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. Vorbereitung der Linker-Kassetten (LC) <ol><li> Mischungs 40 ul LC1 Oligonukleotid (Tabelle 1), 40 ul Oligonukleotid LC2 (Tabelle 1, mit der richtigen Restriktionsenzym Hang), 110 &amp;mgr; l Tris-HCl (100 mM, pH 7,5) und 10 ul 250 mM MgCl 2. </li><li> Inkubation bei 95 ° C für 5min und ließ die Reaktion abkühlen langsam auf Raumtemperatur. In 300 ul H 2 O und konzentrieren dsLinker-DNA auf einem Zentrifugation Filter. In 80 ul H 2 O zu den aliquoten Eluat und 10 ul bereit Linker-Kassette in 0,2 PCR-Röhrchen. </li></ol> 2. Vorverstärkung der Vektor-Genom-Junctions <ol><li> Für jede zu analysierende Probe bereiten ein 50 ul PCR-Reaktion. <ol><li> Bestimmen Konzentration der DNA-Proben. Pipette x ul (1-1000 ng für LAM/100-1, 000 ng für nrLAM) von DNA in eine 0,2 ml PCR-Röhrchen. Volumen der DNA sollte in jeder Probe und in der ran gleich seinge von 0,5 bis 25 &amp;mgr;. </li><li> Vorbereitung PCR Master Mix, wie in Tabelle 2 beschrieben Mix (50 - x). &amp;mgr; L Mastermix mit jeder DNA-Probe in 0,2 ml PCR-Röhrchen. </li></ol></li><li> Preamplify Vektorgenom Kreuzungen mit PCR-Bedingungen in Tabelle 2 veranschaulicht. Nach Abschluss der PCR mit 0,5 ul Taq-Polymerase zu jedem PCR-Röhrchen und erneut PCR-Programm. PCR-Produkte können bei 4 ° C für bis zu 4 Tage oder langfristig bei -20 º C gelagert werden </li></ol> 3. Magnetische Trennung von PCR-Produkt <ol><li> Vorbereitung der Magnetic Beads <ol><li> Je 20 ul (200 ug) von Streptavidin-beschichteten magnetischen Kügelchen in ein 1,5-ml-Röhrchen und setzen für 1 min auf dem Magnet Partikelabscheider (MPS) bei Raumtemperatur. Überstand verwerfen. </li><li> Röhrchen aus MPS und resuspendieren magnetischen Kügelchen in 40 ul PSB / 0,1% BSA (pH 7,5). Expose MPS für 1 min und Überstand verwerfen. Wiederholen Sie diesen Schritt einmal. </li><li> Wash Perlen witen 20 ul 3 M LiCl-Lösung (3 M LiCl, 10 mM Tris-HCl, 1 mM EDTA) aussetzen MPS für 1 min und Überstand verwerfen. Resuspendieren Perlen in 50 ul 6 M LiCl-Lösung (6 M LiCl, 10 mM Tris-HCl, 1 mM EDTA). </li></ol></li><li> Mix gesamten PCR-Reaktion aus Schritt 2.2 mit 50 ul hergestellten magnetischen Kügelchen. Inkubieren auf einem Horizontalschüttler (300 rpm) mindestens 2 h bei Raumtemperatur. Dieser Schritt ermöglicht die Bindung von biotinylierten PCR-Produkt zu beschichteten Kügelchen (DNA-Bead-Komplex) Streptavidin. DNA-Bead-Komplex auf dem Schüttler über Nacht inkubiert und bei 4 ° C für 4 Tage aufbewahrt werden. </li><li> Expose DNA-Bead komplex MPS für 1 min bei Raumtemperatur entfernen Überstand und resuspendieren DNA-Bead-Komplex in 100 ul H 2 O. Sofort mit Schritt 4 für LAM oder Schritt 5 für nrLAM fortzufahren. </li></ol> 4. LAM-Verfahren <ol><li> Doppelstrang-DNA (dsDNA) Synthese (LAM nur) <ol><li> Expose DNA-Bead-Komplex aus Schritt 3,3 bis 1 min und dis MPSKartenstand. Hinzufügen 8,25 ul H &amp;sub2; O, 1 &amp;mgr; l 10 × Puffer Hexanukleotid, 0,25 ul dNTPs (10 mM) und 0,5 ul (2 U) Klenow-Polymerase. Inkubieren bei 37 ° C für 1 Stunde. </li><li> Hinzufügen von 90 &amp;mgr; l H 2 O und aussetzen MPS für 1 min. Überstand verwerfen und resuspendieren DNA-Bead-Komplex in 100 ul H 2 O. </li></ol></li><li> Beschränkung Digest <ol><li> Expose DNA-Bead komplex MPS für 1 min und Überstand verwerfen. Add 8,5 ul H &amp;sub2; O, 1 ul 10x Restriktionsenzym-Puffer und 0,5 ul Restriktionsenzym inkubieren Reaktion für 1 Stunde. Wiederholen Sie Schritt 4.1.2. HINWEIS: Inkubieren Reaktion bei der Temperatur, die vom Hersteller des Restriktionsenzyms empfohlen. Stellen Sie sicher, dass es innerhalb von nicht vorhanden oder nach der Primer-Bindungsstelle für die Vorverstärkung in der DNA von Interesse verwendet Restriktionsstelle. Für die Wahl des geeigneten Restriktionsenzymen / Restriktionsenzym-Kombinationen beziehen sich auf neun. </li></ol></li><li> Ligation von ds Linker (LK) <ol><li> Expose DNA-Bead komplex MPS für 1 min und Überstand verwerfen. Mit 5 &amp;mgr; l H 2 O, 1 ul 10x Fastlink-Puffer, 1 ul ATP (10 mM), 2 &amp;mgr; l Linker Kassette aus Schritt 1.2 und 1 ul Fast-Link-DNA-Ligase (2 U / ul). Inkubieren bei Raumtemperatur für 5 min. Wiederholen Sie Schritt 4.1.2. </li></ol></li><li> Denaturierung von Synthesizer-dsDNA <ol><li> Expose DNA-Bead komplex MPS für 1 min und Überstand verwerfen. Resuspendieren DNA-Bead-Komplex in 5 ul 0,1 N NaOH. Inkubiere 5 min bei Raumtemperatur auf einem Horizontalschüttler. </li><li> Expose DNA-Bead komplex MPS für 1 min und sammeln vorverstärktes Vektor-Genom-Übergang Überstand in neue 1,5 ml Röhrchen mit. Sofort mit Schritt 6 oder speichern Stand gehen bei -20 ° C </li></ol></li></ol> 5. NrLAM-Verfahren <ol><li> Ligation von Einzelstrang-Linker (SSLC) (nrLAM nur) <ol><li> Expose DNA-Bead-Komplex aus Schritt 3.3 MPSfür 1 min und Ablagestand. Add 6,5 ul H &amp;sub2; O, CircLigase 1 ul 10x Reaktionspuffer, 0,5 &amp;mgr; l MnCl 2 (50 mM), 0,5 ul ATP (1 mM), 1 ul ssLinker Oligonukleotid und 0,5 ul CircLigase (100 U / ul). Inkubieren bei 60 ° C für 1 Stunde. </li><li> Hinzufügen von 90 &amp;mgr; l H 2 O und aussetzen MPS für 1 min. Überstand verwerfen und waschen DNA-Bead-Komplex in 100 ul H 2 O. Wieder zu MPS für 1 min, Ablagestand und resuspendieren DNA-Bead-Komplex in 10 ul H 2 O. aussetzen </li></ol></li></ol> 6. Exponential Amplification ich <ol><li> Für jede zu analysierende Probe bereiten ein 50 ul PCR-Reaktion. <ol><li> Pipette 2 ul Template-DNA (aus Schritt 4.4.2 (LAM) oder 5.1.2 (nrLAM)) in ein 0,2 ml PCR-Röhrchen. </li><li> Bereiten PCR Master Mix, wie in Tabelle 3 beschrieben. Hinzufügen 48 ul der Master-Mix zu jeder Probe aus Schritt 6.1.1 und verstärken Vektorgenom Gänge durch PCR conditions in Tabelle 3 veranschaulicht. PCR-Produkte bei 4 ° C für bis zu 4 Tage oder langfristig bei -20 º C gelagert werden, </li></ol></li></ol> 7. Magnetische Trennung von PCR-Produkt <ol><li> Bereiten magnetischen Kügelchen wie in den Schritten 3.1.1 Beispiel - 3.1.3. Resuspendieren Perlen in 20 ul (LAM) oder 50 &amp;mgr; l (für nrLAM) von 6 M LiCl-Lösung (6 M LiCl, 10 mM Tris-HCl, 1 mM EDTA). Mischungs 20 ul (LAM) oder 50 ul (nrLAM) der PCR-Reaktion aus Schritt 6.1.2 mit vorbereiteten magnetischen Kügelchen (Schritt 7.1) und Inkubation auf einem Horizontalschüttler (300 rpm) für 2 h bei Raumtemperatur. DNA-Bead-Komplex auf dem Schüttler über Nacht inkubiert und bei 4 ° C für 4 Tage aufbewahrt werden. </li><li> Expose DNA-Bead komplex MPS für 1 min, entfernen Stand und resuspendieren DNA-Bead-Komplex in 100 ul H 2 O. Expose DNA-Bead komplex MPS für 1 min und Überstand verwerfen. </li><li> Resuspendieren DNA-Bead-Komplex in 20 ul (LAM) oder 5 ul (nrLAM) 0,1 N NaOH. IncuDebatte für 10 Minuten bei Raumtemperatur auf einem Horizontalschüttler, aussetzen MPS für 1 min und sammeln amplifizierte DNA-haltige Überstand in neues 1,5-ml-Tube. Sofort mit Schritt 8.1 oder speichern Stand gehen bei -20 ° C </li></ol> 8. Exponential Amplification II <ol><li> Für jede zu analysierende Probe bereiten ein 50 ul PCR-Reaktion. <ol><li> Pipette 2 &amp;mgr; l Matrizen-DNA (aus Stufe 7.3) in 0,2 ml PCR-Röhrchen. </li><li> Vorbereitung PCR Master Mix, wie in Tabelle 4 beschrieben. In 48 &amp;mgr; l Mastermix zu jeder Probe aus Schritt 8.1.1 und verstärken Vektorgenom Gänge von PCR-Bedingungen in Tabelle 4 veranschaulicht. PCR-Produkte bei 4 ° C für bis zu 4 gespeichert werden, Tage-oder langfristig bei -20 ° C </li></ol></li><li> Zu visualisieren (nr) LAM-PCR-Produkte, Last 10 ul des PCR-Produkts aus Schritt 8.1.2 auf einem 2% Agarosegel. Wenn Banden sichtbar sind, zu analysieren 10 ul der LAM-PCR-Produkt auf hochauflösenden Gel. ACHTUNG: Ethidium Bromid ist mutagen. Arbeiten Sie sehr sorgfältig und immer eine geeignete Schutzhandschuhe. </li></ol> 9. Vorbereitung für die Hochdurchsatz-Sequenzierung <ol><li> Reinigung von (nr) LAM-PCR-Produkte <ol><li> Mischen Sie 40 ul PCR-Produkt von Schritt 8.1.2 mit 44 ul der Raumtemperatur AMPure XP Magnetic Beads. Inkubiere 5 min bei Raumtemperatur ausgesetzt werden, um MPS für weitere 2 min. </li><li> Überstand verwerfen und zweimal waschen mit 200 ul 70% EtOH auf den MPS. </li><li> Überstand verwerfen und resuspendieren DNA-Bead-Komplex in 30 ul H 2 O. 1 min inkubieren und Überstand in frische 0,2 ml-Tube. Bestimmen Konzentration von DNA gereinigt. </li></ol></li><li> Fusionprimer PCR-Sequenzierung, um spezifische Adapter hinzufügen. <ol><li> Für jede zu analysierende Probe bereiten ein 50 ul PCR-Reaktion. </li><li> Pipette x ul (40 ng) von DNA in eine 0,2 ml PCR-Röhrchen. Volumen von DNA sollte in jeder Probe und in dem Bereich von 0,5 bis 25 &amp;mgr; gleich sein. </li><li> Bereiten PCR Master Mix, wie in Tabelle 5 beschrieben hinzufügen (50 - x). Ul Master Mix zu jeder Probe aus Schritt 9.2.2 und Sequenzierung einzuführen Adapter (nr) LAM-PCR-Produkte von PCR-Bedingungen in Tabelle 5 PCR-Produkte veranschaulicht. bei 4 ° C für bis zu 4 Tage oder langfristig bei -20 º C gelagert werden </li><li> Um Fusionprimer-PCR-Produkte sichtbar Last 10 ul des PCR-Produkts aus Schritt 9.2.3 auf einem 2% Agarosegel. Reinige verbleibenden PCR-Produkt wie in den Schritten 9.1.1 - 9.1.3. Analysieren 1 ul gereinigte PCR-Produkt aus Schritt 9.4 auf einem automatisierten Hochauflösung Elektrophorese-Gerät genau zu quantifizieren, Konzentration und Fragmentgröße von Fusionprimer-PCR-Produkte. </li></ol></li></ol>

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The authors have nothing to disclose.

Die LAM-PCR-Technik ermöglicht die Identifizierung von unbekannten DNA-Sequenzen, die eine bekannte DNA-Region flankieren. Wegen der hohen Empfindlichkeit von Vorverstärkung der Verbindungen mit spezifischen Primern hybridisiert in der bekannten DNA-Sequenz ergeben, ist es möglich, zu verstärken und zu detektieren, auch seltene Kreuzungen auf Einzelzellebene. Gegenteil, in einem polyklonalen Situation LAM-PCR ist in der Lage, Tausende von verschiedenen Verbindungen in einer einzigen Reaktion zu verstärken. <p c...

Linear-PCR-vermittelte Amplifikation (LAM-PCR) ermöglicht die Identifizierung und Charakterisierung von unbekannten flankierenden DNA benachbart zu bekannten DNA beliebigen Ursprungs. Genauer gesagt, LAM-PCR wurde zur viralen Vektor Integrationsstellen zu lokalisieren (IS) innerhalb des Wirtsgenoms 1,2. Genetische Elemente wie Transposons oder Retroviren integrieren ihr Genom in das Wirtsgenom in einer (halb-) zufälliger Weise 3-6. In vielen Fällen ist es entscheidend, genau die Position, wo diese Vektoren integriert kennen. LAM-PCR nachgewiesen worden überlegen alternative Techniken wie PCR-vermittelte Ligation 7 und seiner Varianten oder inversen PCR-8 sein. Die Empfindlichkeit und Robustheit dieses Verfahrens ergibt sich aus der anfänglichen Vorverstärkung der Vektor-Genom Gänge und magnetische Auswahl amplifizierte PCR-Produkte. Wie die genannten alternativen Verfahren beruht LAM-PCR auf der Verwendung von Restriktionsenzymen, die Einführung eines Bias in Abrufkapazität 9-11. Folglichnur ein Teil des IS-Repertoire (die integrome) in einer Reaktion nachgewiesen werden. Diese Vorspannung wird durch die parallele Analyse einer gegebenen Probe mit optimalen Kombinationen von Restriktionsenzymen 9 minimiert. Kürzlich wurde eine Variante der Technik bezeichnet nicht einschränkenden LAM-PCR (nrLAM-PCR) entwickelt worden, die die Verwendung von Restriktionsenzymen umgeht und ermöglicht unvoreingenommene genomweiten Analyse einer Probe in einem einzigen Reaktions 9,12. In der Vergangenheit LAM-PCR wurde zur Identifizierung der Verursacher retroviralen IS, die zu Leukämie bei einigen Patienten in der klinischen Gentherapie-Versuche 13-15. Seitdem LAM-PCR wurde angepasst, um zu identifizieren, ist von anderen Integrationsvektoren (lentiviralen Vektoren, Transposons) sowie Integrationsmuster passiv integrierende Vektoren wie Adeno-assoziierten Vektoren (AAV) oder Integrase-defekt lentiviralen Vektoren (IDLV) identifizieren 16 -21. Anwendungen der LAM-PCR sind weit verbreitet: traditionellely, die Technik ist weit verbreitet, um die klonale Zusammensetzung von Gen-veränderten Zellen bei Patienten, die Gen-Therapie unterzogen haben oder studieren, um die biologische Sicherheit von neuartigen Vektorsysteme mit der Entschlüsselung ihrer Integration 15,16,22-24 Verhalten zu beurteilen. Kürzlich LAM-PCR aktiviert Bestimmung Spezifität und Off-Target-Aktivität von Designer-Nukleasen durch eine IDLV Trapping-Assay 25. Darüber hinaus ermöglicht LAM-PCR, um leicht folgen das Schicksal einer Zelle transduziert im Laufe der Zeit in einem Organismus. Dies erlaubt es, Proto-Onkogene als auch Tumor-Suppressor-Gene zu identifizieren und auch zu Blutbildung oder Krebsstammzellbiologie 26-28 studieren. Nicht zuletzt, LAM-PCR wurde an T-Zell-Rezeptor-Diversity beim Menschen 29 (unveröffentlichte Daten) zu studieren. Die Eigenleistung der Technik wird durch die Verknüpfung der Methode, um tiefe Sequenzierungstechnologien, die Charakterisierung von Millionen von unbekannten flankierenden DNA mit Einzel-Nukleotid-r erlauben verstärkteSOLUTION in ganzer Genome. In dem folgenden Protokoll beschreiben wir Schritt für Schritt die Amplifikation und Identifizierung von unbekannten DNA-flankierenden exemplarisch zu identifizieren Lentivirusvektor IST. Oligonukleotide in dem Protokoll verwendet wird, sind in Tabelle 1 aufgeführt. Heraus DNA oder cDNA von jeder Quelle kann als DNA-Matrize für LAM-PCR und nrLAM-PCR verwendet werden.

<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-Amplifikation resultiert in der Vektor-Genom-Übergänge mit einer definierten Fragmentgröße für jede Kreuzung. Die Größe der einzelnen PCR-Fragmente hängt von der Entfernung zwischen dem Ort der bekannten DNA in das Genom und dem nächsten Restriktionsenzym-Erkennungsstelle. Dies ermöglicht die Visualisierung der Vielfalt der amplifizierten Gänge in analysierten Proben durch Gelelektrophorese, zB. Wenn nur einzelne (monoklonal), mehrere (oligoklonale) oder mehrere (polyklonal) Banden auf dem Ge...

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

Linear-vermittelte Amplifikation (LAM)-PCR ist eine Abwicklung, die genauen Positionen der Integration von viraler Vektoren in das Genom zu identifizieren Verfahren. Die Technik hat sich weiterentwickelt, um die überlegene Methode zur klonalen Dynamik in der Gentherapie-Patienten, biologische Sicherheit von neuartigen Technologien Vektor, T-Zell-Vielfalt, die Krebsstammzellen Modelle usw. studieren

Linear Amplification vermittelte PCR - Lokalisierung von genetischen Elementen und Charakterisierung von Unbekannt flankierenden DNA-

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 Aufrufe.  National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ).  Linear-vermittelte Amplifikation (LAM)-PCR ist eine Abwicklung, die genauen Positionen der Integration von viraler Vektoren in das Genom zu identifizieren Verfahren. Die Technik hat sich weiterentwickelt, um die überlegene Methode zur klonalen Dynamik in der Gentherapie-Patienten, biologische Sicherheit von neuartigen Technologien Vektor, T-Zell-Vielfalt, die Krebsstammzellen Modelle usw. studieren 

Serie: 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.

Doppel Whole Mount in situ Hybridisierung von Anfang Hühnerembryonen

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

Forschung

Biologie

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.

Genetic Studies of Human DNA-Reparatur-Proteinen unter Verwendung von Hefe als Modellsystem

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.

Adenovirus-vermittelte genetische Entfernung von Signalmolekülen in kultivierten primären embryonalen Maus-Fibroblasten

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.

DNA-Extraktion aus in Paraffin eingebettete Material für genetische und epigenetische Analysen

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.

Bestimmung genetischer Expressionsprofile in C. elegans Verwenden Microarray-und 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

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.

Die Kombination von Einzelmolekül-Imaging und Manipulation für das Studium der Protein-DNA-Wechselwirkungen

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.

DNA Elektroporation, Isolierung und Imaging von Muskelfasern

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.

In - vivo - Alkaline Comet Assay und enzymmodifizierten Alkaline Comet Assay zur Messung von DNA - Strangbrüchen und oxidative DNA - Schäden in der Rattenleber

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.

Amplification, Next-Generation-Sequenzierung und genomischer DNA Mapping von Retrovirale Integration Seiten

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.