Lineær forstærkning Mediated PCR - Lokalisering af genetiske elementer og karakterisering af Ukendt flankerende DNA
Summary
Lineær-forstærkning medieret (LAM)-PCR er en metode udviklet til at identificere de præcise positioner for at integrere virale vektorer i genomet. Teknikken har udviklet sig til at være den overlegne metode til at studere klonale dynamik i patienter genterapi, biosikkerhed af nye vektor teknologier, T-celle mangfoldighed cancer stamceller modeller osv.
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
Lineær amplifikation medieret PCR (LAM-PCR) kan identificere og karakterisere ukendt flankerende DNA tilstødende til kendte DNA af enhver oprindelse. Mere specifikt har LAM-PCR er blevet udviklet til at lokalisere viral vector integration sites (IS) i værtsgenomet 1,2. Genetiske elementer som retrovirus eller transposoner integrerer deres genom i værtens genom i en (semi-) tilfældig måde 3-6. I mange tilfælde er det afgørende at vide præcis den position, hvor disse vektorer integreret. LAM-PCR har vist sig at være overlegen i forhold til alternative teknikker som ligatur-medieret PCR 7 og dens varianter eller omvendt PCR 8. Følsomheden og robusthed denne metode hidrører fra første forstærkertrinene af vektor-genom kryds og magnetisk udvælgelse af amplificerede PCR-produkter. Ligesom de alternative metoder, der er nævnt, LAM-PCR bygger på anvendelsen af restriktionsenzymer, indførelse af en skævhed i hentning kapacitet IS 9-11. Såledeskun en delmængde af IS repertoiret (det integrome) kan detekteres i en reaktion. Denne skævhed er minimeret ved den parallelle analyse af en given prøve at bruge optimale kombinationer af restriktionsenzymer 9. For nylig, en variant af teknologien betegnes ikke-begrænsende LAM-PCR (nrLAM-PCR) er blevet udviklet, omgår anvendelsen af restriktionsenzymer og tillader objektiv genom-dækkende analyse af en prøve i en enkelt reaktion 9,12.
I fortiden, har LAM-PCR er blevet brugt til at identificere den sygdomsfremkaldende retroviral giver anledning til leukæmi hos nogle få patienter i kliniske forsøg med genterapi 13-15. Siden da har LAM-PCR er blevet tilpasset til at identificere IS fra andre integrerende vektorer (lentivirale vektorer, transposoner) og også til at identificere integration mønstre af passivt integrere vektorer som adenoassocierede vektorer (AAV) eller integrase defekt lentivirusvektorer (IDLV) 16 -21. Anvendelser af LAM-PCR er udbredt: traditionelly, er teknikken udbredt til at studere klonal sammensætning af gen modificerede celler hos patienter, der har gennemgået genterapi eller vurdere biosikkerhed af nye vektorsystemer ved optrevling deres integration adfærd 15,16,22-24. For nylig LAM-PCR aktiveret bestemme specificitet og off-target aktivitet designer nucleaser ved en IDLV fældefangst assay 25.
Desuden LAM-PCR gør det muligt at nemt følge skæbnen for en transduceret celle over tid i en organisme. Dette gør det muligt at identificere protoonkogener samt tumorsuppressorgener og også til at studere hæmatopoiese eller cancer stamcellebiologi 26-28. Sidst men ikke mindst, er LAM-PCR tilpasset til at undersøge T-celle-receptor mangfoldighed i mennesker 29 (og upublicerede data).
Den iboende kraft af teknologien er forstærket ved at knytte den metode til dyb sekventering teknologier, der tillader karakterisere millioner af ukendte flankerende DNA med en enkelt nukleotid resolution i hele genomer. I det følgende protokol beskriver vi trin-trin forstærkning og identifikation af flankerende ukendt DNA eksemplarisk at identificere lentivirusvektor IS. Oligonukleotider, der anvendes i protokollen, er anført i tabel 1.. Ekstraherede DNA eller cDNA af enhver kilde kan anvendes som DNA-template for LAM-PCR og nrLAM-PCR.
Protocol
Representative Results
Discussion
LAM-PCR teknik tillader at identificere ukendte DNA-sekvenser, der flankerer en kendt DNA-region. På grund af den høje følsomhed som følge af forforstærkning af kryds med specifikke primere, der hybridiserer i kendte DNA-sekvens, er det muligt at amplificere og påvise selv sjældne kryds ned til enkelt celle niveau. Derimod er der i et polyklonalt situation, LAM-PCR er i stand til at forstærke tusindvis af forskellige knudepunkter i en enkelt reaktion.
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Disclosures
The authors have nothing to disclose.
Acknowledgements
Funding was provided by the Deutsche Forschungsgemeinschaft (SPP1230, grant of the Tumor Center Heidelberg/Mannheim), by the Bundesministerium für 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.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| 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 |
| 6M 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 |
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