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 tüplerine hazırlanan bağlayıcı kaset eluat ve kısım 10 ul 80 ul H2O ekleyin. </li></ol> Vektör Genom EklemlerininTünelleme 2. Preamplification <ol><li> Analiz edilecek her bir örnek, 50 | il PCR reaksiyonu hazırlamak için. <ol><li> DNA örneklerinin konsantrasyonunu belirleyin. Pipet x ul (1-1000 ng LAM/100-1 nrLAM için, 000 ng), bir 0.2 ml PCR tüpüne DNA. DNA hacmi her bir örnek olarak ve RAN eşit olmalıdır0,5-25 | il ge. </li><li> Tablo 2 &#39;de tarif edildiği gibi PCR Master Mix karışım hazırlayın (- 50 x). 0.2 ml PCR tüplerine her bir DNA numunesi ile ana karışımı ul. </li></ol></li><li> PCR tamamlandıktan sonra., Tablo 2&#39;de örneklendiği PCR koşulları kullanılarak Preamplify vektör genom bağlantıları her PCR tüpü ile tekrar işleme PCR programı için Taq Polimeraz ul 0.5 ekleyin. PCR ürünleri, -20 ° C&#39;de 4 güne kadar ya da uzun bir süre için 4 ° C&#39;de saklanabilir </li></ol> PCR Ürünü 3. Manyetik Ayırma <ol><li> Manyetik Boncuk hazırlanması <ol><li> Pipet 20, bir 1.5 ml tüp içine streptavidin kaplı manyetik boncuklar ul (200 ug) ile, oda sıcaklığında, manyetik parçacık ayırıcı (MPS) 1 dakika boyunca maruz kalmaktadır. Süpernatant atın. </li><li> MPS dan tüpünü çıkarın ve 40 ul PSB /% 0.1 BSA (pH 7.5) manyetik boncuklar tekrar süspansiyon. 1 dakika boyunca MPS maruz ve supernatant atın. Kez bu adımı yineleyin. </li><li> Yıkama wi boncuk3 M LiCl çözeltisi 20 ul th (3 M LiCİ, 10 mM Tris-HCI, 1 mM EDTA) 1 dakika süre ile maruz MPS ve supernatant atın. 6 M LiCl çözeltisi (6 M LiCİ, 10 mM Tris-HCI, 1 mM EDTA), 50 ul içinde süspanse boncuk. </li></ol></li><li> Hazırlanmış manyetik boncuk tanelerin 50 ul aşama 2.2 &#39;den tüm PCR reaksiyonu karıştırın. En az bir oda sıcaklığında 2 saat boyunca yatay bir çalkalayıcı (300 rpm) üzerinde inkübe edin. Bu adım, kaplı boncuklar (DNA-boncuk kompleksi) streptavidin biyotinile edilmiş PCR ürününün bağlanması sağlar. DNA boncuk kompleksi, gece boyunca çalkalayıcıda inkübe veya en fazla 4 gün süreyle 4 ° C&#39;de saklanabilir. </li><li> Oda sıcaklığında 1 dakika boyunca MPS DNA-Boncuk kompleksi Açığa, süpernatant kaldırmak ve 100 ul H2O içinde DNA-boncuk kompleksi tekrar süspansiyon Hemen LAM veya nrLAM için adım 5 için adım 4 ile devam edin. </li></ol> 4. LAM-Prosedürü <ol><li> Çift sicimli DNA (dsDNA) Synthesis (LAM için) <ol><li> 1 dakika ve dis için MPS adım 3.3 den DNA-Boncuk kompleksini Açığakart yüzer. 8.25 H2O ul, 1 ul 10x hexanucleotide tampon, 0.25 ul dNTP (10 mM) ve 0.5 ul (2 U) Klenow polimerazı ekleyin. 1 saat boyunca 37 ° C&#39;de inkübe edilir. </li><li> H 2 O 90 ul ilave edilir ve 1 dakika süre ile maruz MPS. 100 ul H 2 O. süpernatant ve tekrar süspansiyon DNA-Boncuk kompleksini atın </li></ol></li><li> Kısıtlama Digest <ol><li> 1 dakika boyunca MPS DNA-Boncuk kompleksini Açığa ve süpernatant atın. 8.5 ul 2 H, O, 1 ul 10x kısıtlayıcı enzim tamponu ve sınırlama enzimi, 0.5 ul ilave edin ve 1 saat boyunca bir reaksiyon inkübe edin. 4.1.2 adımı tekrarlayın. Not: kısıtlama enzimi üretici tarafından tavsiye edilen sıcaklıkta reaksiyonu inkübe edin. Herhangi bir kısıtlama alanı içinde mevcut ya da ilgi duyulan DNA amplifikasyon için kullanılan primer bağlanma yerinin alt olduğundan emin olun. Uygun kısıtlama enzimleri / sınırlama enzim kombinasyonlarının seçimi için 9&#39;a bakın. </li></ol></li><li> Ligasyon ds Linker (LK) <ol><li> 1 dakika boyunca MPS DNA-Boncuk kompleksini Açığa ve süpernatant atın. 5 ul 2 H, O, 1 ul 10x FastLink tamponu, 1 ul ATP (10 mM), aşama 1.2 &#39;den 2 ul Bağlayıcı kaset ve 1 ul Fast-Link DNA Ligaz (2 U / ml) eklenir. 5 dakika boyunca oda sıcaklığında inkübe edin. 4.1.2 adımı tekrarlayın. </li></ol></li><li> Sentezlenen dsDNA denatürasyonu <ol><li> 1 dakika boyunca MPS DNA-Boncuk kompleksini Açığa ve süpernatant atın. 5 ul 0.1 N NaOH içinde süspanse DNA-Boncuk kompleksi. Yatay çalkalayıcı üzerinde oda sıcaklığında 5 dakika inkübe edin. </li><li> 1 dakika boyunca MPS DNA-Boncuk kompleksini ortaya çıkarmak ve yeni bir 1.5 ml tüp içeren yüzer preamplified vektör genom kavşak toplamak. Hemen -20 ° C&#39;de adım 6 veya mağaza süpernatant ile devam </li></ol></li></ol> 5. NrLAM-Prosedürü <ol><li> Tek şeritli bağlayıcının ligasyon (SSLC) (nrLAM için) <ol><li> MPS adım 3.3 den DNA-Boncuk kompleksini Açığa1 dakika ve atma süpernatant için. 6.5 ul H2O ekleme, 1 ul 10x reaksiyon tamponu, 0.5 ul CircLigase MnCI2 (50 mM), 0.5 ATP (1 mM) ul, 1 ul ssLinker oligonükleotid ve 0.5 ul CircLigase (100 U / ml). 1 saat boyunca 60 ° C&#39;de inkübe edin. </li><li> H 2 O 90 ul ilave edilir ve 1 dakika süre ile maruz MPS. Süpernatant atın ve 100 ul H 2 O DNA-Boncuk kompleksini yıkayın Yine 10 ul H 2 O 1 dakika, ıskarta süpernatant ve tekrar süspansiyon DNA-Boncuk kompleksi için MPS maruz </li></ol></li></ol> 6.. Üstel Amplifikasyon I <ol><li> Analiz edilecek her bir örnek, 50 | il PCR reaksiyonu hazırlamak için. <ol><li> Pipet 2 ul şablonu (adım 4.4.2 (LAM) veya 5.1.2 (nrLAM)) DNA, bir 0.2 ml PCR tüp içine. </li><li> Tablo 3 &#39;de tarif edildiği gibi PCR master karışımı hazırlayın. Aşama 6.1.1 her örnek için ana karışımı 48 ul eklenir ve PCR tarafından vektör genom conditio kavşakları yükseltmekTablo 3&#39;de örneklenen ns. PCR ürünleri, -20 ° C&#39;de 4 güne kadar ya da uzun bir süre için 4 ° C&#39;de saklanabilir </li></ol></li></ol> PCR Ürünü 7. Manyetik Ayırma <ol><li> 3.1.3 - 3.1.1 adımda de örneklendiği gibi, manyetik boncuklar hazırlayın. (LAM için), 20 ul ya da 6 M LiCl çözeltisi (6 M LiCİ, 10 mM Tris-HCI, 1 mM EDTA) içinde (nrLAM için), 50 ul içinde süspanse boncuk. 20 ul (LAM) veya hazırlanmış manyetik boncuklar ile adım 6.1.2 ikinci PCR reaksiyonunda (adım 7.1), 50 ul (nrLAM) karıştırınız ve oda sıcaklığında 2 saat boyunca yatay bir çalkalayıcıda (300 rpm) üzerinde inkübe edilir. DNA boncuk kompleksi, gece boyunca çalkalayıcıda inkübe veya en fazla 4 gün süreyle 4 ° C&#39;de saklanabilir. </li><li> 1 dakika boyunca MPS DNA-Boncuk kompleksini Açığa, süpernatant kaldırmak ve 100 ul H 2 O DNA-Boncuk kompleksini tekrar süspansiyon 1 dakika boyunca MPS DNA-Boncuk kompleksini Açığa ve süpernatant atın. </li><li> 20 ul (LAM) ya da 5 ul (nrLAM) 0.1 N NaOH içinde süspanse DNA-boncuk kompleksi. Incuyatay karıştırıcısı üzerinde oda sıcaklığında 10 dakika boyunca kesmek, 1 dakika boyunca MPS ortaya çıkarmak ve yeni bir 1.5 ml bir tüp içinde büyütülmüş DNA ihtiva eden süpernatan toplamak. Hemen -20 ° C&#39;de adım 8.1 veya mağaza süpernatant ile devam </li></ol> 8. Üstel Amplifikasyon II <ol><li> Analiz edilecek her bir örnek, 50 | il PCR reaksiyonu hazırlamak için. <ol><li> Bir 0.2 ml PCR tüpüne (adım 7.3) Pipet 2 ul DNA şablonu. </li><li> Tablo 4 &#39;de tarif edildiği gibi PCR master karışımı hazırlayın. Aşama 8.1.1 her örnek için ana karışımı 48 ul ekleyin ve Tablo 4&#39;te örneklenen PCR koşullarına göre vektör genom kavşaklar amplifiye. PCR ürünleri kadar 4 için 4 ° C&#39;de saklanabilir -20 ° C&#39;de gün veya uzun vadeli </li></ol></li><li> Görselleştirmek için (nr) LAM-PCR ürünleri,% 2 agaroz jeli üzerinde adım 8.1.2 elde edilen PCR ürünü yükü 10 ul. Bantlar görünür durumdaysa, yüksek çözünürlüklü jel üzerinde LAM-PCR ürünü 10 ul analiz. DİKKAT: Ethidium bromür mutajenik değildir. Çok dikkatli çalışmak ve her zaman uygun eldivenler giyin. </li></ol> Yüksek sekanslama için 9. Hazırlanması <ol><li> (Nr) LAM-PCR ürünlerinin saflaştırılması <ol><li> Oda sıcaklığı AMPure XP Manyetik Boncuk 44 ul ile adım 8.1.2 40 ul PCR-Ürün karıştırın. Oda sıcaklığında 5 dakika inkübe edin ve 2 dakika boyunca ilave MPS maruz. </li><li> Süpernatant atın ve MPS 200 ul% 70 EtOH ile iki kez yıkayın. </li><li> 30 ul H 2 O. süpernatant ve tekrar süspansiyon DNA-Boncuk kompleksini atın Taze 0.2 ml tüp 1 dakika ve transfer süpernatant inkübe. Saflaştınldı DNA konsantrasyonu tayin edilir. </li></ol></li><li> Fusionprimer PCR özel adaptörleri sıralanmasıyla eklemek. <ol><li> Analiz edilecek her bir örnek, 50 | il PCR reaksiyonu hazırlamak için. </li><li> Bir 0.2 ml PCR tüpüne DNA Pipet x ul (40 ng). DNA hacmi her bir örnek olarak ve 0,5-25 ul aralığında eşit olmalıdır. </li><li> Tablo 5&#39;te tarif edildiği gibi PCR ana karışımı hazırlayın ekle (50 - x). Aşama 9.2.2 her örnek için ana karışımı ul ve Tablo 5&#39;te PCR ürünleri örneklenen PCR koşullarının (nr) LAM-PCR ürünlerine Sequencing adaptörleri tanıtmak. -20 ° C&#39;de 4 güne kadar ya da uzun bir süre için 4 ° C&#39;de saklanabilir </li><li> Fusionprimer-PCR ürünleri yüklemek bir% 2 agaroz jeli üzerinde adım 9.2.3 ikinci PCR ürününün 10 ul görselleştirmek için. 9.1.3 - 9.1.1 adımda tarif edildiği gibi geriye kalan PCR ürünü arındırın. Doğru Fusionprimer-PCR ürünleri konsantrasyon ve parça boyutu ölçmek için otomatik bir yüksek çözünürlüklü elektroforez aygıtı, adım 9.4 1 ul saflaştırılmış PCR ürünü analiz edin. </li></ol></li></ol>

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<li>Martins, V. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thymus-autonomous+T+cell+development+in+the+absence+of+progenitor+import%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">Thymus-autonomous T cell development in the absence of progenitor import.</a> J Exp Med. 209, 1409-1417 (2012).</li>
<li>Arens, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bioinformatic+clonality+analysis+of+next-generation+sequencing-derived+viral+vector+integration+sites%5BTitle%5D)+AND+%22Hum+Gene+Ther+Methods%22%5BJournal%5D)">Bioinformatic clonality analysis of next-generation sequencing-derived viral vector integration sites.</a> Hum Gene Ther Methods. 23, 111-118 (2012).</li>
</ol>

The authors have nothing to disclose.

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><tbody><tr><td>Taq DNA Polymerase</td><td>Genaxxon Bioscience GmbH</td><td>M3001.5000</td><td>Alternative Taq Polymerases may be used</td></tr><tr><td>PCR Buffer</td><td>Qiagen</td><td>201203</td><td>Use of this buffer is recommended</td></tr><tr><td>dNTP-Mixture</td><td>Genaxxon Bioscience GmbH</td><td>M3015.4020</td><td>or any other dNTPs</td></tr><tr><td>Oligonucleotides (Primers)</td><td>MWG Biotech</td><td></td><td>HPLC purified</td></tr><tr><td>Dynabeads M-280 Streptavidin&amp;nbsp;</td><td>Invitrogen</td><td>11206D</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>14190-086</td><td>0.1% wt/vol BSA</td></tr><tr><td>6 M LiCl</td><td>Roth</td><td>3739.1</td><td>10 mM Tris-HCl (pH 7.5)/1 mM EDTA</td></tr><tr><td>Tris-HCl, pH 7.5</td><td>USB Corporation&amp;nbsp;</td><td>22637</td><td>or any other supplier</td></tr><tr><td>EDTA</td><td>Applichem</td><td>A1103,0250</td><td>or any other supplier</td></tr><tr><td>Klenow Polymerase</td><td>Roche Diagnostics</td><td>10104523001</td><td></td></tr><tr><td>Hexanucleotide mixture</td><td>Roche Diagnostics</td><td>11277081001</td><td></td></tr><tr><td>Restriction endonuclease</td><td>NEB</td><td></td><td>or any other supplier</td></tr><tr><td>Fast-Link DNA ligation kit</td><td>Epicentre Biotechnologies</td><td>LK11025</td><td></td></tr><tr><td>CircLigase ssDNA Ligase Kit</td><td>Epicentre Biotechnologies</td><td>CL4111K</td><td></td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>72068</td><td>or any other supplier</td></tr><tr><td>Agarose LE</td><td>Roche Diagnostics</td><td>11685660001</td><td>or any other supplier</td></tr><tr><td>TBE buffer</td><td>Amresco</td><td>0658</td><td>or any other supplier</td></tr><tr><td>Ethidium bromide</td><td>Applichem</td><td>A2273,0005</td><td>Ethidium bromide is mutagenic</td></tr><tr><td>100 bp DNA Ladder</td><td>Invitrogen</td><td>15628-050</td><td>or any other DNA ladder</td></tr><tr><td>20 mM NaCl</td><td>Sigma-Aldrich</td><td>71393-1L</td><td>or any other supplier</td></tr><tr><td>Magna-Sep Magnetic Particle Separator&amp;nbsp;</td><td>Life Technologies</td><td>K158501</td><td>for use with 1.5 ml Tubes</td></tr><tr><td>Magna-Sep Magnetic Particle Separator</td><td>Life Technologies</td><td>K158696</td><td>for use with 96-well plates</td></tr><tr><td>Amicon Ultra-0.5, Ultracel-30 membrane</td><td>Millipore</td><td>UFC503096</td><td></td></tr><tr><td>PerfectBlue Gelsystem Midi S</td><td>PeqLab</td><td>40-1515</td><td>or other electrophoresis system&amp;nbsp;</td></tr><tr><td>TProfessional 96</td><td>Biometra</td><td>050-551</td><td>or other Thermocycler for 96-well plates</td></tr><tr><td>Orbital shaker KS 260 basic</td><td>IKA</td><td>2980200</td><td>or other horizontal shaker</td></tr><tr><td>PCR softtubes 0.2 ml</td><td>Biozym Scientific GmbH</td><td>711082</td><td>or other 0.2 ml PCR tubes</td></tr><tr><td>1.5 ml tubes</td><td>Eppendorf</td><td>12682</td><td>or other 1.5 ml tubes</td></tr><tr><td>Gel documentation system</td><td>PeqLab</td><td></td><td>or any other gel documentation system</td></tr><tr><td>Nanodrop ND-1000 spectrophotometer</td><td>Thermo Scientific</td><td>ND-1000</td><td></td></tr><tr><td>Spreadex EL1200 precast gel</td><td>Elchrom Scientific</td><td>3497</td><td></td></tr><tr><td>Submerged gel electrophoresis apparatus SEA 2000&amp;nbsp;</td><td>Elchrom Scientific</td><td>2001E</td><td></td></tr><tr><td>2100 Electrophoresis Bioanalyzer</td><td>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 &#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA

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

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

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

linear-amplification-mediated-pcr-localization-genetic-elements

Research

JoVE Journal

Biology

30.2K Görüntüleme.  National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ).  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 

Video: Linear Amplification Mediated PCR – Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

Tek damlacık dijital polimeraz zincir tepkimesi mutasyonlar Hotspot bölgelerde kapsamlı ve eşzamanlı algılanması için

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

Genetik

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an upstream 3&#39; splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

We clone and analyze reporter genes generating circular RNAs. These reporter genes are larger than constructs to analyze linear splicing and contain Alu elements. To investigate the circular RNAs, the constructs are transfected into cells and resulting RNA is analyzed using RT-PCR after removal of linear RNA.

Dairesel RNA'ları Analiz Etmek Için Minigen İçeren Alu Elementin Kullanımı

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

Biyokimya

2D and 3D Chromosome Painting in Malaria Mosquitoes

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.

Chromosome painting is a useful method for studying organization of the cell nucleus and evolution of the karyotype. Here, we demonstrate an approach to isolate and amplify specific regions of interest from single polytene chromosomes that are subsequently used for two- and three-dimensional fluorescent in situ hybridization (FISH).

Sıtma Sivrisineklerinde 2D ve 3D Kromozom Boyama

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting ...

İmmünoloji ve Enfeksiyon

Kanser Dokusu Örneklerinde Onkojenik Etkenler için FISH Analizinin Geliştirilmesi

Robust Detection of Gene Amplification in Formalin-Fixed Paraffin-Embedded Samples by Fluorescence In Situ Hybridization

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While sequencing-based methodologies enable an unbiased identification of ecDNA, cytogenetic-based techniques, such as fluorescence in situ hybridization (FISH), remain time and cost-effective for identifying ecDNA in clinical specimens. The application of FISH in formalin-fixed paraffin-embedded (FFPE) tissue samples offers a unique avenue for detecting amplified genes, particularly when viable specimens are not available for karyotype examination. However, there is a lack of consensus procedures for this technique. This protocol provides comprehensive, fully optimized, step-by-step instructions for conducting FISH to detect gene amplification, including ecDNA, in FFPE tissue samples which present unique challenges that this protocol aims to overcome and standardize. By following this protocol, researchers can reproducibly acquire high-quality imaging data to assess gene amplification.

Yazar Spotlight: Kanser Örneklerinde Hassas Gen Amplifikasyon Değerlendirmesi için Bir Araç Olarak FISH

This protocol provides a reproducible method to visualize gene amplification in formalin-fixed paraffin-embedded (FFPE) tissue specimens.

Floresan In Situ Hibridizasyon ile Formalinle Sabitlenmiş Parafine Gömülü Numunelerde Gen Amplifikasyonunun Sağlam Tespiti

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While ...

Kanser Araştırmaları

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

Genetik Dernekleri Fonksiyonel Dayanak Test bir Allel özel Gen İfadesi Testi

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Biyoloji

Associated Chromosome Trap for Identifying Long-range DNA Interactions

Genetic information encoded by DNA is organized in a complex and highly regulated chromatin structure. Each chromosome occupies a specific territory, that may change according to stage of development or cell cycle. Gene expression can occur in specialized transcriptional factories where chromatin segments may loop out from various chromosome territories, leading to co-localization of DNA segments which may exist on different chromosomes or far apart on the same chromosome. The Associated Chromosome Trap (ACT) assay provides an effective methodology to identify these long-range DNA associations in an unbiased fashion by extending and modifying the chromosome conformation capture technique. The ACT assay makes it possible for us to investigate mechanisms of transcriptional regulation in trans, and can help explain the relationship of nuclear architecture to gene expression in normal physiology and during disease states.

The associated chromosome trap (ACT) assay is a novel unbiased method for identifying long-range DNA interactions. The characterization of long range DNA interactions will allow us to determine the relationship of nuclear architecture to gene expression in both normal physiology and in diseased states.

Uzun menzilli DNA Etkileşimleri belirlenmesi için İlişkili Kromozom Tuzak

Genetic information encoded by DNA is organized in a complex and highly regulated chromatin structure.  Each chromosome occupies a specific territory, ...

Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction followed by probe hybridization on a nylon membrane, which is re-usable. Probes are 5' amine modified to allow fixation to the membrane. Primers are 5' biotin modified which allows detection of hybridized PCR products using streptavidin-peroxidase and a chemiluminescent substrate via photosensitive film. With low setup and consumable costs, this technique is inexpensive (approximately US$2 per sample), high throughput (multiple membranes can be processed simultaneously) and has a short turnaround time (approximately 10 hours). 
The technique can be utilized in a number of ways. Multiple probes can be designed to detect sequence variation within a single amplified product, or multiple products can be amplified
simultaneously, with one (or more) probes used for subsequent detection. A combination of both approaches can also be used within a single assay. The ability to include multiple probes for a single target sequence makes the assay highly specific.
Published applications of mPCR/RLB include detection of antibiotic resistance genes1,2, typing of methicillin-resistant Staphylococcus aureus3-5 and Salmonella sp6, molecular serotyping of Streptococcus pneumoniae7,8, Streptococcus agalactiae9 and enteroviruses10,11, identification of Mycobacterium sp12, detection of genital13-15 and respiratory tract16 and other17 pathogens and detection and identification of mollicutes18. However, the versatility of the technique means the applications are virtually limitless and not restricted to molecular analysis of micro-organisms.
The five steps in mPCR/RLB are a) Primer and Probe design, b) DNA extraction and PCR amplification c) Preparation of the membrane, d) Hybridization and detection, and e) Regeneration of the Membrane.

Multiplex PCR and Reverse Line Blot Hybridization Assay mPCR/RLB

An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.

Multiplex PCR ve Reverse Line Blot Hibridizasyon Testi (mPCR / RLB)

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction ...

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (<a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a>). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

SPLINTER kullanarak Pooled Sıralama gelen Nadir Genomik Variantların Algılama

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...

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

Özet

Daha Fazla Video Keşfet

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