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.

链接器的磁带1。准备（LC） <ol><li>混合40微升LC1寡核苷酸（ 表1），40微升LC2寡核苷酸（ 表1中 ，用适当的限制性内切酶悬），110微升Tris-盐酸（100毫米，pH值7.5），和10微升250毫摩尔MgCl 2组成。 </li><li>孵育在95℃下5分钟，让反应冷却下来慢慢至室温。加入300微升H 2 O和集中dsLinker-DNA在离心过滤器。加入80微升H 2 O至洗出液和分装10微升的准备盒式连接器在0.2 PCR管。 </li></ol>载体基因组结2。前置放大<ol><li>对于每个待分析样品制备50μl的PCR反应。 <ol><li>确定DNA样品的浓度。移液器x微升DNA插入0.2 ml的PCR管（1-1000纳克的LAM/100-1，000毫微克的nrLAM）。的DNA的量应该等于在每个样品，并在然GE的0.5至25微升。 </li><li>如表2中所述制备PCR主混合物混合（50 - x）的微升主混合物用在0.2ml PCR管中各DNA样品。 </li></ol></li><li>使用例示于表2中的PCR条件。PCR完成后Preamplify载体基因组结微升Taq酶添加0.5到每个PCR管中，然后重新运行PCR程序。 PCR产物可被储存在4℃下4天或长期于-20℃。 </li></ol> PCR产物的3。磁选<ol><li>磁珠的制备<ol><li>移液管20微升（200微克）的链亲和素包被的磁珠到1.5ml试管并暴露，以磁性颗粒分离器（MPS），在室温下放置1分钟。弃上清液。 </li><li>从MPS除去管，重悬磁珠在40微升PSB / 0.1％BSA（pH 7.5）中。暴露在MPS 1分钟，弃上清。一旦重复此步骤。 </li><li>洗珠无线第20微升的3M氯化锂溶液（3M氯化锂，10毫米的Tris-HCl，1 mM EDTA）中暴露于MPS 1分钟，弃上清。重悬珠在50μl6M的氯化锂溶液（6M的LiCl，10mM的Tris-盐酸，1mM EDTA）中。 </li></ol></li><li>混合来自步骤2.2与50微升制备的磁珠整个PCR反应。孵育在水平振荡器上（转速300rpm）至少2​​小时，在室温下进行。这个步骤可让生物素化的PCR产物链霉包被的磁珠（DNA-珠复合物）的结合。 DNA的磁珠复合物可以在摇床上过夜孵育，或储存在4℃下4天。 </li><li>暴露的DNA-珠复合体的MPS，在室温下放置1分钟，除去上清，重悬DNA-珠复合于100μlH 2 O。立即着手与LAM或nrLAM第5步第4步。 </li></ol> 4，LAM-手续<ol><li>双链DNA（dsDNA）的合成（林只） <ol><li>暴露的DNA-珠复杂步骤3.3至总薪级第1分钟和DIS卡上清液。加入8.25微升H 2 O，1微升10X六核苷酸缓冲液，0.25μL的dNTP（10毫米）和0.5微升（2学分）Klenow聚合酶。在37℃下1小时。 </li><li>加入90微升H 2 O和暴露在MPS 1分钟。弃上清，重悬DNA-珠复杂的100微升H 2 O </li></ol></li><li>限制文摘<ol><li>暴露的DNA-珠复杂，MPS 1分钟，弃上清。加入8.5微升H 2 O，1微升10X限制性内切酶缓冲液和0.5μL限制性内切酶和孵化1小时反应。重复步骤4.1.2。 注意：孵育反应物在所建议的限制酶的制造商的温度。确保有内没有限制性位点存在，或在感兴趣的DNA用于预扩增的引物结合位点的下游。对于所选择的合适的限制性内切酶/限制性内切酶的组合是指9。 </li></ol></li><li> DS结扎链接器（LK） <ol><li>暴露的DNA-珠复杂，MPS 1分钟，弃上清。加入5微升H 2 O，1微升10X FastLink缓冲液，1微升ATP（10毫米），从步骤1.2 2微升连接器卡带，和1微升快速连接DNA连接酶（2 U /μL）。在室温下孵育5分钟。重复步骤4.1.2。 </li></ol></li><li>合成的双链DNA变性<ol><li>暴露的DNA-珠复杂，MPS 1分钟，弃上清。重悬DNA-珠复杂的5微升0.1N NaOH中。孵育5分钟，在室温下在水平摇动器。 </li><li>暴露的DNA-珠复合体的MPS为1分钟，并收集上清液含于新的1.5毫升管预扩增载体基因组的交界处。立即在-20°C进行第6步或存储上清液</li></ol></li></ol> 5，nrLAM-手续<ol><li>单链连接器的连接（SSLC）（nrLAM只） <ol><li>暴露的DNA-珠复杂步骤3.3至公安部1分钟，弃上清。加入6.5微升H 2 O，1微升CircLigase 10X反应缓冲液，0.5μL的MnCl 2（50毫米），0.5微升ATP（1毫米），1微升ssLinker寡核苷酸和0.5微升CircLigase（100 U /μL）。孵育在60℃下1小时。 </li><li>加入90微升H 2 O和暴露在MPS 1分钟。弃上清，洗DNA-珠复杂的100微升H 2 O再次暴露在MPS 1分钟，弃上清，重悬DNA-珠复杂的10微升H 2 O </li></ol></li></ol> 6，指数扩增我<ol><li>对于每个待分析样品制备50μl的PCR反应。 <ol><li>吸取2μL模板DNA（从步骤4.4.2（LAM）或5.1.2（nrLAM））到0.2 ml的PCR管。 </li><li>制备PCR主混合物如表3所述。加入48μl反应混合液到每个样品从步骤6.1.1和通过PCR扩增的天气下载体基因组结纳秒列举于表3。PCR产物可以被储存在4℃下4天或长期于-20℃。 </li></ol></li></ol> PCR产物7。磁分离<ol><li>制备磁珠所例示的步骤3.1.1 - 3.1.3。重悬珠在20μl（对于LAM）或6米的LiCl溶液（6M的LiCl，10mM的Tris-盐酸，1mM EDTA）中加入50μl（对于nrLAM）。混合20微升（LAM）或从步骤6.1.2 PCR反应以制备的磁性小珠（步骤7.1）的50微升（nrLAM）孵育在水平振荡器上（转速300rpm）搅拌2小时，在室温下。 DNA的磁珠复合物可以在摇床上过夜孵育，或储存在4℃下4天。 </li><li>暴露的DNA-珠复合体的MPS为1分钟，除去上清，重悬DNA-珠复合于100μlH 2 O。暴露的DNA-珠复杂，MPS 1分钟，弃上清。 </li><li>重悬DNA-珠复杂的20微升（LAM）或5微升（nrLAM）0.1N氢氧化钠。培育大怒，在室温下在水平摇床10分钟，暴露在MPS 1分钟，并收集在新的1.5 ml管扩增含DNA的上清液。立即在-20°C继续执行步骤8.1或存储上清液</li></ol> 8，指数扩增二<ol><li>对于每个待分析样品制备50μl的PCR反应。 <ol><li>移液管加入2μl模板DNA（来自步骤7.3）到0.2 ml的PCR管中。 </li><li>制备PCR主混合物，如表4所述。加入48μl反应混合液到每个样品从步骤8.1.1并通过举例说明在表4中的PCR条件扩增载体基因组结点。PCR产物可以被储存在4℃下进行长达4在-20℃天或长期</li></ol></li><li>可视化（NR）LAM-PCR产物，负载10微升的PCR产物从2％琼脂糖凝胶步8.1.2。如果带不可见，分析10微升的LAM-PCR产物的高分辨率凝胶。注意：逸hidium溴化物是致突变物。工作很认真，总是穿戴适当的手套。 </li></ol> 9。用于制备高通量测序<ol><li>的（NR）LAM-PCR产物纯化<ol><li>从步骤8.1.2用44μL室温AMPure XP的磁珠混合40微升的PCR产物。孵育5分钟，在室温下暴露于MPS额外的2分钟。 </li><li>弃上清，用200μl70％乙醇洗两次在MPS。 </li><li>弃上清，重悬DNA-珠复杂的30微升H 2 O孵育1分钟，上清液转移至新鲜0.2毫升管。确定纯化的DNA的浓度。 </li></ol></li><li> Fusionprimer PCR加测序专用适配器。 <ol><li>对于每个待分析样品制备50μl的PCR反应。 </li><li>移液器x微升（40毫微克）的DNA为0.2 ml的PCR管。的DNA的量应该等于在每个样品和在0.5至25微升的范围内。 </li><li>制备PCR主混合物，如表5中所述添加（50 - x）的μl反应混合液到每个样品从步骤9.2.2和通过列举在表5中的PCR产物的PCR条件引入测序适配器（NR）LAM-PCR产物。可以被保存在4℃下4天或长期在-20℃下</li><li>形象化Fusionprimer-PCR产物负载10微升的PCR产物从2％琼脂糖凝胶步骤9.2.3。 9.1.3 - 按照步骤9.1.1中所述净化剩余的PCR产物。从自动高分辨率电泳设备准确量化的Fusionprimer-PCR产物的浓度和片段大小的步骤9.4分析1微升纯化PCR产物。 </li></ol></li></ol>

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The authors have nothing to disclose.

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?...

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。

<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结果在载体基因组路口放大与定义的片段大小为每个结。个体的PCR片段的大小取决于在基因组中的已知的DNA的位置与最近的限制性内切酶识别位点之间的距离。这允许通过凝胶电泳， 例如可视化扩增结的多样性分析的样品中。如果只有单一的（单克隆），几个（寡克隆）或多个（多克隆）频带上存在的凝胶。 LAM-PCR的结果，最好是由高分辨率凝胶电泳（ 图2A）被观看，但...

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

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

线性扩增介导的PCR  - 未知侧翼DNA的遗传物质和表征本地化

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 次观看.  National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ). 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

视频: 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.

单液滴数字聚合酶链反应对热点地区突变的综合和同步检测

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

科研

遗传学

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

患者生物流体中肿瘤相关循环DNA的检测与监测

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

癌症研究

Detection of Retrotransposition Activity of Hot LINE-1s by Long-Distance Inverse PCR

Long interspersed nuclear elements 1 (LINE-1s) are the only family of mobile genetic elements in the human genome that can move autonomously. They do so by a process called retrotransposition wherein they transcribe to form an mRNA intermediate which is then consequently inserted into the genome by reverse transcription. Despite being silent in normal cells, LINE-1s are highly active in different epithelial tumors. De novo LINE-1 insertions can potentially drive tumorigenesis, and hence it is important to systematically study LINE-1 retrotransposition in cancer. Out of ~150 retrotransposition-competent LINE-1s present in the human genome, only a handful of LINE-1 loci, also referred to as &#34;hot&#34; LINE-1s, account for the majority of de novo LINE-1 insertion in different cancer types. We have developed a simple polymerase chain reaction (PCR)-based method to monitor retrotransposition activity of these hot LINE-1s. This method, based on long-distance inverse (LDI)-PCR, takes advantage of 3&#180; transduction, a mechanism by which a LINE-1 mobilizes its flanking non-repetitive region, which can subsequently be used to identify de novo LINE-1 3&#180; transduction events stemming from a particular hot LINE-1.

This article outlines a simple PCR-based assay to monitor the activity of an active LINE-1 retrotransposon and to map de novo retrotranspositions in a given genome. Using the MCF7 cell line, we demonstrate herein how this method can be applied to detect activity of a LINE-1 located at 22q12.1.

长距离反向PCR对热线1的逆变活性的检测

Long interspersed nuclear elements 1 (LINE-1s) are the only family of mobile genetic elements in the human genome that can move autonomously. They do so ...

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.

使用含有微型基因的Alu元素来分析圆形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 ...

生物化学

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).

疟疾蚊子中的二维和三维染色体绘画

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

免疫与感染

增强癌症组织样本中致癌驱动因素的 FISH 分析

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.

作者聚焦：FISH 作为癌症标本中精确基因扩增评估的工具

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

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

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

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.

确定远距离的DNA相互作用的相关染色体陷阱

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

生物学

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.

罕见的基因变异检测从集资使用斯普林特测序

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

线性扩增介导的PCR - 未知侧翼DNA的遗传物质和表征本地化

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此视频中的章节

单液滴数字聚合酶链反应对热点地区突变的综合和同步检测

患者生物流体中肿瘤相关循环DNA的检测与监测

长距离反向PCR对热线1的逆变活性的检测

使用含有微型基因的Alu元素来分析圆形RNA

疟疾蚊子中的二维和三维染色体绘画

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

确定远距离的DNA相互作用的相关染色体陷阱

罕见的基因变异检测从集资使用斯普林特测序