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 the eluate and aliquot 10 &#181;l of prepared linker cassette in 0.2 PCR tubes.</li>
</ol>
2. Preamplification of Vector Genome Junctions
<ol>
	<li>For every sample to be analyzed prepare a 50 &#181;l PCR reaction.
	<ol>
		<li>Determine concentration of the DNA samples. Pipette x &#181;l (1-1,000 ng for LAM/100-1,000 ng for nrLAM) of DNA into a 0.2 ml PCR tube. Volume of DNA should be equal in each sample and in the range of 0.5 to 25 &#181;l.</li>
		<li>Prepare PCR master mix as described in Table 2. Mix (50 - x) &#181;l of the master mix with each DNA sample in 0.2 ml PCR tubes.</li>
	</ol>
	</li>
	<li>Preamplify vector genome junctions using PCR conditions exemplified in Table 2. After completion of PCR add 0.5 &#181;l Taq Polymerase to each PCR tube and rerun PCR program. PCR products can be stored at 4 &#176;C for up to 4 days or long term at -20 &#176;C.</li>
</ol>
3. Magnetic Separation of PCR Product
<ol>
	<li>Preparation of Magnetic Beads
	<ol>
		<li>Pipette 20 &#181;l (200 &#181;g) of streptavidin coated magnetic beads into a 1.5 ml tube and expose for 1 min on the magnetic particle separator (MPS) at room temperature. Discard supernatant.</li>
		<li>Remove tube from MPS and resuspend magnetic beads in 40 &#181;l PSB/ 0.1% BSA (pH 7.5). Expose to MPS for 1 min and discard supernatant. Repeat this step once.</li>
		<li>Wash beads with 20 &#181;l of 3 M LiCl solution (3 M LiCl, 10 mM Tris-HCl, 1 mM EDTA) expose to MPS for 1 min and discard supernatant. Resuspend beads in 50 &#181;l of 6 M LiCl solution (6 M LiCl, 10 mM Tris-HCl, 1 mM EDTA).</li>
	</ol>
	</li>
	<li>Mix entire PCR reaction from step 2.2 with 50 &#181;l of prepared magnetic beads. Incubate on a horizontal shaker (300 rpm) at least for 2 hr at room temperature. This step allows binding of biotinylated PCR product to streptavidin coated beads (DNA-Bead complex). DNA bead complex can be incubated on the shaker overnight or stored at 4 &#176;C for up to 4 days.</li>
	<li>Expose DNA-Bead complex to MPS for 1 min at room temperature, remove supernatant, and resuspend DNA-Bead complex in 100 &#181;l H2O. Immediately proceed with step 4 for LAM or step 5 for nrLAM.</li>
</ol>
4. LAM-Procedure
<ol>
	<li>Double Strand DNA (dsDNA) Synthesis (LAM only)
	<ol>
		<li>Expose DNA-Bead complex from step 3.3 to MPS for 1 min and discard supernatant. Add 8.25 &#181;l of H2O, 1 &#181;l 10x hexanucleotide buffer, 0.25 &#181;l dNTPs (10 mM) and 0.5 &#181;l (2 U) Klenow polymerase. Incubate at 37 &#176;C for 1 hr.</li>
		<li>Add 90 &#181;l of H2O and expose to MPS for 1 min. Discard supernatant and resuspend DNA-Bead complex in 100 &#181;l H2O.</li>
	</ol>
	</li>
	<li>Restriction Digest
	<ol>
		<li>Expose DNA-Bead complex to MPS for 1 min and discard supernatant. Add 8.5 &#181;l H2O, 1 &#181;l 10x restriction enzyme buffer and 0.5 &#181;l of restriction enzyme and incubate reaction for 1 hr. Repeat step 4.1.2. 
		NOTE: Incubate reaction at the temperature recommended by the manufacturer of the restriction enzyme. Make sure that there is no restriction site present within or downstream of the primer binding site used for preamplification in the DNA of interest. For choice of suitable restriction enzymes/restriction enzyme combinations refer to 9.</li>
	</ol>
	</li>
	<li>Ligation of ds Linker (LK)
	<ol>
		<li>Expose DNA-Bead complex to MPS for 1 min and discard supernatant. Add 5 &#181;l H2O, 1 &#181;l 10x FastLink buffer, 1 &#181;l ATP (10 mM), 2 &#181;l Linker cassette from step 1.2, and 1 &#181;l Fast-Link DNA Ligase (2 U/&#181;l). Incubate at room temperature for 5 min. Repeat step 4.1.2.</li>
	</ol>
	</li>
	<li>Denaturation of Synthesized dsDNA
	<ol>
		<li>Expose DNA-Bead complex to MPS for 1 min and discard supernatant. Resuspend DNA-Bead complex in 5 &#181;l 0.1 N NaOH. Incubate 5 min at room temperature on a horizontal shaker.</li>
		<li>Expose DNA-Bead complex to MPS for 1 min and collect preamplified vector-genome junction containing supernatant in new 1.5 ml tube. Immediately proceed with step 6 or store supernatant at -20 &#176;C.</li>
	</ol>
	</li>
</ol>
5. nrLAM-Procedure
<ol>
	<li>Ligation of single stranded linker (ssLC) (nrLAM only)
	<ol>
		<li>Expose DNA-Bead complex from step 3.3 to MPS for 1 min and discard supernatant. Add 6.5 &#181;l H2O, 1 &#181;l CircLigase 10x Reaction Buffer, 0.5 &#181;l MnCl2 (50 mM), 0.5 &#181;l ATP (1 mM), 1 &#181;l ssLinker oligonucleotide and 0.5 &#181;l CircLigase (100 U/&#181;l). Incubate at 60 &#176;C for 1 hr.</li>
		<li>Add 90 &#181;l of H2O and expose to MPS for 1 min. Discard supernatant and wash DNA-Bead complex in 100 &#181;l H2O. Again expose to MPS for 1 min, discard supernatant and resuspend DNA-Bead complex in 10 &#181;l H2O.</li>
	</ol>
	</li>
</ol>
6. Exponential Amplification I
<ol>
	<li>For every sample to be analyzed prepare a 50 &#181;l PCR reaction.
	<ol>
		<li>Pipette 2 &#181;l template DNA (from step 4.4.2 (LAM) or 5.1.2 (nrLAM)) into a 0.2 ml PCR tube.</li>
		<li>Prepare PCR master mix as described in Table 3. Add 48 &#181;l of master mix to each sample from step 6.1.1 and amplify vector genome junctions by PCR conditions exemplified in Table 3. PCR products can be stored at 4 &#176;C for up to 4 days or long term at -20 &#176;C.</li>
	</ol>
	</li>
</ol>
7. Magnetic Separation of PCR Product
<ol>
	<li>Prepare magnetic beads as exemplified in steps 3.1.1 - 3.1.3. Resuspend beads in 20 &#181;l (for LAM) or 50 &#181;l (for nrLAM) of 6 M LiCl solution (6 M LiCl, 10 mM Tris-HCl, 1 mM EDTA). Mix 20 &#181;l (LAM) or 50 &#181;l (nrLAM) of PCR reaction from step 6.1.2 with prepared magnetic beads (step 7.1) and incubate on a horizontal shaker (300 rpm) for 2 hr at room temperature. DNA bead complex can be incubated on the shaker overnight or stored at 4 &#176;C for up to 4 days.</li>
	<li>Expose DNA-Bead complex to MPS for 1 min, remove supernatant and resuspend DNA-Bead complex in 100 &#181;l H2O. Expose DNA-Bead complex to MPS for 1 min and discard supernatant.</li>
	<li>Resuspend DNA-Bead complex in 20 &#181;l (LAM) or 5 &#181;l (nrLAM) 0.1 N NaOH. Incubate for 10 min at room temperature on a horizontal shaker, expose to MPS for 1 min and collect amplified DNA containing supernatant in new 1.5 ml tube. Immediately proceed with step 8.1 or store supernatant at -20 &#176;C.</li>
</ol>
8. Exponential Amplification II
<ol>
	<li>For every sample to be analyzed prepare a 50 &#181;l PCR reaction.
	<ol>
		<li>Pipette 2 &#181;l template DNA (from step 7.3) into a 0.2 ml PCR tube.</li>
		<li>Prepare PCR master mix as described in Table 4. Add 48 &#181;l of master mix to each sample from step 8.1.1 and amplify vector genome junctions by PCR conditions exemplified in Table 4. PCR products can be stored at 4 &#176;C for up to 4 days or long term at -20 &#176;C.</li>
	</ol>
	</li>
	<li>To visualize (nr)LAM-PCR products, load 10 &#181;l of the PCR product from step 8.1.2 on a 2% agarose gel. If bands are visible, analyze 10 &#181;l of the LAM-PCR product on high-resolution gel. CAUTION: Ethidium bromide is mutagenic. Work very carefully and always wear appropriate gloves.</li>
</ol>
9. Preparation for High-throughput Sequencing
<ol>
	<li>Purification of (nr)LAM-PCR products
	<ol>
		<li>Mix 40 &#181;l PCR-Product from step 8.1.2 with 44 &#181;l of room temperature AMPure XP Magnetic Beads. Incubate 5 min at room temperature and expose to MPS for additional 2 min.</li>
		<li>Discard supernatant and wash twice with 200 &#181;l 70% EtOH on the MPS.</li>
		<li>Discard supernatant and resuspend DNA-Bead complex in 30 &#181;l H2O. Incubate 1 min and transfer supernatant to fresh 0.2 ml tube. Determine concentration of purified DNA.</li>
	</ol>
	</li>
	<li>Fusionprimer PCR to add sequencing specific adaptors.
	<ol>
		<li>For every sample to be analyzed prepare a 50 &#181;l PCR reaction.</li>
		<li>Pipette x &#181;l (40 ng) of DNA into a 0.2 ml PCR tube. Volume of DNA should be equal in each sample and in the range of 0.5 to 25 &#181;l.</li>
		<li>Prepare PCR master mix as described in table 5. Add (50 - x) &#181;l of master mix to each sample from step 9.2.2 and introduce Sequencing adaptors to (nr)LAM-PCR products by PCR conditions exemplified in Table 5. PCR products can be stored at 4 &#176;C for up to 4 days or long term at -20 &#176;C.</li>
		<li>To visualize Fusionprimer-PCR products load 10 &#181;l of the PCR product from step 9.2.3 on a 2% agarose gel. Purify remaining PCR product as described in steps 9.1.1 - 9.1.3. Analyze 1 &#181;l purified PCR product from step 9.4 on an automated high-resolution electrophoresis device to accurately quantify concentration and fragment size of Fusionprimer-PCR products.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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

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

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

LAM-PCR 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 Ã¢â‚¬â€œ Localization of Genetic Elements and Characterization of Unknown Flanking DNA at JoVE.com

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

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

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

Research

JoVE Journal

Biology

30.1K Views.  National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ).  Lineaire amplificatie-gemedieerde (LAM)-PCR is een methode ontwikkeld om de exacte positie van integratie virale vectoren in het genoom te identificeren. De techniek heeft zich ontwikkeld tot de superieure methode om klonale dynamiek in gentherapie patiënten, bioveiligheid van nieuwe vector technologieën, diversiteit T-cel, kanker stamcellen modellen, enz. te bestuderen 

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

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

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

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

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

Genetics

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

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

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

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

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

Biochemistry

2D and 3D Chromosome Painting in Malaria Mosquitoes

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

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

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

Immunology and Infection

Enhancing FISH Analysis for Oncogenic Drivers in Cancer Tissue Samples

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

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

Author Spotlight: FISH as a Tool for Precise Gene Amplification Assessment in Cancer Specimens

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

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

Cancer Research

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

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

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

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

Associated Chromosome Trap for Identifying Long-range DNA Interactions

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

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

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

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

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

Multiplex PCR and Reverse Line Blot Hybridization Assay mPCR/RLB

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

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

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

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

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

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

Enhanced Genetic Analysis of Single Human Bioparticles Recovered by Simplified Micromanipulation from Forensic &#8216;Touch DNA&#8217; Evidence

DNA profiles can be obtained from &#8216;touch DNA&#8217; evidence, which comprises microscopic traces of human biological material. Current methods for the recovery of trace DNA employ cotton swabs or adhesive tape to sample an area of interest. However, such a &#8216;blind-swabbing&#8217; approach will co-sample cellular material from the different individuals, even if the individuals&#8217; cells are located in geographically distinct locations on the item. Thus, some of the DNA mixtures encountered in touch DNA samples are artificially created by the swabbing itself. In some instances, a victim&#8217;s DNA may be found in significant excess thus masking any potential perpetrator&#8217;s DNA.
In order to circumvent the challenges with standard recovery and analysis methods, we have developed a lower cost, &#8216;smart analysis&#8217; method that results in enhanced genetic analysis of touch DNA evidence. We describe an optimized and efficient micromanipulation recovery strategy for the collection of bio-particles present in touch DNA samples, as well as an enhanced amplification strategy involving a one-step 5 &#181;l microvolume lysis/STR amplification to permit the recovery of STR profiles from the bio-particle donor(s). The use of individual or few (i.e., &#8220;clumps&#8221;) bioparticles results in the ability to obtain single source profiles. These procedures represent alternative enhanced techniques for the isolation and analysis of single bioparticles from forensic touch DNA evidence. While not necessary in every forensic investigation, the method could be highly beneficial for the recovery of a single source perpetrator DNA profile in cases involving physical assault (e.g., strangulation) that may not be possible using standard analysis techniques. Additionally, the strategies developed here offer an opportunity to obtain genetic information at the single cell level from a variety of other non-forensic trace biological material.

Here we describe an optimized and efficient removal strategy for the collection of bio-particles present in &#8216;touch DNA&#8217; samples, together with an enhanced amplification protocol involving a one-step 5 &#181;l micro-volume lysis/STR amplification, to permit the recovery of short tandem repeat (STR) profiles of the bio-particle donor(s).

DNA profiles can be obtained from &#8216;touch DNA&#8217; evidence, which comprises microscopic traces of human biological material. Current methods for ...

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.

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

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

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