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. Preparation of Linker Cassettes (LC)
<ol>
	<li>Mix 40 &#181;l of LC1 oligonucleotide (Table 1), 40 &#181;l of LC2 oligonucleotide (Table 1, with proper restriction enzyme overhang), 110 &#181;l Tris-HCl&#160;(100 mM, pH 7.5), and 10 &#181;l 250 mM MgCl2.</li>
	<li>Incubate at 95 &#176;C for 5min and let the reaction cool down slowly to room temperature. Add 300 &#181;l H2O and concentrate dsLinker-DNA on a centrifugation filter. Add 80 &#181;l H2O to...

<ol>
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The LAM-PCR technique allows identifying unknown DNA sequences that flank a known DNA region. Because of the high sensitivity resulting from preamplification of the junctions with specific primers hybridizing in the known DNA sequence, it is possible to amplify and detect even rare junctions down to the single cell level. Contrary, in a polyclonal situation LAM-PCR is able to amplify thousands of different junctions in one single reaction.
However, due to the use of restriction enzymes only a ...

Linear-amplification mediated PCR (LAM-PCR) allows identifying and characterizing unknown flanking DNA adjacent to known DNA of any origin. More specifically, LAM-PCR has been developed to localize viral vector integration sites (IS) within the host genome1,2. Genetic elements like retroviruses or transposons integrate their genome into the host genome in a (semi-) random manner3-6. In many cases it is decisive to know exactly the position where these vectors integrated. LAM-PCR has been proven to be superior to alternative techniques like ligation-mediated PCR7 and its variants or inverse PCR8. The sensitivity and robustness of this method arises from the initial preamplification of the vector-genome junctions and magnetic selection of amplified PCR products. Like the alternative methods mentioned, LAM-PCR relies on the use of restriction enzymes, introducing a bias into retrieval capacity of IS9-11. Thus, only a subset of the IS repertoire (the integrome) can be detected in one reaction. This bias is minimized by the parallel analysis of a given sample using optimal combinations of restriction enzymes9. Recently, a variant of the technology termed non-restrictive LAM-PCR (nrLAM-PCR) has been developed that circumvents the use of restriction enzymes and allows unbiased genome-wide analysis of a sample in a single reaction9,12.
In the past, LAM-PCR has been used to identify the causative retroviral IS giving rise to leukemia in a few patients in clinical gene therapy trials13-15. Since then, LAM-PCR has been adapted to identify IS from other integrating vectors (lentiviral vectors, transposons) and also to identify integration patterns of passively integrating vectors like adeno-associated vectors (AAV) or integrase-defective lentiviral vectors (IDLV)16-21. Applications of LAM-PCR are wide spread: traditionally, the technique is widely used to study the clonal composition of gene modified cells in patients that have undergone gene therapy or to assess the biosafety of novel vector systems by unraveling their integration behavior15,16,22-24. Recently, LAM-PCR enabled determining specificity and off-target activity of designer nucleases by an IDLV trapping assay25.
Moreover, LAM-PCR allows to easily follow the fate of a transduced cell over time in an organism. This allows to identify proto-oncogenes as well as tumor suppressor genes and also to study hematopoiesis or cancer stem cell biology26-28. Last but not least, LAM-PCR was adapted to study T-cell receptor diversity in humans29 (and unpublished data).
The intrinsic power of the technology is reinforced by linking the method to deep sequencing technologies that allow characterizing millions of unknown flanking DNA with single nucleotide resolution in whole genomes. In the following protocol, we describe step-by step the amplification and identification of flanking unknown DNA exemplarily to identify lentiviral vector IS. Oligonucleotides used in the protocol are listed in Table 1. Extracted DNA or cDNA of any source can be used as DNA template for LAM-PCR and 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 results in amplification of vector genome junctions with a defined fragment size for each junction. The size of individual PCR fragments depends on the distance between the location of the known DNA in the genome and the closest restriction enzyme recognition site. This allows visualizing the diversity of amplified junctions in analyzed samples by gel electrophoresis, e.g., if only single (monoclonal), several (oligoclonal), or multiple (polyclonal) bands are present on the gel. The results of LAM-PCR ar...

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-amplification mediated (LAM)-PCR is a method developed to identify the exact positions of integrating viral vectors in the genome. The technique has evolved to be the superior method to study clonal dynamics in gene therapy patients, biosafety of novel vector technologies, T-cell diversity, cancer stem cell models, etc.

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 ...

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

Research

JoVE Journal

Biology

29.8K 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 