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 robustnes....

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....

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