资助由美国国家卫生研究院R00-HL116234，U19 AI117941和R56 HL126544提供;国家科学基金会授予DMS-1516675;韩国国家研究基金会（NRF-2011-0030049，NRF-2014M3C9A3064552）;和KRIBB倡议计划。

1.生成上游（左） - 和下游（右） - 连接序列库<ol><li> DNA接头制备: <ol><li>通过加入2μL100μM的LINKER_A寡核苷酸（最终的20μM），2μL的100μM的LINKER_B寡核苷酸（最终的20μM），2μL的5M的NaCl（最终的1M），加入2μL的10μL的接头DNA溶液PCR管中4μL无核酸酶的水。接头序列见表1 。 </li><li>在PCR仪器中将接头DNA溶液在95℃孵育5分钟，停止运行程序，并关闭PCR仪器电源。将接头溶液留在仪器中30分钟以缓慢冷却接头DNA。接头DNA溶液可以在4℃下储存。 </li></ol></li><li> LTR特异性生物素引物延伸: <ol><li>使用UV-Vis分光光度计来确定体内基因组DNA的DNA浓度和260/280 nm值或体外实验。使用无核酸酶的水稀释1-2μg样品基因组DNA至终体积为170μL的基因组DNA溶液。 </li><li>通过混合10μMHIV-1特异性生物素引物（ 表1中的 L-BP和R-BP:四种慢病毒特异性引物共10μL），将20μL10X热稳定剂DNA聚合酶缓冲液，4μL10mM dNTPs（最终每个dNTP200μM），3μL2.5U /μL热稳定DNA聚合酶和163μL基因组DNA。 注意:对于gammaretroviral载体（pMX载体），使用两个10μMpMX特异性生物素引物（L-BP和R-BP:总共为10μL）的5μL，而不是上述四种HIV-1特异性生物素引物。 </li><li>将溶液分成四个PCR管（每个50μL），并在以下条件下进行单个延伸循环:94℃5分钟，56℃3分钟，72℃5分钟min和4℃储存。 </li><li>将所有四个PCR反应物合并到2 mL微量离心管中，并按照PCR纯化试剂盒的PCR纯化程序进行。用50μL洗脱缓冲液（试剂盒中提供的5倍水稀释洗脱缓冲液）洗脱。立即进行步骤1.3.1或将洗脱的DNA储存在-20℃。 </li></ol></li><li> RsaI和CviQI消化 <ol><li>通过加入50μL的DNA（来自步骤1.2.4），10μL的10x缓冲液A和2μL（20U）的RsaI酶，准备100μL的消化反应。在PCR仪中37℃孵育1小时。 </li><li>向反应中加入1μL（10U）CviQI酶，并在PCR仪中于25℃温育30分钟。立即进行第1.4.1节</li></ol></li><li> 钝的结局: <ol><li>制备含有2.5μLDNA聚合酶I大（klenow）片段和2μL10 mM dNTP的4.5μL混合液。转移181;将来自步骤1.3.2的DNA样品的混合物的L。总体积为102μL。通过涡旋混合并在25℃下在PCR仪中孵育1小时。立即进行步骤1.6.1。 </li></ol></li><li> 链霉亲和素珠的制备: <ol><li>简单地涡旋链霉亲和素珠溶液，并将50μL转移到新的2 mL微量离心管中。使用磁性支架取出上清液，用200μL的结合溶液洗涤珠子。 </li><li>将珠子悬浮在100μL的结合溶液中，并将管放置在离磁架上。立即进行步骤1.6.1。 </li></ol></li><li> 链霉抗生物素蛋白珠结合: <ol><li>将100μL样品DNA（步骤1.4.1）转移到100μL再悬浮珠粒溶液（步骤1.5.2），并通过移液小心混合，以避免溶液发生任何发泡。在旋转轮或滚筒上，室温孵育3小时。 </li><li>使用磁性支架捕获珠子并丢弃上清液。在400μL洗涤溶液中洗涤珠子两次，并在400μL1x T4 DNA连接酶缓冲液（从不含核酸酶的水稀释的10X溶液）中洗涤两次。 </li><li>将珠子悬浮于200μL的1×T4 DNA连接酶缓冲液中（使用不含核酸酶的水从10X溶液中稀释），并将其放置在磁性架上。立即继续执行步骤1.7.1。 </li></ol></li><li> 连接器结扎: <ol><li>通过混合0.5μL的DNA接头（步骤1.1.2），10μL的10×T4 DNA连接酶缓冲液，20μL的5X T4 DNA连接酶缓冲液（含有25％的聚乙烯），制备400μL的连接反应溶液于2mL的微量离心管中乙二醇），5μLT4 DNA连接酶，164.5μL无核酸酶的水和200μL再悬浮的珠（步骤1.6.3）。将反应管放在旋转轮上，并在RT（22℃）下孵育3小时（或16°CO / N）。 </li&gt; <li>用洗涤溶液洗涤珠子两次，并用1X耐热DNA聚合酶缓冲液（使用不含核酸酶的水从10x溶液稀释）使用磁性支架两次。 </li><li>将珠子重悬于50μL的1×热稳定DNA聚合酶PCR缓冲液中，并将其放置在离磁架上。立即进行第1.8.1步，或在4℃下储存长达一天。 </li></ol></li><li> 左和右连接DNA的预扩增: <ol><li>通过加入10μL10μM1L引物，10μL10μM1R引物，20μL10μM引物Link1（表1），10μL10X热稳定DNA聚合酶缓冲液，4μL 10mM dNTPs（终体积:每个dNTP为200μM），8μL（20U）热稳定DNA聚合酶和88μL无核酸酶的水加入来自步骤1.7.3的再悬浮珠子（50μL）中。 </li><li>将反应混合物等分成4个PCR管（每个50μL1; L）并进行PCR，具有以下条件:94℃孵育2分钟; 94℃20秒，56℃25秒，72℃2分钟25个循环;最后在72℃延伸5分钟。 </li><li>将所有4个PCR反应混合到2 mL微量离心管中，并按照PCR纯化试剂盒的PCR纯化程序进行。用50μL洗脱缓冲液洗脱。 </li><li>使用UV-Vis分光光度计测定DNA浓度和260/280-nm值; DNA可以保存在-20°C直到准备下一步。继续执行步骤1.9.1 / 1.10.1（可选）或直接执行步骤1.11.1 / 1.12.1。 </li></ol></li><li> 取出左侧内部DNA扩增子（可选）: <ol><li>将高达100ng PCR DNA产物从步骤1.8.4转移到2mL微量离心管中，并使用不含核酸酶的水将体积调节至10μL。 </li><li>准备专门针对左侧内部D的限制酶反应NA扩增子。 注意:反应条件可能因酶的选择而异。例如，当从基于NL4.3的慢病毒载体中除去左侧内部DNA时，加入1μL的pvuII ，2μL的缓冲液B和7μL的无核酸酶的水。在PCR仪中37℃孵育1小时。立即进行步骤1.11.1或存储在-20°C。 </li></ol></li><li> 取出右侧内部DNA扩增子（可选）: <ol><li>继续与步骤1.9.1相同。 </li><li>准备特异性靶向右侧内部DNA扩增子的限制酶反应。 注意:例如，从基于NL4.3的慢病毒载体中取出右侧内部DNA时，加入1μLsfoI ，2μL缓冲液B和7μL无核酸酶的水。在PCR仪中37℃孵育1小时。立即进行步骤1.12.1或储存于-20°C。 </li></ol></li><li> 剩下结特异性扩增: <ol><li>通过混合来自步骤1.8.4（或选自步骤1.9.2）的5μLDNA，5μL10μM2L引物（终体积:1μM），5μL10μM引物Link2，制备50μLPCR反应（最终为1μM），5μL10×耐热DNA聚合酶缓冲液，1μL10mM dNTP（终体积:每个dNTP200μM），2μL（5U）热稳定DNA聚合酶和27μL无核酸酶水。 </li><li>进行以下条件的PCR:94℃孵育3分钟; 94℃20s，56℃25s，72℃2分钟8-15个循环;最后在72℃延伸5分钟。 注:扩增的DNA可以储存在-20°C。 *建议循环次数优化。 </li><li>按照PCR纯化试剂盒的PCR纯化程序。用50μL洗脱缓冲液洗脱。确定DNA浓度和260/280 nm值。 </li></ol></li><li><st荣&gt;右结特异性扩增: <ol><li>所有方法与"左结特异性扩增"相同（步骤1.11.1-1.11.3），不同之处在于使用不同的引物;在该步骤中使用右侧特异性引物（2R-引物， 参见表1 ）。 </li></ol></li><li> PCR扩增子长度变异试验: <ol><li>通过进行2％琼脂糖凝胶电泳或毛细管电泳分析PCR扩增子长度变化（ 图2 ）。 注意:这是确认完成测定程序并基于PCR带图案粗略评估IS模式的重要步骤。来自步骤1.11.3和1.12.3的纯化的PCR扩增子可用于各种DNA测序平台。继续进行经典链终止（Sanger）测序或下一代测序的适当样品制备程序。 DNA可以保存在-20°;C。 </li></ol></li></ol> 2.计算IS序列分析<ol><li> 数据文件的准备: <ol><li>准备三个输入数据文件:fasta格式序列数据文件（补充数据中的Test_data.fa），用于向量和链接器序列的搜索图案的tsv文件（补充数据中的Demultiplexing_Trimming_blunt_GTAC.tsv）和限制酶信息的tsv文件（Enzyme.tsv补充资料）。 注意:这些文件是解复用，修剪向量和链接器序列，以及删除内部向量序列所必需的（ 图3A ）。在补充数据中的README.txt文件中提供了实现计算工作流程的详细分步说明。 </li></ol></li><li> 计算分析: <ol><li>运行解复用和修剪脚本。 注意:原始序列将被处理用于解复用和trimming的载体，接头和引物序列（见补充README.txt文件中的STEP-1）。 </li><li>运行映射命令（参见README.txt文件中的STEP-2），使用类似BLAST的对齐工具（BLAT; www.genome.ucsc.edu）将处理的序列映射到参考基因组上。 </li><li>运行定量IS分析脚本（请参阅README.txt文件中的STEP-3）。 注意:将生成两个输出文件（Initial_count_without_homopolymer_correction.txt和Final_count.txt）。有关映射和序列枚举策略的更多详细信息，请参见"README.txt文件中的补充数据和以前的出版物2,15。 </li></ol></li></ol>

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双向测定能够同时分析上游（左）和下游（右）载体 - 宿主DNA连接序列，并且可用于许多基因治疗，干细胞和癌症研究应用。与以前的基于TCGA基序的酶（ TaqαI ）为基础的检测2,15和其他方法相比，使用GTAC基序酶（RsaI和CviQI）和双向PCR方法显着提高了检测内含子（或克隆群体）的机会单向LM-PCR方法5 。双向测定改善了IS的分析，特别是在临床或小动物模型研究中经常?...

逆转录病毒将其基因组DNA插入各个位点的宿主基因组。这种独特的性质可能有助于癌症和其他形式的病毒发病机制的发展，具有使这些病毒高度适应细胞工程用于基因治疗和基础生物学研究的讽刺意义。病毒整合位点（IS） - 整合了外来DNA（病毒）的宿主基因组上的位置 - 对整合的病毒和宿主细胞的命运具有重要意义。 IS测定已经用于各种生物和临床研究环境，以研究逆转录病毒整合位点选择和发病机理，癌症发展，干细胞生物学和发育生物学1,2,3,4 。检测灵敏度低，数据重现性差，交叉污染频繁将IS测定应用限制在当前和计划研究中的关键因素。 已经开发了许多IS分析技术。包括连接介导的（LM）聚合酶链反应（PCR） 5 ，逆PCR 6和线性扩增介导（LAM）PCR 7的限制性酶基整合位点测定法是最广泛使用的。然而，使用位点特异性限制性酶在IS的检索期间产生偏倚，仅允许在限制性位点附近恢复4个整合到宿主基因组内的整合子（集成到宿主基因组中的外来DNA） 4 。最近几年也引入了更综合评估载体IS的分析技术。这些测定采用各种策略，包括Mu转座子介导的PCR 8 ，非限制性（nr）-LAM PCR 9 ，II型限制性内切酶缓冲消化10 ，机械剪切11和随机六聚体基因PCR（Re-free PCR） 12 ，以克隆基因组DNA并扩增IS。目前的技术具有不同程度的检测灵敏度，基因组覆盖率，靶特异性，高通量能力，测定程序的复杂性以及检测目标位点相对频率的偏差。鉴于现有测定的不同质量和可以使用的各种目的，应仔细选择最佳测定方法。 该工作提供了详细的实验程序和双向测定的计算数据分析工作流程，通过同时分析整合的靶DNA的上游和下游IS，显着提高检测率和序列定量准确度（参见图1的测定程序示意图）。这种方法也提供了是表征逆转录病毒整合过程的手段（例如，靶位点重复的保真度和上游和下游插入的基因组序列的变化）。其他双向方法主要用于克隆和测序目标DNA 11,13,14的两端。使用公认的LM-PCR方法和计算分析映射以及用于量化上游和下游连接点，广泛优化用于载体标记克隆的高通量和可重复定量的测定。已经证明使用TaqαI酶的双向分析对于干细胞基因治疗临床前研究中的高通量克隆定量是有用的。本文介绍了一种使用更频繁的切割器（RsaI / CviQI-主题:GTAC）的修改方法，使两倍与基于TaqαI的测定相比，他检测到整合素的机会。描述了使用GTAC基序酶用于慢病毒（NL4.3及其衍生物）和γ-逆转录病毒（pMX载体）载体IS分析的详细实验和数据分析程序。测定中使用的寡核苷酸列于表1中 。用于IS序列分析的内部编程脚本在补充文档中提供。

<table><tbody><tr><td>Thermostable DNA polymerase</td><td>Agilent</td><td>600424</td><td>PicoMaxx Polymerase</td></tr><tr><td>Thermostable DNA polymerase buffer</td><td>Agilent</td><td>600424</td><td>PicoMaxx Polymerase buffer</td></tr><tr><td>Deoxynucleotide (dNTP) solution mix</td><td>New England Biolabs</td><td>N0447L</td><td>dNTP solution mix (10 mM each)</td></tr><tr><td>PCR tubes</td><td>VWR International</td><td>53509-304</td><td>PCR Strip Tubes With Individual Attached Caps</td></tr><tr><td>2 mL microcentrifuge tube</td><td>Molecular Bioproducts</td><td>3453</td><td>microcentrifuge tubes</td></tr><tr><td>PCR purification kit</td><td>Qiagen</td><td>28106</td><td></td></tr><tr><td>RsaI&amp;nbsp;</td><td>New England Biolabs</td><td>R0167L</td><td>restriction enzyme</td></tr><tr><td>CviQI</td><td>New England Biolabs</td><td>R0639L</td><td>restriction enzyme</td></tr><tr><td>Buffer A</td><td>New England Biolabs</td><td>B7204S</td><td>NEB CutSmart buffer</td></tr><tr><td>DNA Polymerase I large (klenow) fragment&amp;nbsp;</td><td>New England Biolabs</td><td>M0210L</td><td>Blunting</td></tr><tr><td>streptavidin beads solution</td><td>Invitrogen&amp;nbsp;</td><td>60101</td><td>Dynabeads kilobaseBINDER kit</td></tr><tr><td>Binding Solution</td><td>Invitrogen&amp;nbsp;</td><td>60101</td><td>Dynabeads kilobaseBINDER kit</td></tr><tr><td>Washing Solution</td><td>Invitrogen&amp;nbsp;</td><td>60101</td><td>Dynabeads kilobaseBINDER kit</td></tr><tr><td>magnetic stand</td><td>ThermoFisher</td><td>12321D</td><td>DynaMag&amp;trade;-2 Magnet</td></tr><tr><td>T4 DNA ligase</td><td>New England Biolabs</td><td>M0202L</td><td>T4 DNA ligase</td></tr><tr><td>10X T4 DNA ligase buffer</td><td>New England Biolabs</td><td>B0202S</td><td>T4 DNA ligase reaction buffer</td></tr><tr><td>5X T4 DNA ligase buffer</td><td>Invitrogen&amp;nbsp;</td><td>46300-018</td><td>T4 DNA ligase buffer with polyethylene glycol-8000</td></tr><tr><td>UV-Vis spectrophotometer</td><td>Fisher Scientific</td><td>S06497</td><td>Nanodrop 2000</td></tr><tr><td>pvuII</td><td>new England Biolabs</td><td>R0151L</td><td>restriction enzyme</td></tr><tr><td>sfoI</td><td>new England Biolabs</td><td>R0606L</td><td>restriction enzyme</td></tr><tr><td>Buffer B</td><td>new England Biolabs</td><td>B7203S</td><td>NEB buffer 3.1</td></tr><tr><td>Nuclease free water</td><td>Integrated DNA Technologies</td><td>11-05-01-14</td><td></td></tr><tr><td>Capillary electrophoresis</td><td>Qiagen</td><td>9001941</td><td>QIAxcel capillary electrophoresis</td></tr><tr><td>Veriti 96-well Fast Thermal Cycler</td><td>Thermo Fisher Scientific</td><td>4375305</td><td>PCR Instrument</td></tr><tr><td>Rotating wheel (or Roller)</td><td>Eppendorf</td><td>M10534004</td><td>Cell Culture Roller Drums</td></tr><tr><td>DNA size marker</td><td>Qiagen</td><td>929559</td><td>QX size marker (100 - 2,500 bp)</td></tr><tr><td>DNA size marker</td><td>Qiagen</td><td>929554</td><td>QX size marker (50 - 1,500 bp)</td></tr><tr><td>DNA alignment markers</td><td>Qiagen</td><td>929524</td><td>QX DNA Alignment Marker</td></tr><tr><td>genomc DNA</td><td>Not Available</td><td>Not Available</td><td>Sample genomc DNA from in vivo or in vitro experiments</td></tr></tbody></table>

bidirectional retroviral integration site pcr methodology and quantitative data analysis workflow

整合位点（IS）测定是逆转录病毒整合位点研究的关键组成部分及其生物学意义。在最近的逆转录病毒基因治疗研究中，IS测定结合下一代测序已被用作细胞跟踪工具来表征共享相同IS的克隆干细胞群体。为了准确比较不同样品内和跨越不同样品的干细胞克隆，检测灵敏度，数据重现性和高通量能力均为最重要的检测标准之一。这项工作为双向IS分析提供了详细的协议和数据分析工作流程。双向测定可以同时排列上游和下游载体 - 宿主结。与常规单向IS测序方法相比，双向方法显着提高了IS检测率和t两端积分事件的表征他瞄准DNA。这里描述的数据分析流程通过多个比较步骤准确地识别和列举相同的IS序列，将IS序列映射到参考基因组上并确定测序误差。使用优化的测定程序，我们最近在恒河猴猕猴移植后发表了数千个造血干细胞（HSC）克隆的详细重新填充模式，首次证实了HSC重新增殖的精确时间点和HSC在功能异质性中的功能异质性灵长类动物系统。以下协议描述了准确识别和量化相同的IS序列的逐步实验过程和数据分析工作流程。

双向IS测定为上游（左）和下游（右）载体宿主结（ 图2 ）产生不同大小的PCR扩增子。 PCR扩增子的大小取决于最后的GTAC基序上游和下游的位置。该测定还产生内部DNA PCR扩增子:分别在左侧和右侧连接PCR期间在多巴胺附近的逆转录病毒序列和引物结合位点同时扩增。通过毛细管或琼脂糖（2％）电泳可以显示PCR扩增子条带。 <p class="jove_content" fo:keep-to...

Watch this Scientific Journal Video about Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow at JoVE.com

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

该手稿描述了可以同时分析上游和下游载体 - 宿主连接DNA的双向整合位点测定的实验程序和软件分析。双向PCR产品可用于任何下游测序平台。所得数据对于综合DNA靶标的高通量，定量比较是有用的。

双向逆转录病毒整合位点PCR方法和定量数据分析工作流程

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

bidirectional-retroviral-integration-site-pcr-methodology

Research

JoVE Journal

Genetics

10.7K 次观看.  University of California at Los Angeles (UCLA). 该手稿描述了可以同时分析上游和下游载体 - 宿主连接DNA的双向整合位点测定的实验程序和软件分析。双向PCR产品可用于任何下游测序平台。所得数据对于综合DNA靶标的高通量，定量比较是有用的。 

视频: Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 nucleotides apart result in either the inclusion (S) or exclusion (&#916;S) of a single residue, Serine-701. Further downstream, splicing at a pair of alternative acceptor splice sites result in transcripts encoding either the 55 terminal residues of the transactivation domain (&#945;) or a truncated transactivation domain with 7 unique residues (&#946;). As outlined in this manuscript, measuring the proportions of STAT3's four spliced transcripts (S&#945;, S&#946;, &#916;S&#945; and &#916;S&#946;) was possible using absolute qPCR (quantitative polymerase chain reaction). The protocol therefore distinguishes and measures highly similar splice variants. Absolute qPCR makes use of calibrator plasmids and thus specificity of detection is not compromised for the sake of efficiency. The protocol necessitates primer validation and optimization of cycling parameters. A combination of absolute qPCR and efficiency-dependent relative qPCR of total STAT3 transcripts allowed a description of the fluctuations of STAT3 splice variants' levels in eosinophils treated with cytokines. The protocol also provided evidence of a co-splicing interdependence between the two STAT3 splicing events. The strategy based on a combination of the two qPCR techniques should be readily adaptable to investigation of co-splicing at other tandem splicing sites.

Tandem splicing events occur at sites less than 12 nucleotides apart. Quantifying ratios of such splice variants is feasible using an absolute quantitative PCR approach. This manuscript describes how splice variants of the gene STAT3, in which two splicing events results in Serine-701 inclusion/exclusion and &#945;/&#946; C-termini, can be quantified.

合并绝对和相对定量PCR数据来量化STAT3剪接变体成绩单

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 ...

科研

遗传学

Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been investigated in various organs and tissues in rat. However, standard methods for the purification of miRNAs and detection of their expression in rat peritoneal membrane have not been well established. We have developed an effective and reliable method to purify and quantify miRNAs using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) in rat peritoneal membrane. This protocol consists of four steps: 1) purification of peritoneal membrane sample; 2) purification of total RNA including miRNA from peritoneal membrane sample; 3) reverse transcription of miRNA to produce cDNA; and 4) qRT-PCR to detect miRNA expression. Using this protocol, we successfully determined that the expression of six miRNAs (miRNA-142-3p, miRNA-21-5p, miRNA-221-3p, miRNA-223-3p, miRNA-327, and miRNA-34a-5p) increased significantly in the peritoneal membrane of a rat peritoneal fibrosis model compared with those in control groups. This protocol can be used to study the profile of miRNA expression in the peritoneal membrane of rats in many pathological conditions.

Here we present a protocol for the detection of microRNA expression in rat peritoneal membrane using quantitative real-time reverse-transcription polymerase chain reaction. This method is suitable for studying the microRNA expression profile in rat peritoneal membrane in several pathological conditions.

使用定量实时PCR检测大鼠腹膜中的微小RNA表达

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been ...

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs.
The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.

Here, we describe a protocol for genome-wide mapping of the integration sites of Moloney murine leukemia virus-based retroviral vectors in human cells.

逆转录病毒扫描:映射MLV集成站点以定义细胞特异性监管区域

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and host cell proteins on viral DNA synthesis. Here we address the lack of a cell-based, high-coverage, and high-resolution assay that is capable of defining retroviral reverse transcription intermediates within the physiological context of virus infection. The described method captures the 3'-termini of nascent complementary DNA (cDNA) molecules within HIV-1 infected cells at single nucleotide resolution. The protocol involves harvesting of whole cell DNA, targeted enrichment of viral DNA via hybrid capture, adaptor ligation, size fractionation by gel purification, PCR amplification, deep sequencing, and data analysis. A key step is the efficient and unbiased ligation of adaptor molecules to open 3'-DNA termini. Application of the described method determines the abundance of reverse transcripts of each particular length in a given sample. It also provides information about the (internal) sequence variation in reverse transcripts and thereby any potential mutations. In general, the assay is suitable for any questions relating to DNA 3'-extension, provided that the template sequence is known.

Here we present a deep sequencing approach that provides an unbiased determination of nascent 3&#39;-termini as well as mutational profiles of single-stranded DNA molecules. The main application is the characterization of nascent retroviral complementary DNAs (cDNAs), the intermediates generated during the process of retroviral reverse transcription.

hiv-1 反向转录过程中鼻部单链病毒 dna 分子的 3 '-特米尼和序列的测定

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and ...

InSET: A Novel Tool for Unbiased Genome-Wide Functional Element Discovery

Identification of Functionally-Relevant Lentivirus Integration Sites in an Insertional Mutagenesis Cell Library

The extent of functional sequences within the human genome is a pivotal yet debated topic in biology. Although high-throughput reverse genetic screens have made strides in exploring this, they often limit their scope to known genomic elements and may introduce non-specific effects. This underscores the urgent need for novel functional genomics tools that enable a deeper, unbiased understanding of genome functionality. This protocol introduces the Insertion-based Screen for functional Elements and Transcripts (InSET), a method for identifying lentivirus integration sites within a lentivirus-based insertional mutagenesis cell library. InSET facilitates the capture of genome-wide lentiviral integration sites, with next-generation sequencing used to detect and quantify flanking sequences. InSET's design enables the analysis of integration site abundance variations in phenotypic screens on a large scale, establishing it as a robust tool for forward genetics and for identifying functional genomic elements. A key benefit of InSET is its capacity to reveal previously unidentified genomic elements, including novel functional exons of both protein-coding and non-coding RNAs, independent of prior annotation. Overall, InSET holds significant value in studying the intricate complexity of the human genome and transcriptome, where many genomic elements await functional characterization.

Insertional mutagenesis is an essential tool in forward genetics for identifying functional genomic elements. Here, we describe the Insertion-based Screen for functional Elements and Transcripts (InSET), a method for detecting lentivirus integration sites within a lentivirus-based insertional mutagenesis cell library.

The extent of functional sequences within the human genome is a pivotal yet debated topic in biology. Although high-throughput reverse genetic screens ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

测量<em&gt;体外</em&gt; HIV-1 Preintegration配合物的整合活动

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...

免疫与感染

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1&#8211;2 copies) integration using inverse nested PCR.

乙型肝炎体外感染形成的低拷贝数综合病毒 dna 的检测

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...

<meta charset="utf8"></head><body>使用数字液滴 PCR 进行病毒基因组定量的标准化方案

Quantification of Adeno-Associated Viral Genomes in Purified Vector Samples by Digital Droplet Polymerase Chain Reaction

Adeno-associated virus (AAV) is a non-pathogenic virus used as a delivery vehicle to transfer therapeutic genes into patients. Accurate quantification of AAV genome copy number in vector preparations is essential for bioprocess optimization and dosage calculation in both preclinical and clinical studies of AAV-based gene therapy products. Currently, a consensus protocol for AAV viral genome titration is lacking. Here, we present a digital droplet PCR (dd_PCR) protocol for the quantification of viral genomes in purified vector samples. Samples are treated with DNase I to eliminate unpackaged contaminant DNA. DNase-treated samples are then mixed with an appropriate primer-probe set (designed according to the target AAV genome) and PCR reagents, and then loaded into a droplet generator. The prepared droplets are transferred into a PCR plate, where PCR amplification is performed and analyzed. The viral genome titer is calculated based on the concentration (copies/&#181;L), accounting for sample dilutions. A successful measurement shows a clear separation of the positive and negative droplet clouds, has at least 10,000 accepted droplets, shows a value between 10 copies/&#181;L and 10,000 copies/&#181;L, and has a coefficient of variation (CV) between repeats lower than 20%. Reliable viral genome titration will aid in the development of safe and effective AAV-based gene therapy products.

<meta charset="utf8"></head><body>作者聚焦:优化数字微滴 PCR 方法以实现准确的腺相关病毒基因组定量

Precise quantification of adeno-associated virus (AAV) vector genome copies is critical, but a standardized protocol has yet to be established. This protocol describes a validated method for preparing purified AAV samples and conducting digital droplet polymerase chain reaction (dd_PCR) to reliably quantify the viral genome titer.

通过数字液滴聚合酶链反应定量纯化载体样品中的腺相关病毒基因组

Adeno-associated virus (AAV) is a non-pathogenic virus used as a delivery vehicle to transfer therapeutic genes into patients. Accurate quantification of ...

双向逆转录病毒整合位点PCR方法和定量数据分析工作流程

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合并绝对和相对定量PCR数据来量化STAT3剪接变体成绩单

使用定量实时PCR检测大鼠腹膜中的微小RNA表达

逆转录病毒扫描:映射MLV集成站点以定义细胞特异性监管区域

hiv-1 反向转录过程中鼻部单链病毒 dna 分子的 3 '-特米尼和序列的测定

Identification of Functionally-Relevant Lentivirus Integration Sites in an Insertional Mutagenesis Cell Library

测量

乙型肝炎体外感染形成的低拷贝数综合病毒 dna 的检测

通过数字液滴聚合酶链反应定量纯化载体样品中的腺相关病毒基因组

通过数字液滴聚合酶链反应定量纯化载体样品中的腺相关病毒基因组

通过数字液滴聚合酶链反应定量纯化载体样品中的腺相关病毒基因组