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.

1. Preparación del vinculador Cassettes (LC) <ol><li> Mezclar 40 l de LC1 oligonucleótido (Tabla 1), 40 l de LC2 oligonucleótido (Tabla 1, con la enzima de restricción apropiada voladizo), 110 l de Tris-HCl (100 mM, pH 7,5), y 10 l 250 mM de MgCl 2. </li><li> Incubar a 95 ° C durante 5 minutos y dejar que la reacción se enfríe lentamente a temperatura ambiente. Añadir 300 l de H 2 O y concentrarse dsLinker-ADN en un filtro de centrifugación. Añadir 80 l H 2 O al eluato y alícuotas de 10 l de casete de engarce dispuesto en 0.2 tubos de PCR. </li></ol> 2. Preamplificación del genoma del vector Uniones <ol><li> Para cada muestra a analizar preparar una reacción de PCR de 50 l. <ol><li> Determinar la concentración de las muestras de ADN. Pipeta x l (1-1.000 ng para LAM/100-1, 000 ng para nrLAM) de ADN en un tubo de PCR de 0,2 ml. Volumen de ADN debe ser igual en cada muestra y en la RANGE de 0,5 a 25 l. </li><li> Preparar mezcla maestra de PCR como se describe en la Tabla 2 de la mezcla (50 - x). L de la mezcla maestra con cada muestra de ADN en 0,2 ml tubos de PCR. </li></ol></li><li> Preamplificar vector uniones genoma utilizando condiciones de PCR ejemplificados en la Tabla 2. Después de la terminación de la PCR añaden 0,5 l de Taq polimerasa a cada tubo de PCR y vuelva a ejecutar el programa de PCR. Los productos de PCR se pueden almacenar a 4 ° C durante un máximo de 4 días o largo plazo a -20 ° C. </li></ol> 3. Separación Magnética de la PCR Producto <ol><li> Preparación de perlas magnéticas <ol><li> Pipetear 20 l (200 g) de perlas magnéticas recubiertas con estreptavidina en un tubo de 1,5 ml y se exponga durante 1 min en el separador de partículas magnéticas (MPS) a temperatura ambiente. Desechar el sobrenadante. </li><li> Saque el tubo del MPS y volver a suspender las perlas magnéticas en 40 l PSB / BSA al 0,1% (pH 7,5). La exposicion a MPS durante 1 min y desechar el sobrenadante. Repita este paso una vez. </li><li> Lavar perlas wiº 20 l de solución de LiCl 3 M (3 M LiCl, 10 mM de Tris-HCl, 1 mM EDTA) exponer a MPS durante 1 minuto y desechar el sobrenadante. Resuspender las perlas en 50 l de solución 6 M de LiCl (6 M de LiCl, mM de Tris-HCl 10, EDTA 1 mM). </li></ol></li><li> Mezcla de reacción PCR entera de la etapa 2.2 con 50 l de perlas magnéticas preparadas. Incubar en un agitador horizontal (300 rpm) a menos de 2 horas a temperatura ambiente. Este paso permite la unión del producto de PCR biotinilado a estreptavidina perlas recubiertas (complejo ADN-Bead). Complejo de grano de ADN se puede incubar en un agitador durante la noche o se almacenó a 4 ° C durante hasta 4 días. </li><li> La exposicion complejo ADN-Bead para MPS durante 1 minuto a temperatura ambiente, quite el sobrenadante y resuspender complejo ADN-bolas en 100 l de H 2 O. Proceder inmediatamente con el paso 4 para LAM o el paso 5 para nrLAM. </li></ol> 4. LAM-Procedimiento <ol><li> ADN de doble cadena (dsDNA) Síntesis (LAM solamente) <ol><li> La exposicion complejo ADN-Bead desde el paso 3.3 para MPS por 1 min y dissobrenadante tarjeta. Añadir 8,25 l de H 2 O, 1 l de tampón 10x hexanucleótido, 0,25 mu l dNTPs (10 mM) y 0,5 l (2 U) Klenow polimerasa. Incubar a 37 ° C durante 1 hora. </li><li> Añadir 90 l de H 2 O y se exponga a MPS durante 1 min. Desechar el sobrenadante y complejo ADN-Bead resuspender en 100 l de H 2 O. </li></ol></li><li> Digestión de restricción <ol><li> La exposicion complejo ADN-Bead para MPS por 1 min y desechar el sobrenadante. Añadir 8,5 l de H 2 O, 1 l de 10x tampón de enzima de restricción y 0,5 l de enzima de restricción y se incuba la reacción durante 1 hora. Repita el paso 4.1.2. NOTA: Se incuba la reacción a la temperatura recomendada por el fabricante de la enzima de restricción. Asegúrese de que no hay ningún sitio de restricción presentes dentro o aguas abajo del sitio de unión del cebador utilizado para la preamplificación en el ADN de interés. Para la elección de combinaciones de enzimas enzimas de restricción / limitación adecuados referirse a 9. </li></ol></li><li> La ligación de DS Linker (LK) <ol><li> La exposicion complejo ADN-Bead para MPS por 1 min y desechar el sobrenadante. Añadir 5 l de H 2 O, 1 l de tampón 10x FastLink, 1 l de ATP (10 mM), 2 l vinculador casete desde el paso 1.2 y 1 l Fast-Link ADN ligasa (2 U / l). Incubar a temperatura ambiente durante 5 min. Repita el paso 4.1.2. </li></ol></li><li> La desnaturalización de ADN de doble cadena sintetizado <ol><li> La exposicion complejo ADN-Bead para MPS por 1 min y desechar el sobrenadante. Resuspender complejo ADN-bolas en 5 l 0,1 N NaOH. Incubar 5 min a temperatura ambiente en un agitador horizontal. </li><li> Exponer complejo de ADN-Talón a MPS durante 1 min y recoger unión vector de genoma-preamplificada que contiene sobrenadante en nuevo tubo de 1,5 ml. Proceder de inmediato con el paso 6 o tienda sobrenadante a -20 ° C. </li></ol></li></ol> 5. NrLAM-Procedimiento <ol><li> La ligación del enlazador de cadena sencilla (SSLC) (nrLAM solamente) <ol><li> La exposicion complejo ADN-Bead desde el paso 3.3 para MPSdurante 1 min y el sobrenadante de descarte. Añadir 6,5 l de H 2 O, 1 l CircLigase 10x tampón de reacción, 0,5 l de MnCl2 (50 mM), 0,5 l de ATP (1 mM), 1 l de oligonucleótido ssLinker y 0,5 l CircLigase (100 U / l). Incubar a 60 ° C durante 1 hora. </li><li> Añadir 90 l de H 2 O y se exponga a MPS durante 1 min. Desechar el sobrenadante y lavar complejo ADN-bolas en 100 l de H 2 O. Una vez más exponer a MPS por 1 min, el sobrenadante y resuspender descarte complejo ADN-Bead de 10 l H 2 O. </li></ol></li></ol> 6. Exponencial de amplificación me <ol><li> Para cada muestra a analizar preparar una reacción de PCR de 50 l. <ol><li> Pipetear 2 ADN molde l (del paso 4.4.2 (LAM) o 5.1.2 (nrLAM)) en un tubo de PCR de 0,2 ml. </li><li> Preparar mezcla maestra de PCR como se describe en la Tabla 3. Añadir 48 l de mezcla maestra a cada muestra de la etapa 6.1.1 y amplificar las uniones genoma del vector por PCR conditions ejemplificados en la Tabla 3. productos de la PCR se pueden almacenar a 4 ° C durante un máximo de 4 días o largo plazo a -20 ° C. </li></ol></li></ol> 7. Separación Magnética de la PCR Producto <ol><li> Preparar perlas magnéticas como se ejemplifica en los pasos 3.1.1 - 3.1.3. Resuspender las perlas en 20 l (para LAM) o 50 l (para nrLAM) de solución 6 M de LiCl (6 M de LiCl, mM de Tris-HCl 10, EDTA 1 mM). Mezclar 20 l (LAM) o 50 l (nrLAM) de reacción de PCR de la etapa 6.1.2 con perlas magnéticas preparadas (paso 7,1) e incubar en un agitador horizontal (300 rpm) durante 2 horas a temperatura ambiente. Complejo de grano de ADN se puede incubar en un agitador durante la noche o se almacenó a 4 ° C durante hasta 4 días. </li><li> La exposicion complejo ADN-Bead para MPS por 1 min, retirar el sobrenadante y resuspender complejo ADN-bolas en 100 l de H 2 O. La exposicion complejo ADN-Bead para MPS por 1 min y desechar el sobrenadante. </li><li> Resuspender complejo ADN-Bead de 20 l (LAM) o 5 l (nrLAM) 0,1 N NaOH. Incubate durante 10 min a temperatura ambiente en un agitador horizontal, exponga a MPS durante 1 min y recoger el ADN que contiene sobrenadante amplificado en nuevo tubo de 1,5 ml. Proceder de inmediato con el paso 8.1 o tienda sobrenadante a -20 ° C. </li></ol> 8. Exponencial de amplificación II <ol><li> Para cada muestra a analizar preparar una reacción de PCR de 50 l. <ol><li> Pipetear 2 l de ADN plantilla (de la etapa 7.3) en un tubo de PCR de 0,2 ml. </li><li> Preparar mezcla maestra de PCR como se describe en la Tabla 4. Añadir 48 l de mezcla maestra a cada muestra de la etapa 8.1.1 y amplificar las uniones genoma del vector por las condiciones de PCR ejemplificados en la Tabla 4. Productos de la PCR se pueden almacenar a 4 ° C durante hasta 4 días o largo plazo a -20 ° C. </li></ol></li><li> Para visualizar (NR) productos LAM-PCR, carga de 10 l del producto de PCR de la etapa 8.1.2 en un gel de agarosa al 2%. Si las bandas son visibles, analizar 10 l de producto de LAM-PCR en gel de alta resolución. PRECAUCIÓN: Etbromuro hidium es mutagénico. Trabaje con mucho cuidado y siempre use guantes apropiados. </li></ol> 9. Preparación para la secuenciación de alto rendimiento <ol><li> Purificación de (nr) productos LAM-PCR <ol><li> Mezclar 40 l PCR-producto del paso 8.1.2 con 44 l de temperatura ambiente AMPure XP perlas magnéticas. Incubar 5 minutos a temperatura ambiente y exponerlo a MPS durante 2 min. </li><li> Desechar el sobrenadante y lavar dos veces con 200 l 70% de EtOH en las MPS. </li><li> Desechar el sobrenadante y complejo ADN-Bead resuspender en 30 l H 2 O. Incubar 1 min y transferir el sobrenadante a fresco tubo 0,2 ml. Determinar la concentración de ADN purificado. </li></ol></li><li> Fusionprimer PCR para añadir la secuenciación de adaptadores específicos. <ol><li> Para cada muestra a analizar preparar una reacción de PCR de 50 l. </li><li> Pipeta x l (40 ng) de ADN en un tubo de PCR de 0,2 ml. Volumen de ADN debe ser igual en cada muestra y en el intervalo de 0,5 a 25 l. </li><li> Prepare la mezcla maestra de PCR como se describe en la tabla 5 Agregar (50 - x). L de mezcla maestra a cada muestra a partir del paso 9.2.2 e introducir adaptadores de secuenciación a (nr) productos LAM-PCR por PCR condiciones ejemplificados en la Tabla 5 los productos de PCR. puede almacenarse a 4 ° C durante un máximo de 4 días o largo plazo a -20 ° C. </li><li> Para visualizar Fusionprimer-PCR productos de carga 10 l de producto de PCR de la etapa 9.2.3 en un gel de agarosa al 2%. Purificar el producto remanente PCR como se describe en los pasos 9.1.1 - 9.1.3. Analizar 1 l producto de PCR purificado de la etapa 9.4 en un dispositivo de electroforesis de alta resolución automatizado para cuantificar con precisión la concentración y el fragmento de tamaño de los productos Fusionprimer-PCR. </li></ol></li></ol>

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The authors have nothing to disclose.

La técnica de LAM-PCR permite la identificación de secuencias de ADN desconocidas que flanquean una región de ADN conocida. Debido a la alta sensibilidad resultante de preamplificación de las uniones con cebadores específicos de hibridación en la secuencia de ADN conocida, es posible amplificar y detectar incluso uniones raras hasta el nivel de una sola célula. Contrariamente, en una situación policlonal LAM-PCR es capaz de amplificar miles de diferentes uniones en una sola reacción. <p class="jove_content"...

Lineal-amplificación mediada por PCR (LAM-PCR) permite la identificación y caracterización de ADN que flanquea desconocida adyacente a ADN conocido de cualquier origen. Más específicamente, LAM-PCR se ha desarrollado para localizar los sitios de integración de vectores virales (IS) dentro del genoma del huésped 1,2. Los elementos genéticos como retrovirus o transposones integrar su genoma en el genoma huésped en un (semi-) de forma aleatoria 3-6. En muchos casos es decisivo para saber exactamente la posición en que estos vectores integrados. LAM-PCR se ha demostrado ser superior a las técnicas alternativas como la ligadura mediada por PCR 7 y sus variantes o PCR inversa 8. La sensibilidad y la robustez de este método surge de la etapa previa de amplificación inicial de las uniones de vector y el genoma de la selección magnética de los productos de PCR amplificados. Al igual que los métodos alternativos mencionados, LAM-PCR se basa en el uso de enzimas de restricción, la introducción de un sesgo en la capacidad de recuperación de IS 9-11. Por lo tanto,sólo un subconjunto del repertorio ESTÁ (la integrome) se puede detectar en una reacción. Este sesgo se minimiza por el análisis paralelo de una muestra dada usando combinaciones óptimas de las enzimas de restricción 9. Recientemente, una variante de la tecnología denomina no restrictiva LAM-PCR (nrLAM-PCR) se ha desarrollado que evita el uso de enzimas de restricción y permite el análisis de todo el genoma imparcial de una muestra en una sola reacción 9,12. En el pasado, LAM-PCR se ha utilizado para identificar el causante retroviral está dando lugar a la leucemia en algunos pacientes en los ensayos clínicos de terapia génica 13-15. Desde entonces, LAM-PCR ha sido adaptado para identificar es de otros vectores de integración (vectores lentivirales, transposones) y también para identificar patrones de integración de la integración de pasivamente vectores como vectores adeno-asociados (AAV) o vectores lentivirales-integrasa defectuosa (IDLV) 16 -21. Aplicaciones de la LAM-PCR son de amplia expansión: tradicionalLY, la técnica se usa ampliamente para estudiar la composición clonal de células de genes modificados en los pacientes que han sido sometidos a la terapia génica o para evaluar la seguridad de la biotecnología de sistemas de vectores novedosos por desentrañar su comportamiento integración 15,16,22-24. Recientemente, LAM-PCR permitió determinar la especificidad y la actividad fuera del objetivo de las nucleasas de diseño mediante un ensayo de captura de IDLV 25. Por otra parte, LAM-PCR permite seguir fácilmente el destino de una célula transducida con el tiempo en un organismo. Esto permite identificar los proto-oncogenes, así como genes supresores de tumores y también para estudiar la hematopoyesis o madre de cáncer biología celular 26-28. Por último, pero no menos importante, LAM-PCR fue adaptado para estudiar la diversidad de receptores de células T en seres humanos (29 y datos no publicados). La potencia intrínseca de la tecnología se ve reforzada por la vinculación el método a las tecnologías de secuenciación profundas que permiten la caracterización de millones de ADN desconocido que flanquea con solo nucleótido resolución de genomas enteros. En el siguiente protocolo, se describe paso a paso la amplificación e identificación de ADN flanqueante desconocido a modo de ejemplo para identificar vector lentiviral ES. Los oligonucleótidos utilizados en el protocolo se enumeran en la Tabla 1. Extrajeron el ADN o el ADNc de cualquier fuente se pueden utilizar como plantilla de ADN para LAM-PCR y nrLAM-PCR.

<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 resulta en la amplificación de las uniones genoma de vector con un tamaño de fragmento definido para cada unión. El tamaño de los fragmentos individuales de PCR depende de la distancia entre la ubicación de la ADN conocida en el genoma y el sitio de reconocimiento de enzima de restricción más cercano. Esto permite la visualización de la diversidad de uniones amplificados en muestras analizadas por electroforesis en gel, por ejemplo., Aunque sólo sea sola (monoclonales), varios (oligoclonales), ...

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

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

Lineal-amplificación mediada (LAM)-PCR es un método desarrollado para identificar las posiciones exactas de los vectores de integración viral en el genoma. La técnica ha evolucionado a ser el método superior para estudiar la dinámica clonales en los pacientes de terapia génica, la bioseguridad de las tecnologías de nuevo vector, la diversidad de células T, los modelos de células madre de cáncer, etc

Lineal Amplificación Mediada por PCR - Localización de elementos genéticos y caracterización de ADN que flanquean Desconocido

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

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

29.9K vistas.  National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ).  Lineal-amplificación mediada (LAM)-PCR es un método desarrollado para identificar las posiciones exactas de los vectores de integración viral en el genoma. La técnica ha evolucionado a ser el método superior para estudiar la dinámica clonales en los pacientes de terapia génica, la bioseguridad de las tecnologías de nuevo vector, la diversidad de células T, los modelos de células madre de cáncer, etc 

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

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Monte doble entero hibridación in situ de embriones de pollo temprano

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

Investigación

Biología

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

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

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

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Los estudios genéticos de proteínas humanas de reparación del ADN utilizando la levadura como un sistema modelo

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

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.

In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.

Mediada por adenovirus eliminación de moléculas de señalización genética en cultivos de fibroblastos de embriones de ratón primaria

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

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

Extracción de ADN a partir de material incluido en parafina para análisis genéticos y epigenéticos

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

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, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

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

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

La determinación de perfiles genéticos de expresión en C. elegans Utilización de microarrays y PCR en tiempo real

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

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

Combinando Manipulación sola molécula y de imagen para el estudio de las interacciones proteína-ADN

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

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

ADN electroporación, aislamiento e Imagen de miofibras

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

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

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

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

En Vivo alcalina Comet Assay y Comet Assay alcalina modificada con enzimas para la medición de roturas en el ADN Strand y daño oxidativo del ADN en el hígado de rata

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

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Amplificación, secuenciación de próxima generación, y Genómica Mapeo de ADN retrovirales sitios de integración

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

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and activates the mixed lineage kinase-like domain pseudokinase, MLKL, to rupture or permeabilize the plasma membrane. Necroptosis is an inflammatory pathway associated with multiple pathologies including autoimmunity, infectious and cardiovascular diseases, stroke, neurodegeneration, and cancer. Here, we describe protocols that can be used to characterize MLKL as the executioner of plasma membrane rupture in necroptosis. We visualize the process of necroptosis in cells using live-cell imaging with conventional and confocal fluorescence&#160;microscopy, and in fixed cells using electron microscopy, which together revealed the redistribution of MLKL from the cytosol to the plasma membrane prior to induction of large holes in the plasma membrane. We present in vitro nuclear magnetic resonance (NMR) analysis using lipids to identify putative modulators of MLKL-mediated necroptosis. Based on this method, we identified quantitative lipid-binding preferences and phosphatidyl-inositol phosphates (PIPs) as critical binders of MLKL that are required for plasma membrane targeting and permeabilization in necroptosis.

We report methods for characterization of MLKL-mediated plasma membrane rupture in necroptosis including conventional and confocal live-cell microscopy imaging, scanning electron microscopy, and NMR-based lipid binding.