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

Summary

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.

Abstract

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 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’- and 5’- sequences adjacent to the integrated lentiviral vector.

Introduction

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 genome^1,2. Genetic elements like retroviruses or transposons integrate their genome into the host genome in a (semi-) random manner^3-6. 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 IS^9-11. 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 reaction^9,12.

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 trials^13-15. 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 behavior^15,16,22-24. 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 biology^26-28. 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.

Protocol

1. Preparation of Linker Cassettes (LC)

1. Mix 40 µl of LC1 oligonucleotide (Table 1), 40 µl of LC2 oligonucleotide (Table 1, with proper restriction enzyme overhang), 110 µl Tris-HCl (100 mM, pH 7.5), and 10 µl 250 mM MgCl₂.
2. Incubate at 95 °C for 5min and let the reaction cool down slowly to room temperature. Add 300 µl H₂O and concentrate dsLinker-DNA on a centrifugation filter. Add 80 µl H₂O to the eluate and aliquot 10 µl of prepared linker cassette in 0.2 PCR tubes.

2. Preamplification of Vector Genome Junctions

1. For every sample to be analyzed prepare a 50 µl PCR reaction.
   1. Determine concentration of the DNA samples. Pipette x µl (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 µl.
   2. Prepare PCR master mix as described in Table 2. Mix (50 - x) µl of the master mix with each DNA sample in 0.2 ml PCR tubes.
2. Preamplify vector genome junctions using PCR conditions exemplified in Table 2. After completion of PCR add 0.5 µl Taq Polymerase to each PCR tube and rerun PCR program. PCR products can be stored at 4 °C for up to 4 days or long term at -20 °C.

3. Magnetic Separation of PCR Product

1. Preparation of Magnetic Beads
   1. Pipette 20 µl (200 µg) 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.
   2. Remove tube from MPS and resuspend magnetic beads in 40 µl PSB/ 0.1% BSA (pH 7.5). Expose to MPS for 1 min and discard supernatant. Repeat this step once.
   3. Wash beads with 20 µl 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 µl of 6 M LiCl solution (6 M LiCl, 10 mM Tris-HCl, 1 mM EDTA).
2. Mix entire PCR reaction from step 2.2 with 50 µl 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 °C for up to 4 days.
3. Expose DNA-Bead complex to MPS for 1 min at room temperature, remove supernatant, and resuspend DNA-Bead complex in 100 µl H₂O. Immediately proceed with step 4 for LAM or step 5 for nrLAM.

4. LAM-Procedure

1. Double Strand DNA (dsDNA) Synthesis (LAM only)
   1. Expose DNA-Bead complex from step 3.3 to MPS for 1 min and discard supernatant. Add 8.25 µl of H₂O, 1 µl 10x hexanucleotide buffer, 0.25 µl dNTPs (10 mM) and 0.5 µl (2 U) Klenow polymerase. Incubate at 37 °C for 1 hr.
   2. Add 90 µl of H₂O and expose to MPS for 1 min. Discard supernatant and resuspend DNA-Bead complex in 100 µl H₂O.
2. Restriction Digest
   1. Expose DNA-Bead complex to MPS for 1 min and discard supernatant. Add 8.5 µl H₂O, 1 µl 10x restriction enzyme buffer and 0.5 µl 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.
3. Ligation of ds Linker (LK)
   1. Expose DNA-Bead complex to MPS for 1 min and discard supernatant. Add 5 µl H₂O, 1 µl 10x FastLink buffer, 1 µl ATP (10 mM), 2 µl Linker cassette from step 1.2, and 1 µl Fast-Link DNA Ligase (2 U/µl). Incubate at room temperature for 5 min. Repeat step 4.1.2.
4. Denaturation of Synthesized dsDNA
   1. Expose DNA-Bead complex to MPS for 1 min and discard supernatant. Resuspend DNA-Bead complex in 5 µl 0.1 N NaOH. Incubate 5 min at room temperature on a horizontal shaker.
   2. 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 °C.

5. nrLAM-Procedure

1. Ligation of single stranded linker (ssLC) (nrLAM only)
   1. Expose DNA-Bead complex from step 3.3 to MPS for 1 min and discard supernatant. Add 6.5 µl H₂O, 1 µl CircLigase 10x Reaction Buffer, 0.5 µl MnCl₂ (50 mM), 0.5 µl ATP (1 mM), 1 µl ssLinker oligonucleotide and 0.5 µl CircLigase (100 U/µl). Incubate at 60 °C for 1 hr.
   2. Add 90 µl of H₂O and expose to MPS for 1 min. Discard supernatant and wash DNA-Bead complex in 100 µl H₂O. Again expose to MPS for 1 min, discard supernatant and resuspend DNA-Bead complex in 10 µl H₂O.

6. Exponential Amplification I

1. For every sample to be analyzed prepare a 50 µl PCR reaction.
   1. Pipette 2 µl template DNA (from step 4.4.2 (LAM) or 5.1.2 (nrLAM)) into a 0.2 ml PCR tube.
   2. Prepare PCR master mix as described in Table 3. Add 48 µl 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 °C for up to 4 days or long term at -20 °C.

7. Magnetic Separation of PCR Product

1. Prepare magnetic beads as exemplified in steps 3.1.1 - 3.1.3. Resuspend beads in 20 µl (for LAM) or 50 µl (for nrLAM) of 6 M LiCl solution (6 M LiCl, 10 mM Tris-HCl, 1 mM EDTA). Mix 20 µl (LAM) or 50 µl (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 °C for up to 4 days.
2. Expose DNA-Bead complex to MPS for 1 min, remove supernatant and resuspend DNA-Bead complex in 100 µl H₂O. Expose DNA-Bead complex to MPS for 1 min and discard supernatant.
3. Resuspend DNA-Bead complex in 20 µl (LAM) or 5 µl (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 °C.

8. Exponential Amplification II

1. For every sample to be analyzed prepare a 50 µl PCR reaction.
   1. Pipette 2 µl template DNA (from step 7.3) into a 0.2 ml PCR tube.
   2. Prepare PCR master mix as described in Table 4. Add 48 µl 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 °C for up to 4 days or long term at -20 °C.
2. To visualize (nr)LAM-PCR products, load 10 µl of the PCR product from step 8.1.2 on a 2% agarose gel. If bands are visible, analyze 10 µl of the LAM-PCR product on high-resolution gel. CAUTION: Ethidium bromide is mutagenic. Work very carefully and always wear appropriate gloves.

9. Preparation for High-throughput Sequencing

1. Purification of (nr)LAM-PCR products
   1. Mix 40 µl PCR-Product from step 8.1.2 with 44 µl of room temperature AMPure XP Magnetic Beads. Incubate 5 min at room temperature and expose to MPS for additional 2 min.
   2. Discard supernatant and wash twice with 200 µl 70% EtOH on the MPS.
   3. Discard supernatant and resuspend DNA-Bead complex in 30 µl H₂O. Incubate 1 min and transfer supernatant to fresh 0.2 ml tube. Determine concentration of purified DNA.
2. Fusionprimer PCR to add sequencing specific adaptors.
   1. For every sample to be analyzed prepare a 50 µl PCR reaction.
   2. Pipette x µl (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 µl.
   3. Prepare PCR master mix as described in table 5. Add (50 - x) µl 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 °C for up to 4 days or long term at -20 °C.
   4. To visualize Fusionprimer-PCR products load 10 µl 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 µl 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.

Results

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...

Discussion

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 ...

Materials

List of materials used in this article

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