaciğer DNA Strand Breaks ve Oksidatif DNA Hasar Ölçüm Vivo Alkali Comet Assay ve Enzim-modifiye Alkali Comet Tahlili

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviral Entegrasyon Siteleri Amplifikasyon, Yeni Nesil Sıralama ve Genomik DNA Haritalama

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...