作者感谢加拿大自然科学和（NSERC）工程研究理事会资助这项工作。

1.媒体和文化的制备<ol><li>对于LB +放大器:制成500毫升之的Luria-BERTANI肉汤（LB）的使用在121℃高压釜中进行30分钟灭菌。补充有氨苄青霉素至4℃，得到的100微克/毫升氨苄青霉素和存储的终浓度直到需要。 </li><li>对于TB +放大器:准备大（500毫升以上）ministring生产量。制备使用在121℃高压釜中进行30分钟灭菌600毫升无菌了不起肉汤（TB）的。补充有氨苄青霉素至4℃，得到的100微克/毫升氨苄青霉素和存储的终浓度直到需要。 </li><li>对于LB琼脂+放大器:由制备添加了15克/升琼脂LB +安培准备无菌的LB琼脂平板上。等分约15 - 20ml如无菌培养皿中并储存倒挂在4℃直至需要。 </li><li>准备W3NN [PDMS]的连胜板经无菌裸奔W3NN [PDMS]在LB琼脂+放大器细胞。孵育板overni向右在30℃下，直到单个菌落可以观察到。不要孵化高于此温度W3NN避免derepressing protelomerase表达。 </li><li>无菌地转移5ml的LB +安培到无菌试管中。接种此体积与来自划线平板的单个菌落制备W3NN [PDMS]的过夜培养。孵育在摇床于30℃培养过夜以230-250转。 </li></ol> 2.成长阶段<ol><li>无菌地将10毫升LB培养基+安培的进入无菌125毫升Erlenmeyer烧瓶，并用100微升过夜W3NN [PDMS]培养到准备LB +安培，使得1接种:100稀释。 <ol><li>对于更大的体积，加入500μl过夜培养物于50ml TB +安培的在250毫升Erlenmeyer烧瓶中，代替LB +安培。 </li><li>另外，使用如步骤2.2第二个"代表"文化。 </li></ol></li><li>在30℃和250rpm孵育在振荡培养箱直到尽头TURE达到中期到晚对数期，由光密度（OD）或吸光度表示:A 600 = 0.8。 1-2小时后，将培养基应该开始出现浑浊肉眼。 <ol><li>无菌传送1 ml培养到一个干净的分光光度计小杯中。 </li><li>使用设置为600纳米波长的紫外可见分光光度计测定光吸收。使用1个毫升其中细胞生长至设定空白（LB或TB）的媒体。或者，使用步骤2.1.3代表文化这一步吧。 </li><li>如果培养尚未达到适当的外径，返回到摇动器，并再次检查每半小时。检查OD更频繁的A 600接近0.8。 </li></ol></li><li>一旦培养达到晚期对数期，由A 600 = 0.8所指出的，在无菌条件下转移培养至无菌50ml离心管中。 </li><li>通过以10,000 xg离心离心培养10分钟收集细胞。检查对于PEllet在管的底部。 </li><li>弃去上清液清除到一个合适的生物危险废物的容器。请勿打扰细胞沉淀。 </li><li>重复步骤2.4-2.5，直到所有的细胞已被收集。 </li><li>重悬在100μlLB +安培沉淀。吸出使用微量或使用涡旋混合器。对于更大的ministring生产，再从暂停50毫升TB +放大器文化沉淀在1ml新鲜LB + amp培养基中。 </li></ol> 3. Ministring生产<ol><li>无菌在125毫升的Erlenmeyer烧瓶中，并热加20毫升LB培养基+安培至42℃。更大ministring生产，制成500毫升之LB +安培的，预热至42℃。 </li><li>转移到悬浮预热网上平台。 </li><li>孵育在42℃和250rpm，直到过去数期，由A 600 = 1.0来表示。在42℃，不要离开20毫升文化过去的1小时。 </li><li>降低温度至37℃，并使培养的阿迪TIONAL 90分钟。为500毫升，离开培养90额外诱导期 - 120分钟，并不会降低的温度。 </li><li>降低温度至30℃并保持培养物在200rpm振荡2小时的额外的温度降档期间。对于500毫升，扩展温度降档至4小时或过夜。 </li></ol> 4.收获Ministrings <ol><li>无菌转移文化进入无菌的50ml离心管中。离心在10,000rpm×g离心10分钟，并检查是否有一个小球。 </li><li>倒出上清液到一个合适的生物危险废物的容器，而不会干扰颗粒。重复，直到所有的细胞已收集。 </li><li>保留质粒提取沉淀与在-80℃下提取后液氮存储中的任何商业开采和内毒素去除试剂盒或闪存冻结。 </li></ol>

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作者宣称他们没有竞争的经济利益。

我们在这里描述为生产使用一步法热诱导协议，它不需要特殊的设备，除了这种处理是在典型的细菌的生长和操纵用于LCC DNA ministrings的一个简单的系统。 DNA ministrings是自由扭转，稳定的线性DNA表达盒，缺乏原核的遗传元件。它们可以代替用于基因转移，报道基因的表达，以及在朝向的转基因表达在真核系统中的效率评估的常规方法使用。 DNA ministrings结合生物相容性和线性DNA载体的优越的安全"迷你"载体提高转染效率的好处。我们的健壮一步法体内生产平台迅速和容易产生的DNA ministrings为任何基因转移应用程序，而不需要多个昂贵的体外处理。 有几个关键步骤确保最佳ministring生产（ 图2和3）。热感应是ministring生产中最关键的一步。完全缺乏ministring生产是最有可能是由于缺乏热诱导（保持在30℃的细胞），其中protelomerase表达的抑制防止了前体的质粒的转化为LCC ministring的。以下的热感应协议，预期大约75％的生产效率。而细胞对数生长期热感应是最佳的。虽然目前在固定相当使用细胞（"杂草丛生"， 图3）ministring PE无统计学差异显著，我们确实观察到几乎整个质粒产量下降了50％。产率将取决于所用的质粒提取协议。对于最佳ministring生产，引发热感应而细胞处于对数期。差ministring生产将最有可能是长时间的温度升档或insufficie的结果nt的温度降档。长时间温度升档气馁，因为这会激活E.大肠杆菌热休克反应15，这可能抑制protelomerase活动，并与质粒稳定性造成干扰。因此，定时，温度降档是高效ministring生产另一关键步骤。如果没有在30℃额外孵化时间，ministring生产效率被认为是非常差（"早拆"， 图3）。这可以归因于的时间为电话表达和活性所需的量。始发噬菌体PY54编码电话protelomerase通常感染耶尔森 ，其最佳生长约28℃，12，这表明30℃也可以是用于电话活动更优化。 该DNA minivector生产系统利用体内平台上生成高质量的细菌无序列的DNA minivectors。修改该协议可能我nclude改变生长培养基，以优化的质粒和蛋白质生产，以促进在生长阶段的细胞生长（TB代替，LB），或增加ministring生产和转换效率。对于较大的培养体积（200 ml和更高），温度降档，并在30℃培养可过夜而对ministring PE严重的负面影响进行扩展。我们已在我们的500毫升文化作为一个例子协议的修改。 DNA ministrings纯化可通过原核生物骨干体外内切酶消化加快。有许多内切酶靶位点（ 例如 ，PI-SceI位，PvuⅠ的，ScaI位）上的前体的质粒，其可被切割以暴露开放线性的原核骨干可用结束时，允许在原核骨架的体外降解。从其它载体物种ministrings分离也可以通过阴离子交换膜层析16来实现的。 与LCC DNA载体生产系统相关联的一个电流限制是用于从其它LCC和CCC产品的DNA ministring的纯化的需要。虽然很容易地用标准的质粒分离方法纯化，所述分离步骤还可以在大规模的设置的缺点。所述ministring分离可以是费时使用AGE如果ministring和LCC原核生物骨干在尺寸太相似了。另外，阴离子交换膜层析可以提供从CCC媒介物种16 ministrings更好的分离。温度升档可以是另一种潜在的限制，因为高温也激活在大肠杆菌中的热休克反应大肠杆菌 ，其负面影响重组蛋白产生17，从而减少质粒率ministring转换。我们在其他地方报告生产平台的优化，以规避和/或减轻热休克18的这种影响。我们目前还优化方法，以消除或减少LCC原核生物骨干污染体内 。 LCC的DNA ministring的染色体整合破坏宿主染色体和细胞科目凋亡。这不仅减少插入诱变诸如肿瘤发生和相邻基因的沉默，从而提高其安全性的潜在的负面影响;致死作用也可以作为敲除中和基因置换的研究没有相关的副作用和一体化的损伤的理想方法。的DNA ministring载体可以向基因用于治疗蛋白质，RNA，抗体，或抗原的体内转移和表达成给定的目标应用或更换造成故障或无功能的等位基因。在简单的一步热诱导体内生产系统允许用于临床或工业应用中容易产生的DNA ministring载体。

的目标是产生线性共价闭合使用简单的和高效率的一步热诱导体内 DNA ministring生产系统（LCC）的DNA minivectors的伸缩量。 DNA ministrings提供一个安全，有效的非病毒性的策略来提供的DNA。他们结合的LCC载体的安全性与DNA小环的效率，并且还提供到病毒衍生的载体更安全的替代不影响转染效率。 为了使转基因递送系统是成功的，该DNA载体必须进入靶宿主细胞和表达编码转基因（多个）。有需要在实践中加以克服，特别是在哺乳动物系统几个细胞的障碍。为了通过网状内皮系统，以避免血清核酸酶和免疫检测退化，DNA载体必须是生物与目标系统免疫兼容。未受保护的DNA很快被血浆核酸酶消化和质粒膜由密集的脂蛋白障碍，使该DNA载体必须能够迅速地横过靶细胞的质膜。一旦在细胞中，载体必须遍历细胞质和穿过核膜以输入的转基因表达的核。非病毒基因传递技术侧重于增强组织特异性和细胞靶向。然而，这些技术的使用常规的质粒的降低转染效率由于免疫原性细菌序列的存在，从而迅速沉默基因表达的1,2-。传统的质粒一般带有抗生素抗性基因在原核系统的维护。然而，这些可能产生的人类宿主的潜在不利影响，或传授给通过基因水平转移的效果自然发生主机菌群阻力。常规的质粒还含有二核苷酸CpG基序2，其可以引发有害免疫反应，潜在地减少或沉默转基因的表达。 因为它们仅包含真核表达盒的，DNA minivectors如DNA小环3，是由于它们的减小的尺寸和缺乏免疫原核元件的，因为它们表现出改进的细胞外和细胞内的生物利用度和改进的基因表达的基因传递一个更好的选择4。减小的矢量大小票价相对于电阻与体内给药到靶位5相关联的剪切力更好。每单位质量的矢量的较高拷贝数的需要较少的转染试剂，从而降低毒性。然而，在随机矢量积分的情况下，进宿主染色体，圆共价闭合（CCC）的载体，包括DNA小环和常规质粒，赋予分子的连续性，因此可能导致插入诱变，其可具有破坏性的后果<s达&gt; 6。比较，线性DNA载体整合破坏染色体并启动的细胞死亡途径，从而去除从增殖细胞群体的突变体细胞，并防止插入诱变7。简约免疫定义的基因表达（蚊）载体8和微型线性矢量9（MILV） 在体外开发LCC DNA载体。蠓载体已在体内表现出高达17倍提高转基因表达相对于传统的质粒DNA载体11，并且已经证明，在疫苗10和癌症8的基因治疗有希望的结果。此外，由于将LCC minivector的自由扭转的结构，较少的转染试剂，需要相比于CCC超螺旋的对应，从而降低毒性7。 我们的增强LCC DNA载体，DNA ministrings，都表现出了超强的表达效率和bioavai7不稳定。此外，DNA的ministrings是在一步法热诱导体内生产系统中，一个权宜和具有成本效益的替代蠓和MILV LCC载体，其需要在体外的多个步骤制备。该DNA ministring生产系统待证明是在体内进行的简单热诱导过程中，使其既具有成本效益和易于扩展。该系统利用了小肠结肠炎耶尔森菌噬菌体PY54衍生电话/ PAL protelomerase重组系统12到最小的真核表达盒从原核表达载体主干分开。我们设计的大肠杆菌细胞（W3NN）的热诱导噬菌体λ启动子，CI [TS] 857 13的控制下，表达电话protelomerase。表达时，电话protelomerase作用于"超级顺序"（SS）位于前体质粒网站中存在PAL靶位点得到LCC产品，从其中DNA ministring然后可纯化（ 图1）。在DNA ministring前体质粒的SS网站还编码为其他重组酶包括Cre重组酶（LOX），TELN protelomerase（telRL）和Flp重组（FRT）靶点，从而有利于从一个前体质粒生产等基因LCC和CCC minivectors的。此外，每个SS网站由SV40增强子（SV40e）序列，其用于改善核易位14在两侧。我们已经在别处证明，连续另外SV40e的逐步赋予的转染效率7相应增加。该前体的质粒（质粒DNA MiniString或PDMS）含有多接头（ 图1），以促进在转录融合的绿色荧光报道（GFP）感兴趣的任何期望的基因的插入。 该DNA minivector技术可能代替用于基因转移，报道基因的表达，并朝向转基因表达的在真核系统中的效率评估的常规方法使用。 DNA ministrings结合生物相容性和线性DNA载体的优越的安全"迷你"载体提高转染效率的好处。我们强大的一步法生产平台，快速方便地生成DNA ministrings任何基因转移的应用程序，并提供了更高的安全性。

<table><tbody><tr><td>&lt;em&gt;E. coli&lt;/em&gt; W3NN</td><td>Mediphage</td><td></td><td>strain for ministring processing: Nafissi &amp;amp; Slavcev, 2012</td></tr><tr><td>pDMS.IRES.GFP</td><td>Mediphage</td><td></td><td>parent plasmid for ministring processing: also referred to as pDNA Ministring (pDMS)</td></tr><tr><td>BD Difco Dehydrated Culture Media: LB Broth, Miller (Luria-Bertani)</td><td>Fisher Scientific</td><td>244620</td><td></td></tr><tr><td>Fisher BioReagents Terrific Broth</td><td>Fisher Scientific</td><td>BP2468500</td><td></td></tr><tr><td>BD Bacto Dehydrated Agar</td><td>Fisher Scientific</td><td>214010</td><td></td></tr><tr><td>Ampicilin</td><td>MP Biomedicals LCC</td><td>2190147</td><td></td></tr><tr><td>Ultrapure Milipore Water</td><td>Fisher Scientific&amp;nbsp;</td><td>Z00Q0V0US</td><td>Model: Mili-Q Advantage A10 Water Purification System</td></tr><tr><td>Surgical Gloves</td><td>VWR</td><td>(S) 82026-424, (M)82026-426, (L) 82026-428, (XL) 82026-430&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Fisherbrand Petri Dishes with Clear Lid Raised Ridge (100&amp;nbsp;x&amp;nbsp;15&amp;nbsp;mm)</td><td>Fisher Scientific</td><td>FB0875712</td><td></td></tr><tr><td>30 ml KIMAX Test Tubes</td><td>VWR</td><td>89001-448</td><td></td></tr><tr><td>Spectrophotometer Cuvette</td><td>Sarstedt</td><td>67.74</td><td></td></tr><tr><td>Sterile 15 ml Falcon Tubes</td><td>&amp;nbsp;BD Falcon</td><td>62406-200,</td><td></td></tr><tr><td>Sterile 50 ml Centrifuge Tubes</td><td>&amp;nbsp;FroggaBio</td><td>2930-S0</td><td></td></tr><tr><td>Falcon Disposable Polystyrene Serological Pipets 10 ml</td><td>BD Falcon</td><td>13-675-20</td><td></td></tr><tr><td>Falcon Disposable Polystyrene Serological Pipets 25 ml</td><td>BD Falcon</td><td>13-668-2&amp;nbsp;</td><td></td></tr><tr><td>Micropipettor (100-1,000&amp;nbsp;&amp;mu;l)</td><td>FroggaBio</td><td>C1000-1</td><td></td></tr><tr><td>Micropipettor (20-200&amp;nbsp;&amp;mu;l)</td><td>FroggaBio</td><td>C200-1</td><td></td></tr><tr><td>Sterile Pipette Tips (100-1,250&amp;nbsp;&amp;mu;l)</td><td>VWR</td><td>89079-472&amp;nbsp;</td><td></td></tr><tr><td>Sterile Pipette Tips (1-200&amp;nbsp;&amp;mu;l)</td><td>VWR</td><td>89079-460</td><td></td></tr><tr><td>Fisher Scientific Economy Burner</td><td>Fisher Scientific</td><td>03-917Q</td><td></td></tr><tr><td>Incubator Shaker</td><td>New Brunswick Scientific&amp;nbsp;</td><td>M1352-00000</td><td></td></tr><tr><td>Sterile Erlenmeyer Flask (125 ml)</td><td>Pyrex (Sigma Aldrich)</td><td>CLS4980125 Aldrich&amp;nbsp;</td><td></td></tr><tr><td>Sterile Erlenmeyer Flask (250 ml)</td><td>Pyrex (Sigma Aldrich)</td><td>CLS4980250 Aldrich&amp;nbsp;</td><td></td></tr><tr><td>Sterile Erlenmeyer Flask (500 ml)</td><td>Pyrex (Sigma Aldrich)</td><td>CLS4980500 Aldrich&amp;nbsp;</td><td></td></tr><tr><td>Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>335901</td><td>Model: Genesys 10 Vis</td></tr><tr><td>Floor Centrifuge</td><td>Beckman Coulter&amp;nbsp;</td><td>369001</td><td>Model: Avanti J-E</td></tr><tr><td>Benchtop Centrifuge</td><td>Beckman</td><td>362114</td><td>Model: GS-6R</td></tr><tr><td>Fisher Scientific Analog Vortex Mixer&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>02-215-365</td><td></td></tr><tr><td>1 kb DNA Ladder</td><td>New England BioLabs</td><td>N3232S</td><td></td></tr></tbody></table>

production of double-stranded dna ministrings

我们构建线性共价闭合（LCC）的DNA minivectors经由体内一个简单的平台产生的非病毒基因递送载体的替代，DNA ministrings具有一个升高的安全性，也有效地提供的DNA货物到靶细胞。常规的DNA载体携带不希望的原核序列，包括抗生素抗性基因，CpG基序，和细菌复制起点，这可能导致的宿主免疫应答的刺激。常规DNA载体的生物利用度也受到影响，由于其更大的分子大小。其圆形性质也可赋予染色体整合，导致插入诱变。 细菌序列是从DNA minivectors切除，仅留下感兴趣的（GOI）的和必要的真核表达元件的基因。我们LCC DNA minivectors，或DNA ministrings，是缺乏免疫原性细菌的序列;因此，提高个EIR生物利用度和印度政府表达。在矢量积分的情况下入染色体中，LCC的DNA ministring将致死破坏宿主染色体，从而去除从增殖细胞群的潜在危险的突变体。因此，DNA ministrings提供"小环"DNA的好处，同时消除了不良的载体整合事件的可能性。相比于常规的质粒及其同基因型圆形共价闭合（CCC）的对应的DNA ministrings展示优越的生物利用度，转染效率，以及细胞质动力学 - 因而它们需要较低量为靶细胞的有效转染的阳离子表面活性剂。 我们已经构建了在大肠杆菌中生产的DNA ministrings的是使用简单，快速，可扩展的一步法体内系统诱导的。

DNA提取，残余前体的质粒，LCC原核生物骨干，并且该DNA ministring之后将被恢复。整体的质粒产率和纯度可使用分光光度计进行评估。纯度是使用吸光度的比率在260nm和280nm（A260 / A280），其中的1.9-2.0的比率是最佳的估计。所有LCC和CCC的产品可以通过琼脂糖凝胶电泳（AGE）（ 图2）分开。 AGE形象化ministring生产热诱导后，ministring纯化之前的结果。 ministring生产的定性评估可以在这个阶段比较ministring（2.4 KB）母公司质粒（5.6 KB）（ 图2）带强度进行。 Ministring DNA可以通过以下年龄标准质粒凝胶提取方案回收。 图3总结了可能，同时携带出现在各种条件下ministring生产效率ING出的协议。根据使用制造商的分析程序的DNA带的强度被确定Ministring生产效率。任何类似密度的程序可以被用来分析AGE的结果。 <img alt="图1" src="/files/ftp_upload/53177/53177fig1highres.jpg" width="700" /> 图1:ministring生产概况 （A）的亲本载体含有在转录融合多接头用报道基因，由两个SS站点成帧。骨干编码β-内酰胺酶抗氨苄青霉素，也包含独特的骨干体外消化几截点。主协议的（B）的综述。步骤2）生长阶段和3）Ministring生产的图中被表示，以提供所涉及的步骤的总体示意图，突出协议的简易性。 （C）Ministring感应。热感应失活的温度敏感的阻遏，允许电话表达随后在父质粒SS网站电话活动。 <a href="https://www.jove.com/files/ftp_upload/53177/53177fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53177/53177fig2highres.jpg" width="350" /> 图2:Ministring产量从协议偏离后 。 1:控制，2:未诱导，3:蔓生，4:Overinduced，5:早期切除。 DNA ministring约为2.4 kb的，LCC骨干为3 KB，而pNN9父质粒是5.6 kb的。 ministring和LCC骨干带的带强度与方案偏离下降，表明较低的ministring产量。从左至右依次为:新英格兰生物实验室1 kb梯度;控制，以下协议;未诱导，没有温度升档;蔓生，温度升档都灵克固定相; Overinduced，扩展温度升档;早拆，没有温度降档。采用欧米茄百特EZNA质粒Mini试剂盒提取质粒。 <a href="https://www.jove.com/files/ftp_upload/53177/53177fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53177/53177fig3highres.jpg" width="550" /> 图3:对ministring生产效率（PE）从协议的偏差的影响的结果是3次实验的平均值。 PE是使用HP AlphaImager岁后测量的相对带强度估计。偏差发生在以下的步骤:控制，以下协议;未诱导，在步骤2中没有温度升档;在步骤1.6静止期杂草丛生，温度升档开始; Overinduced，延长在步骤2.3升档温度;早期切除，取出细胞并提取没有在步骤2.3温度降档。 <a href="https://www.jove.com/files/ftp_upload/53177/53177fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Production of Double-stranded DNA Ministrings at JoVE.com

Production of Double-stranded DNA Ministrings

The linear covalently closed (LCC) DNA minivector (DNA ministring) is a non-viral gene delivery vector offering high transfection efficiency and is relevant to any DNA delivery application. The production system is simple, rapid, and scalable in vivo. The following protocol provides visual step-by-step instructions for the production of DNA ministrings.

双链DNA Ministrings的生产

We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ...

production-of-double-stranded-dna-ministrings

Research

JoVE Journal

Biology

10.3K 次观看.  University of Waterloo. The linear covalently closed (LCC) DNA minivector (DNA ministring) is a non-viral gene delivery vector offering high transfection efficiency and is relevant to any DNA delivery application. The production system is simple, rapid, and scalable in vivo. The following protocol provides visual step-by-step instructions for the production of DNA ministrings. 

视频: Production of Double-stranded DNA Ministrings

DNA Curtains Shed Light on Complex Molecular Systems During Homologous Recombination

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in HR are closely associated with a loss of genome integrity and oncogenic transformation in human cells. HR involves coordinated actions of a complex set of proteins, many of which remain poorly understood. The key aspect of the research described here is a technology called "DNA curtains", a technique which allows for the assembly of aligned DNA molecules on the surface of a microfluidic sample chamber. They can then be visualized by total internal reflection fluorescence microscopy (TIRFM). DNA curtains was pioneered by our laboratory and allows for direct access to spatiotemporal information at millisecond time scales and nanometer scale resolution, which cannot be easily revealed through other methodologies. A major advantage of DNA curtains is that it simplifies the collection of statistically relevant data from single molecule experiments. This research continues to yield new insights into how cells regulate and preserve genome integrity.

<meta charset="utf8"></head><body>DNA 窗帘揭示了同源重组过程中的复杂分子系统

DNA curtains present a novel method for visualizing hundreds or even thousands of DNA-binding proteins in real-time as they interact with DNA molecules aligned on the surface of a microfluidic sample chamber.

DNA 窗帘揭示了同源重组过程中的复杂分子系统

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in ...

科研

遗传学

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

使用单分子DNA拉伸含量直接观察DNA复制的酶

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

生物学

<meta charset="utf8"></head><body>通过 ssDNA-RPA 束生长观察到的 CMG 介导的 DNA 解旋

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a dynamic complex of proteins termed the replisome. At the heart of the replisome is the&#160;CDC45-MCM2-7-GINS (CMG) helicase, which separates the two strands of the DNA double helix such that DNA polymerases can copy each strand. During genome duplication, replisomes must overcome a plethora of obstacles and challenges. Each of these threatens genome stability, as failure to replicate DNA completely and accurately can lead to mutations, diseases, or cell death. Therefore, it is of great interest to understand how CMG functions in the replisome during both normal replication and replication stress. Here, we describe a total internal reflection fluorescence (TIRF) microscopy assay using recombinant purified proteins, which allows for real-time visualization of surface-tethered stretched DNA molecules by individual CMG complexes. This assay provides a powerful platform to investigate CMG behavior at the single-molecule level, allowing helicase dynamics to be directly observed with real-time control over reaction conditions.

<meta charset="utf8"></head><body>作者聚焦:通过单分子可视化揭示真核 DNA 复制的动力学

This protocol demonstrates performing a single-molecule assay for live visualization of DNA unwinding by CMG helicase. It describes (1) preparing a DNA substrate, (2) purifying fluorescently labeled Drosophila melanogaster CMG helicase, (3) preparing a microfluidic flow cell for total internal reflection fluorescence (TIRF) microscopy, and (4) the single-molecule DNA unwinding assay.

通过 CMG 解旋酶对 DNA 解旋进行单分子实时可视化

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a ...

生物化学

Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method

Genome duplication is orchestrated by replisome proteins, including helicases, which unwind double-stranded DNA into individual antiparallel single strands, each requiring distinct modes of replication: continuous (leading) and discontinuous (lagging). Understanding the interactions of chromatin-associated proteins with replicating DNA strands in vivo is crucial for elucidating the mechanisms of chromatin assembly and DNA repair coupled to DNA replication. This protocol presents the enrichment and sequencing of protein-associated nascent DNA (eSPAN) method, designed to quantify relative protein levels on nascent leading and lagging DNA strands at replication forks. The eSPAN procedure starts with chromatin immunoprecipitation (ChIP, in yeast) or cleavage under targets and tagmentation (CUT&amp;Tag; in mammalian cells) of a protein of interest, followed by enrichment of the protein-associated nascent DNA by bromodeoxyuridine (BrdU) immunoprecipitation. Strand-specific next-generation sequencing is applied to isolated ssDNA. This technique can be used to determine whether a protein is enriched at leading or lagging replication forks. The eSPAN provides genome-wide strand-preference of chromatin-associated proteins, including histones at replication forks.

Enrichment and sequencing of protein-associated nascent DNA (eSPAN) was developed to detect the relative abundance of a chromatin-associated protein on two replicating DNA strands, thereby revealing molecular insight into chromatin replication and its coupled processes. This protocol describes eSPAN procedures in yeast and mouse embryonic stem (ES) cells.

Genome duplication is orchestrated by replisome proteins, including helicases, which unwind double-stranded DNA into individual antiparallel single ...

The DNA Replication Fork

An organism&#8217;s genome needs to be duplicated in an efficient and error-free manner for its growth and survival. The replication fork is a Y-shaped active region where two strands of DNA are separated and replicated continuously. The coupling of DNA unzipping and complementary strand synthesis is a characteristic feature of a replication fork.&#160;&#160; Organisms with small circular DNA, such as E. coli, often have a single origin of replication; therefore, they have only two replication forks, one in each direction leading away from the location of the initial opening.&#160; In organisms with large genomes, the replication of DNA is not done from a single point of origin but in many distinct, localized replication forks.&#160;
The unhindered progression of the replication fork is necessary for complete DNA replication and genome stability; however, the replication fork is often stalled by internal or external factors that can slow or stop its progression, resulting in replication stress. Replication stress causes genomic instability, which is a hallmark of diseases like cancer. Genomic instability is characterized by genomic alterations and increased frequency of harmful mutations. The movement of the replication fork can stop due to several reasons. For example, the drug hydroxyurea depletes the pool of nucleotides available for incorporation into the daughter strand, stalling the replication fork. Other problems that may hinder the progression of the replication fork include DNA lesions, a collision between a replication fork and a transcription complex, and defects in the enzymes involved in DNA replication.
The cell has a variety of repair mechanisms to reinitiate the stalled replication fork.&#160; S-phase checkpoints do not allow cells to begin mitosis until DNA repair is complete. Additionally, fork repriming can restart DNA synthesis by bypassing a DNA lesion or block. Despite these robust mechanisms, sometimes stalled forks cannot be reinitiated, which leads to the collapse of a fork, halting DNA replication.

DNA 复制叉

An organism&#8217;s genome needs to be duplicated in an efficient and error-free manner for its growth and survival. The replication fork is a Y-shaped ...

教育

JoVE Core

分子生物学

Unit 1

DNA Replication

关键术语与概念

Fixing Double-strand Breaks

The double-stranded structure of DNA has two major advantages. First, it serves as a safe repository of genetic information where one strand serves as the back-up in case the other strand is damaged. Second, the double-helical structure can be wrapped around proteins called histones to form nucleosomes, which can then be tightly wound to form chromosomes. This way, DNA chains up to 2 inches long can be contained within microscopic structures in a cell. A double-stranded break not only damages both copies of genetic information but also disrupts the continuity of DNA, making the chromosome fragile.
In a cell, there are an estimated ten double-strand breaks (DSBs) per day. The primary source of damage is metabolic by-products such as Reactive Oxygen Species and environmental factors such as ionizing radiations. Although less common, malfunctioning nuclear enzymes can also cause DSBs. Failure of enzymes like type II topoisomerases, which cut both strands of DNA and rejoin them while disentangling chromosomes, can inadvertently result in DSBs. Mechanical stress on the DNA duplex can also lead to DSBs. In prokaryotes, prolonged desiccation strains DNA, causing DSBs.
Of the two mechanisms for DNA repair, homologous recombination depends on a sister chromatid being nearby, which happens during the S and G2 phases. Due to this restriction, in the absence of a homology donor, cells have to resort to Nonhomologous end joining (NHEJ), even though it is much less accurate. It has been hypothesized that the reason higher eukaryotes can afford to preferentially utilize NHEJ for DSB repairs is that they have abundant non-coding DNA, which permits nucleotide substitutions, deletions or additions without grievous consequences.

Double-strand Breaks DSB &amp; Nonhomologous End Joining NHEJ

修复双股断裂

The double-stranded structure of DNA has two major advantages. First, it serves as a safe repository of genetic information where one strand serves as the ...

DNA（脱氧核糖核酸）修复和重组

Replication in Prokaryotes

DNA replication has three main steps: initiation, elongation, and termination. Replication in prokaryotes begins when initiator proteins bind to the single origin of replication (ori) on the cell&#39;s circular chromosome. Replication then proceeds around the entire circle of the chromosome in each direction from the two replication forks, resulting in two DNA molecules.
Many Proteins Work Together to Replicate the Chromosome
Replication is coordinated and carried out by a host of specialized proteins. Topoisomerase breaks one side of the double-stranded DNA phosphate-sugar backbone, allowing the DNA helix to unwind more rapidly, while helicase breaks the bonds between base pairs at the fork, separating the DNA into two template strands. Proteins that bind single-stranded DNA molecules stabilize the strands as the replication fork travels along the chromosome. DNA can only be synthesized in the 5&#39; to 3&#39; direction, so one strand of the template&#8212;the leading strand&#8212;is elongated continuously, while the other strand&#8212;the lagging strand&#8212;is synthesized in shorter pieces of 1,000&#8211;2,000 base pairs called Okazaki fragments.
Multiple Polymerases Take Part in Elongation
Much of the research to understand prokaryotic DNA replication has been performed in the bacterium Escherichia coli, a commonly-used model organism. E. coli has five DNA polymerases: Pol I, II, III, IV, and V. Pol III is responsible for the majority of DNA replication. It can polymerize about 1,000 base pairs per second. This astonishing pace allows the machinery present at the two replication forks to duplicate the E. coli chromosome&#8212;4.6 million base pairs&#8212;in roughly 40 minutes. DNA polymerase I is also well-characterized. Its primary role is to process the RNA primers on the lagging strand.
When Division Outpaces Duplication
Under favorable growth conditions, E. coli can divide every 20 minutes, about half the amount of time that it takes to replicate the genome. But how is this possible when both daughter cells must have their own DNA? Scientists found that the bacteria can begin another round of DNA replication from the origin of replication before the first round is complete; this means that daughter cells receive a chromosome that is already in the process of being copied and are prepared to divide again very quickly.

原核生物中的复制

DNA replication has three main steps: initiation, elongation, and termination. Replication in prokaryotes begins when initiator proteins bind to the ...

双链DNA Ministrings的生产

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DNA 窗帘揭示了同源重组过程中的复杂分子系统

DNA 窗帘揭示了同源重组过程中的复杂分子系统

使用单分子DNA拉伸含量直接观察DNA复制的酶

通过 CMG 解旋酶对 DNA 解旋进行单分子实时可视化

通过 CMG 解旋酶对 DNA 解旋进行单分子实时可视化

通过 CMG 解旋酶对 DNA 解旋进行单分子实时可视化

Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method

DNA 复制叉

修复双股断裂

原核生物中的复制