线性扩增介导的PCR - 未知侧翼DNA的遗传物质和表征本地化
Summary
线性扩增介导的（LAM）-PCR方法是开发，以确定基因组中的整合型病毒载体的精确位置的方法。该技术已经发展成为学习中的基因治疗的患者中，新颖的矢量技术，T-细胞的多样性，癌症干细胞模型等生物安全克隆动力学的优越方法
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
线性扩增介导的PCR（LAM-PCR）技术可识别和表征邻近任何来源的已知的DNA未知侧翼的DNA。更具体地，LAM-PCR方法已经开发出来，定位病毒载体整合位点（IS）宿主基因组1,2内。像逆转录病毒或转座子的遗传元件整合自己的基因组入在一个（半）随机方式3-6宿主基因组中。在许多情况下，它是决定性的确切地知道这些矢量一体化的立场。 LAM-PCR方法已被证明是优于像连接介导的PCR 7和它的变体或反向PCR 8的替代技术。该方法的灵敏度和鲁棒性源自载体基因组结和磁性选择的扩增的PCR产物的初始预扩增。像提到的替代方法，LAM-PCR依赖于使用限制性内切酶，引入偏差为9-11的检索能力。因此，可以在一个反应中检测出的IS剧目（在integrome）的一个子集。这种偏差是通过使用限制性内切酶9的合适组合的给定样品的平行分析最小化。最近，该技术的一个变种称为非限制性的LAM-PCR（nrLAM-PCR）已经开发出来，绕过使用限制性内切酶，并允许在单个反应9,12的样品的无偏的全基因组分析。
在过去，LAM-PCR方法已被用于鉴定致病逆转录病毒IS白血病引起在少数患者中的临床基因治疗试验13-15。从那时起，LAM-PCR已被改编以确定从其它整合载体（慢病毒载体，转座子）和还识别的被动结合像腺相关载体（AAV）或整合酶缺陷型慢病毒载体（IDLV）载体的集成模式IS 16 -21。 LAM-PCR方法的应用是广泛的传播：传统LY，该技术被广泛地用于研究基因修饰的细胞的克隆性组合物中已经历基因疗法的患者或解开他们的整合行为15,16,22-24评估新型载体系统的生物安全。近日，LAM-PCR启用确定特异性和脱靶设计师核酸酶的活性由IDLV诱捕法25。
此外，LAM-PCR方法可以非常方便地跟随转导的细胞的命运随着时间的推移在一个有机体。这允许识别原癌基因以及肿瘤抑制基因和还研究造血或癌症干细胞生物26-28。最后但并非最不重要的，LAM-PCR法适于研究T细胞受体多样性的人类29（和未公布的数据）。
该技术的固有功率是由所述方法连接到深度测序技术，使表征百万未知侧翼DNA的单核苷酸Ř增强esolution在整个基因组。在下面的协议中，我们描述了一步一步的扩增和鉴定未知侧翼DNA的模范，以确定慢病毒载体IS的。在协议中使用的寡核苷酸列于表1。提取的DNA的任何来源的或cDNA可以用作用于LAM-PCR和nrLAM-PCR的DNA模板。
Protocol
Representative Results
Discussion
在LAM-PCR技术允许识别侧翼已知的DNA区域未知的DNA序列。因为高灵敏度从具有特异性的引物杂交的已知DNA序列中的结的预扩增得到的，能够扩增并检测甚至罕见路口下至单个细胞水平。相反，在多克隆情况LAM-PCR能够扩增数千种不同的结在一个单一的反应。
然而，由于使用了限制性内切酶的integrome只有一个子部分可以由LAM-PCR检测结与每一个特定的限制性内切酶的存在下进行分?…
Declarações
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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