Name: genome-wide purification of extrachromosomal circular dna from eukaryotic cells
Uploaded: 2016-04-04T13:00:00
Duration: 14 min 26 s
Description: Watch this Scientific Journal Video about Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells at JoVE.com

Thanks to Kenn D. M&#248;ller and Claus Sternberg (DTU) for technical assistance and to Tue S. J&#248;rgensen for quantitative PCR analysis.

注：在环状DNA纯化和测序方法（圆-SEQ）的概述在图1中示出。 1.栽培，收获细胞和细胞膜破坏<ol><li>从接种的O / N培养酵母细胞（例如酿酒酵母 ）到酵母蛋白胨葡萄糖50 ml完全培养基（YPD）。接种在1-3×10 5个细胞/ ml的低的初始细胞密度或约0.01的OD 600的光密度。 <ol><li>孵育所述细胞在30℃下搅拌，在150转每分钟（RPM），直到细胞达到约1×10 10个细胞的最大细胞密度，大约经过24〜48小时，或在OD 600&gt; 10.0的光学密度。 注：培养时间不是关键，因为较低的细胞浓度可以使用。 </li></ol></li><li>转移能再培养到50ml锥形管中，通过离心沉淀细胞800×g离心3分钟并弃上清。 </li><li>用10mM三Cl，1毫摩尔EDTA，pH 8.0的25ml的缓冲溶液洗涤沉淀，离心，在800×g离心3分钟重新沉淀细胞并弃去上清液。 </li><li>重悬在从质粒柱纯化试剂盒提供1.2毫升重悬浮缓冲液的细胞沉淀。 </li><li>可选步骤：添加高度稀释的质粒作为对照的环状DNA元件20的净化。 注意：在当前的数据集，涂布含有10 10个细胞的每个样品7.7微升质粒混合物。质粒库存混合物由在不同浓度的三种质粒;在pBR322的38纳克/样品，pUC19的0.5纳克/样品，并pUG72 0.01纳克/样品。 </li><li>总悬浮液体积的3比：转移细胞悬液分成两2毫升微离心管中，每个以1补充有0.5毫米的玻璃珠。 </li><li>涡流每个管以最大速度为10分钟，以破坏浆细胞膜。沉淀通过离心珠在268×g离心30秒，并从两个离心管转移1.2毫升合并上清液至新管。 注：替代步骤1.6-1.7，用酶解酶扰乱0.6毫升重悬缓冲液的细胞。 10个单位酶解酶可在35℃下破坏1.5小时内5×10 7个细胞。 </li></ol> 2. EccDNA富集柱色谱<ol><li>按照从质粒柱纯化试剂盒的协议。总之，治疗与1.2ml碱性溶液每个样品中，轻轻混匀并在室温下孵育3分钟。 </li><li>添加1.2毫升中和缓冲液，在9650 xg离心轻轻离心混合5分钟。 </li><li>加载溶液涂布用1ml平衡溶液平衡过的柱，并允许液体在重力作用下通过该柱流过。 </li><li>洗用4ml洗涤溶液的列。当溶液通过树脂，小心加0.3ml洗脱所以lution取代大部分0.35 ml柱的空隙体积。 </li><li>洗脱DNA导入用1ml洗脱溶液新的收集管，并通过加入0.8毫升沉淀混合物沉淀DNA。离心在9650 xg离心10分钟。 </li><li>洗用0.5毫升70％的乙醇，离心该DNA沉淀在9650×g离心5分钟，空气干燥5至15分钟，并溶解在25μl的无菌水的纯化的DNA。 注：仅短期储存水的DNA建议。优先，直接进入第3步。 </li></ol>剩余线性染色体DNA的3消解<ol><li>任选的步骤：为方便线性DNA的特定消化通过核酸外切酶，具有罕见切割核酸内切酶如NotI位对待纯化的DNA。为5微克的DNA，可以使用1个单位的NotI，5微升10X消化缓冲液和无菌水到50微升的总体积。孵育在37℃下反应16小时，并热灭活在80℃的内切酶5分钟。 </li><li>添加20单位外切核酸酶（2微升），4微升的ATP（25毫米），34微升无菌水和直接10微升10X反应缓冲液的50微升内切酶切割的DNA达到100微升的1倍的反应体积，使用ATP依赖性核酸外切酶套件。 </li><li>在37℃下进行直链状单链和双链DNA的水解进行5天以上。增加一个额外的4微升ATP（25毫摩尔），0.6微升10×反应缓冲液和20单位的核酸外切酶，每24小时以继续酶的DNA消化在一个1×反应体积。 </li><li>去除线性DNA后，从外切核酸酶处理的溶液样品2微升确认通过定量聚合酶链反应（qPCR的），使用一个染色体标记染色体的线性DNA的消除，如肌动蛋白基因ACT1 20。 <ol><li>每20微升的qPCR反应体积中含有2微升外切核酸酶处理的样品，150纳米ACT1引物5&#39;-TCCGTCTGGATTGGTGGTTCTA-3&#39;和5&#39;-TGGACCACTTTCGTCGTATTC-3&#39;，2％（体积/体积）二甲基亚砜，和10μl绿色荧光母液。 </li><li>使用的反应条件; 3分钟，在95℃，随后在95℃下45个循环15秒，在60℃30秒。 注：ACT1是线性的DNA特别合适的标记，因为这种基因拷贝数变异是有害的21-23所以eccDNA不应该携带ACT1。 </li><li>替代DNA消化分析，通过qPCR是标准PCR（4.3）或碘化丙啶染色（4.4）。 <ol><li>使用2微升外切核酸酶处理的样品与ACT1引物5&#39;-TGGATTCTGGTATGTTCTAGC-3&#39;和5&#39;-GAACGACGTGAGTAACACC-3&#39;PCR的模板。作为阳性对照ACT1，用50-100纳克基因组S.酵母 DNA为模板。 PCR反应条件; 3分钟，在95℃，接着30秒的35个循环，在95℃，在56℃30秒和72℃1分钟。 </li><li>运行PCR反应的凝胶electrophore虫病在1％琼脂糖用0.5微克/毫升溴化乙锭。寻找一个0.8 kb的ACT1带。 </li></ol></li><li>没有或线性DNA的存在也可以通过碘化丙啶染色之前和之后的DNA扩增进行检测。 <ol><li> 20毫米碘化股票1000 H 2 O型稀释液：用1 1卷：在1混合每一个DNA样本。留在黑暗中10-20分钟的溶液在RT和在663-738纳米和5至30秒的曝光时间使用红色激发荧光滤波器在放大100倍荧光显微镜分析DNA染色。由于DNA染色控制，使用O29放大从酵母和/或O29扩增质粒基因组DNA。 </li></ol></li></ol></li><li>热灭活在70℃下30分钟的核酸外切酶溶液。 </li></ol> 4. DNA扩增<ol><li>扩增纯化的以及从与O29 DNA聚合酶24-26符合步骤3.5）富集eccDNAING的聚合酶制造商的协议。 <ol><li>简言之，混合5微升丰富eccDNA与5微升变性缓冲区。 </li><li>在室温下3分钟后，加入10微升和缓冲。轻轻混匀，加入含有29微升反应缓冲液和1微升O29 DNA聚合酶30μl反应混合液。孵育在30℃下反应16小时以上（高达72小时）。热灭活O29 DNA聚合酶在65℃下3分钟。 </li></ol></li></ol> 5.测序和数据分析<ol><li>用聚焦超声发生器300 bp的平均目标峰大小剪放大eccDNA。使用以下设置为130微升DNA样本：450W峰值功率强，60秒的治疗，30％的占空比，每爆200次循环，温度7℃。 </li><li>添加条形码标签，索引和适配器的零散读取的测序文库合成，使用文库制备合适的方法。 </li><li>运行深度测序，例如141个核苷酸的单端上的高通量测序平台的读取。 </li><li>地图读取被调查酵母参考基因组，并允许读取映射到多个区域。例如，使用一个免费提供的工作流系统27,28和短读对齐绘图软件29。 </li><li>识别使用连续读取，例如，多于七个连续读取（&gt; 1 kb的）无间隙20从推定eccDNAs的区域进行读取。 注：软件可用27,28探索读取映射在感兴趣的基因组区域。 </li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Improved+Method+for+Including+Upper+Size+Range+Plasmids+in+Metamobilomes.">An Improved Method for Including Upper Size Range Plasmids in Metamobilomes.</a> PLoS ONE. 9 (8), e104405 (2014).</li><li>Storlazzi, C. T., Lonoce, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+amplification+as+double+minutes+or+homogeneously+staining+regions+in+solid+tumors:+Origin+and+structure.">Gene amplification as double minutes or homogeneously staining regions in solid tumors: Origin and structure.</a> Genome research. 20 (9), 1198-1206 (2010).</li><li>Von Hoff, D. D., Needham-VanDevanter, D. R., Yucel, J., Windle, B. E., Wahl, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplified+human+MYC+localized+to+replicating+submicroscopic+circular+DNA+molecules.">Amplified human MYC localized to replicating submicroscopic circular DNA molecules.</a> Proceedings of the National Academy of Sciences of the United States of America. 85 (13), 4804-4808 (1988).</li><li>Raymond, E., Faivre, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+hydroxyurea+on+extrachromosomal+DNA+in+patients+with+advanced+ovarian+carcinomas.">Effects of hydroxyurea on extrachromosomal DNA in patients with advanced ovarian carcinomas.</a> Clinical cancer research : an official journal of the American Association for Cancer Research. 7 (5), 1171-1180 (2001).</li><li>Shimizu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrachromosomal+Double+Minutes+and+Chromosomal+Homogeneously+Staining+Regions+as+Probes+for+Chromosome+Research.">Extrachromosomal Double Minutes and Chromosomal Homogeneously Staining Regions as Probes for Chromosome Research.</a> Cytogenetic and genome research. 124 (3-4), 3-4 (2009).</li><li>Eckhardt, S. G., Dai, A., Davidson, K. K., Forseth, B. J., Wahl, G. M., Von Hoff, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+differentiation+in+HL60+cells+by+the+reduction+of+extrachromosomally+amplified+c-myc.">Induction of differentiation in HL60 cells by the reduction of extrachromosomally amplified c-myc.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (14), 6674-6678 (1994).</li><li>Vogt, N., Lef&#232;vre, S. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+structure+of+double-minute+chromosomes+bearing+amplified+copies+of+the+epidermal+growth+factor+receptor+gene+in+gliomas.">Molecular structure of double-minute chromosomes bearing amplified copies of the epidermal growth factor receptor gene in gliomas.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (31), 11368-11373 (2004).</li><li>Ahn, K., Gotay, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+rate+of+disease-related+copy+number+variations+in+childhood+onset+schizophrenia.">High rate of disease-related copy number variations in childhood onset schizophrenia.</a> Molecular psychiatry. 19 (5), 568-572 (2013).</li><li>Girirajan, S., Johnson, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+increases+in+both+common+and+rare+copy+number+load+associated+with+autism.">Global increases in both common and rare copy number load associated with autism.</a> Human molecular genetics. 22 (14), 2870-2880 (2013).</li><li>Vogt, N., Gibaud, A., Lemoine, F., de la Grange, P., Debatisse, M., Malfoy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplicon+rearrangements+during+the+extrachromosomal+and+intrachromosomal+amplification+process+in+a+glioma.">Amplicon rearrangements during the extrachromosomal and intrachromosomal amplification process in a glioma.</a> Nucleic Acids Research. , (2014).</li></ol>

The authors declare that they have no competing financial interests.

圆-SEQ方法允许从酵母细胞序列级别分辨率eccDNA的基因组范围的检测。该方法是一种温和的eccDNA纯化不需要密集涡旋或吹打并通过重力利用柱分离以限制eccDNA破损，这将导致外切核酸酶消化在随后的步骤。该方法的这些特征可以是用于检测包含基因序列大eccDNAs至关重要。圈-SEQ检测众多eccDNAs包括全基因（ 数据集1）。它也检测到86 kb的酵母线粒体DNA。因此，该协议有利于大型环状DNA分子的纯化。保持的DNA提取步骤的数目为最小降低eccDNA损失风险和最大化产量。基于结果的控制，尖刺入质粒，圆-SEQ是高度敏感的，2500细胞检测一个环状DNA。此外，去除内源丰富质粒如2μ;质粒或线粒体DNA可能显著提高灵敏度。从酵母培养2μ的固化已经描述30。可替代地，2μ和线粒体DNA去除可能与一个罕见切割核酸内切酶，如SwaI位来实现。然而，限制性内切酶一步可以针对感兴趣的其他eccDNAs和限制总eccDNA产量。 为eccDNA检测关键步骤是去除线性DNA（步骤3）和DNA测序（步骤5）以一适当的深度。从一个细胞群体记录的大多数eccDNAs的，深度测序可能需要20。配对末端测序应该提供eccDNA检测甚至更大的信心，因为环状DNA结被预期产生配对末端拼凑读取地图。这些差异支持环状DNA结构的发现，并且可以潜在地用作一个附加eccDNA检测滤波器。 圆硒Q滤波方法是使用三个独立S.验证酵母 CEN.PK人群。检测序列包括此前报道eccDNAs，内生质粒和尖刺，质粒和数百假定eccDNAs（ 数据集1）的。这些发现支持从以前S.圈-SEQ数据集酵母 S288C 20。几个共同eccDNAs到CEN.PK和S288C群体的发现表明这些位点有一个倾向存在圆形元件（ 图4）。之前我们已经表明，[GAP1 圈子 ]在CEN.PK后台8个氮有限条件下的丰富，虽然在其他应变背景[GAP1 圈 ]的证据还没有找到。从CUP1-1 RSC30，ASP3-1，COS111和HXT6 HXT7位点均S288C和CEN.PK eccDNA的发现表明，对于DNA环化的倾向是骗子酵母菌株之间供应。这还有待证明，如果[HXT6 / 7 圈 ]，[ASP3-1 圈 ]，[COS111 圈 ]和[CUP1-1 RSC30 圈 ]赋予选择性的优势细胞，或者他们的存在仅仅是高利率的效果DNA函证。 两者合计，结果表明，金环-SEQ是非常适合用于检测碱基大小eccDNAs并具有优势用于识别与完整基因eccDNAs。圆-SEQ是使eccDNAs的全基因组规模屏幕从酵母高灵敏度的方法。圆-SEQ方法可以打开旨在阐明eccDNA在产生基因缺失和扩增的作用研究的新领域。给定的DNA结构和结构的主要来自真核酵母保守到高等真核生物，圆-SEQ方法应，在原则上，是applicablE要所有真核细胞，有轻微的修改。目前，该方法不会出现有任何的限制，虽然还没有被示出它的净化兆碱基大小的eccDNAs能力。此外，使用O29 DNA聚合酶，它采用了滚环扩增方法31的，产生向更小eccDNAs使得eccDNA量化更加困难的偏压。圈-SEQ检测eccDNAs大到足以进行全基​​因，使其适用于从人体细胞双分钟，环状DNA研究。双分钟就可以导致癌症时，原癌基因被这些元素放大32-37。在生殖细胞eccDNAs研究可以用来衡量胚系突变率和评估精子质量，例如在牲畜。因此，圆-SEQ有得到分析上市在哪些遗传变异产生的拷贝数变异的形式的速率，并导致的疾病的新的理解是涉及基因的复印的电位数变异38-40。

发现早期或瞬态染色体扩增是困难的，因为它需要识别大量人口细胞在单个DNA分子的改变。染色体拷贝数变化（CNV的）其成立后，一般检测良好，只留下最后的CNV结构生成该变异1,2-机理的证据。检测并恢复在CNV形成的早期阶段外的环状DNA（eccDNA）可能在基因重排阐明正在进行的过程。 先前，eccDNA 从头发现是由电子显微镜照片如图3所示 ，中期染色体4，或双向电泳5的吉姆萨染色。这些方法提供了关于环状DNA的序列很少或没有信息。定位技术，例如Southern印迹6,7，反向PCR 8，或荧光原位 hybridizatioN 9只提供有关特定eccDNA元素的证据。这些方法没有提供所有现有eccDNA类型的细胞群的序列。 在细胞的基因组池分歧可以通过基因组测序和/或瓦片阵列10,11为特征。检测由常规的DNA纯化方法的缺失或扩增通常需要一个突变的等位基因代表了细胞群12,13的至少0.1-1％。偏心eccDNAs有望甚至在细胞培养更短暂，因为它们缺乏着丝粒中，并在复制潜在缺乏DNA合成。因此，由于大多数eccDNAs想必是在低用量和它们的序列相似的基因组中，需要替代的DNA提取方法来检测eccDNAs。 一些环状DNA纯化技术利用染色体和环状DNA之间的结构性差异。例如，高速ultracentrifug通货膨胀在氯化铯梯度被用于从人HeLa癌细胞株14分离350-3000个碱基对（bp）的大eccDNAs。然而，高速可以打破或缺口超螺旋环状DNA结构的中坚力量，改变沉降速度15和eccDNA产量。杜塔和同事开发了从头 ，从小鼠组织以及来自鸡的培养物和人细胞16,17环状DNA的基因组范围的识别的方法。他们的方法是通过蔗糖超速离心后质粒纯化和几轮酶促反应及DNA提取，从组织匀浆细胞核提取。他们的主要协议标识200-400基点eccDNAs，叫microDNAs。杜塔和同事也试图从酿酒酵母 microDNAs的净化，但无法从这种酵母菌种16条microDNA。 我们已经开发出一种新的方法从酵母eccDNA 从头检测称为圈-SEQ。此方法使基因组范围的调查为大到足以进行全基​​因和一样大86千碱基（kb的）线粒体DNA（mtDNA的）环状DNA分子。圆-SEQ方法是从一套行之有效的原核表达载体纯化方法18,19发达，真核酵母细胞进行了优化和深度测序相结合。通过圆弧-SEQ方法1756不同eccDNAs，全部大于1 KB，从十个与检测S.酵母 S288C烽烟20。之所以选择大小截止专注于eccDNA是大到足以进行全基​​因。圈-SEQ是高度敏感;它检测到的内数千个单元20的一个单eccDNA。在目前的研究中，圆-SEQ被用于分离和从另一个S的三次生物学重复确定294 eccDNAs 酵母酵母菌株，CEN.PK.该数据表明，eccDNA是一种常见的遗传elemeNT在S.酵母菌株。

<table>
	<tbody>
		<tr>
			<td>Bacto peptone</td>
			<td>BD Difco</td>
			<td>211677</td>
			<td>Alternative product can be used.</td>
		</tr>
		<tr>
			<td>Brilliant III SYBR Green PCR Master Mix&#160;</td>
			<td>Agilent Technologies</td>
			<td>600882</td>
			<td>For qPCR analysis. Alternative product can be used.</td>
		</tr>
		<tr>
			<td>Dextrose (D-glucose)</td>
			<td>Carl Roth</td>
			<td>HN06.4</td>
			<td>Alternative product can be used.</td>
		</tr>
		<tr>
			<td>Disruptor Beads, 0.5 mm</td>
			<td>Scientific Industries, Inc.</td>
			<td>SI-BG05</td>
			<td>Glass beads to disrupt plasma cell membranes. Alternative product can be used.</td>
		</tr>
		<tr>
			<td>Ethidium bromide</td>
			<td>Carl Roth</td>
			<td>2218.2</td>
			<td>Agarose gel stain for detecting DNA/RNA.</td>
		</tr>
		<tr>
			<td>GeneJet plasmid miniprep kit</td>
			<td>Thermo Fisher</td>
			<td>K0502</td>
			<td>Plasmid purifcation from bacteria. Alternative product can be used&#160;</td>
		</tr>
		<tr>
			<td>NotI, FastDigest</td>
			<td>Life Technologies -&#160; Thermo Fisher Scientific, USA</td>
			<td>FD0594</td>
			<td>Endonuclease. Alternative product can be used.&#160;</td>
		</tr>
		<tr>
			<td>Plasmid Mini AX kit&#160;</td>
			<td>A&#38;A Biotechnology, Poland</td>
			<td>010-50</td>
			<td>Plasmid purifcation kit used to purify eccDNA.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Plasmid-Safe ATP-dependent DNase kit</td>
			<td>Epicentre, USA</td>
			<td>E3105K</td>
			<td>ATP-dependent exonuclease kit. Alternative product can be used.</td>
		</tr>
		<tr>
			<td>Propidium iodide&#160;</td>
			<td>Sigma-Aldrich, USA</td>
			<td>81845</td>
			<td>Alternative product can be used.</td>
		</tr>
		<tr>
			<td>pUG6 plasmid</td>
			<td>EUROSCARF, Germany</td>
			<td>P30114</td>
			<td>Marker gene: loxP-PAgTEF1-kanMX-TAgTEF1-loxP. Plasmid requests: Please contact Dr. Peter Philippsen@unibas.ch</td>
		</tr>
		<tr>
			<td>QIAGEN genomic-tip 100/G&#160;</td>
			<td>Qiagen, USA</td>
			<td>13343</td>
			<td>Genomic DNA purifcation from yeast. Alternative product can be used.</td>
		</tr>
		<tr>
			<td>REPLI-g Mini Kit protocol&#160;</td>
			<td>Qiagen, USA</td>
			<td>150023</td>
			<td>Amplification of eccDNA by the phi29 polymerase&#160;</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>BD Difco</td>
			<td>210929</td>
			<td>Alternative product can be used.</td>
		</tr>
		<tr>
			<td>Zymolyase 100T (Lyticase, Yeast Lytic Enzyme)</td>
			<td>Nordic BioSite, Sweden</td>
			<td>Z1004-3</td>
			<td>Alternative product can be used.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Data access to sequence files</td>
			<td>European Nucleotide Archive&#160;</td>
			<td></td>
			<td>EccDNA dataset from Saccharomyces cerevisiae CEN.PK113-7D. Study accession number PRJEB9684. 2nd accession number is ERP010820. Locus tag prefix is BN2032.&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Saccharomyces cerevisiae CEN.PK113-7D</td>
			<td></td>
			<td>Genotype MATa MAL2-8c SUC2</td>
		</tr>
		<tr>
			<td>Saccharomyces cerevisiae yeast deletion library pool</td>
			<td>EUROSCARF, Germany</td>
			<td></td>
			<td>S288c BY4741 pool of 4400 viable single-gene deletion mutants disrubted by KanMX module. Genotypes MATa his3&#8710;1 leu2&#8710;0 met15&#8710;0 ura3&#8710;0 genexxx::KanMX.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Spectrophotometer&#160;</td>
			<td colspan="2">NanoDrop 1000 Spectrophotometer, Thermo Fisher</td>
			<td>Measuring DNA concentration. Alternative product can be used.</td>
		</tr>
		<tr>
			<td>Fluorescence microscopy</td>
			<td>Nikon Optronics Magnafire. Red excitation fluorescence filter, 663-738 nm.</td>
			<td></td>
			<td>Alternative product can be used.</td>
		</tr>
		<tr>
			<td>Robotic library-build system</td>
			<td>Apollo 324, IntegenX Inc.</td>
			<td></td>
			<td>DNA library preparation. Alternative product can be used.</td>
		</tr>
		<tr>
			<td>Sequencing platform</td>
			<td colspan="2">Illumina HiSeq 2000 platform, Illumina Inc.</td>
			<td>DNA sequencing. Alternative product can be used.</td>
		</tr>
		<tr>
			<td>Ultrasonicator</td>
			<td colspan="2">Covaris LE220, microTUBE AFA Fiber tubes</td>
			<td>Alternative product can be used.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methods</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2% YPD media&#160;</td>
			<td></td>
			<td></td>
			<td>Mix 10 g Dextrose, 10 g Yeast extract, 20 g Bacto peptone and add H2O to a total volume of 1000 ml and autoclave.</td>
		</tr>
		<tr>
			<td>Circle-Seq test on genomic DNA</td>
			<td></td>
			<td></td>
			<td>Genomic DNA was purified (Qiagen) from a pool of the yeast deletion library (Euroscarf). The DNA concentration was measured by nanodrop and 30 &#181;g genomic DNA was pipetted into two micro centrifuge tubes. One micro centrifuge tube was supplemented with 100 nanogram plasmid (pUG6). The DNA samples were purified by Circle-Seq, omitting the protocol steps 1.1-1.3 and 1.5-1.7. The eluted DNA concentrations were measured by nanodrop and the entire DNA yield from sample GD and GD+P was treated with exonuclease for a period of 29 hours. A 10% fraction was collected for phi29-amplification and PCR analysis, while the remaining DNA was subjected to 72 hour exonuclease treatment. The samples were analyzed for linear DNA content by PCR, using the ACT1 gene as chromosomal marker. A 5% fraction of each of the exonuclease treated samples was amplified by the phi29 DNA polymerase for 16 hours (Qiagen). The presence of DNA in each sample was examined by loading an equal amount (7 &#181;l) in wells on an 0.5 &#181;g/ml ethidium-bromide 0.9% agarose gel after running gel-electrophoresis.&#160;</td>
		</tr>
		<tr>
			<td>Mapping software</td>
			<td>Bowtie2 aligner, John Hopkins University</td>
			<td></td>
			<td>Ultrafast short read alignment. Reference: Langmead B, Salzberg S. Fast gapped-read alignment with Bowtie 2. Nature Methods. 2012, 9:357-359.</td>
		</tr>
		<tr>
			<td>Propidium iodide stain</td>
			<td></td>
			<td></td>
			<td>Images of propidium iodine stained DNA were captured by fluorescence microscopy at 100x magnification (100x/1.30 oil, Nikon) in the RFP channel (red excitation fluorescence filter, 663-738 nm) using identical exposition time (5 seconds).&#160;</td>
		</tr>
		<tr>
			<td>Workflow bioinformatic system</td>
			<td>Galaxy, Open source.</td>
			<td></td>
			<td>A free web-based platform for data intensive biomedical research. References: Goecks, J, Nekrutenko, A, Taylor, J and The Galaxy Team. Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol. 2010 Aug 25;11(8):R86. Blankenberg D, Von Kuster G, Coraor N, Ananda G, Lazarus R, Mangan M, Nekrutenko A, Taylor J. &#34;Galaxy: a web-based genome analysis tool for experimentalists&#34;. Current Protocols in Molecular Biology. 2010 Jan; Chapter 19:Unit 19.10.1-21. Giardine B, Riemer C, Hardison RC, Burhans R, Elnitski L, Shah P, Zhang Y, Blankenberg D, Albert I, Taylor J, Miller W, Kent WJ, Nekrutenko A. &#34;Galaxy: a platform for interactive large-scale genome analysis.&#34; Genome Research. 2005 Oct; 15(10):1451-5.</td>
		</tr>
	</tbody>
</table>

genome-wide purification of extrachromosomal circular dna from eukaryotic cells

<html>外的环状DNA的（eccDNAs）是在酿酒酵母中共有遗传元件和在其它真核生物报告为好。 EccDNAs有助于遗传变异在多细胞生物的体细胞中和单细胞真核生物进化。需要检测eccDNA敏感的方法，以澄清这些因素是如何影响基因组的稳定性，如何将环境和生物因素诱导其在真核细胞的形成。此视频介绍了被称为圈-SEQ敏感eccDNA净化方法。该方法包括环状DNA，除去残留的线性染色体DNA，eccDNA的滚环扩增，深度测序和映射的柱纯化。被要求有足够的线性染色体DNA降解广泛核酸外切酶治疗。滚环扩增步骤由INE高度：正常;“&gt;φ29聚合酶丰富超过线性DNA环状DNA上10细胞三维酿酒酵母 CEN.PK人群中圈-SEQ方法验证检测大小数百eccDNA型材超过1千碱基大。ASP3-1，COS111，CUP1，RSC30，HXT6，HXT7基因的环状DNA均S288C和CEN.PK表明，DNA环化是在这些位点菌株间保守的。总之反复调查，圆-SEQ方法具有广阔适用性在真核生物以及用于检测特定eccDNA类型的基因组规模筛选eccDNA。

为了验证圈-SEQ法，三S.之后细胞在YPD分别生长十代1×10 10个细胞的酵母 CEN.PK群体进行筛选。如先前所述20（数据未示出）的染色体的线性DNA消除由不存在的qPCR ACT1信号的确认。纯化和富集eccDNA测序高达68000000读取（141个核苷酸的单端读出），并映射到CEN.PK113-7D参照基因组（版本19 2012年6月）。来自名为C1，C2和C4中的三个样品推定eccDNAs录音被分配到由连续的映射大于1 kb的读取的基因组区域。按10000 Monte Carlo模拟，通过连续的映射每一区域的意义读取长于1kb的估计。从这79，159和56个地区被标注为可能eccDNA序列（P &lt;0.1， 数据集1）。记录contiguo数我们读&gt; 1 kb的增加作为序列深度暗示，如果样本已被测序进一步（ 图2）更eccDNA内容将被记录的功能。正如预期的，圆-SEQ法提取大量从许多公知的环状DNA分子，包括2μ质粒，线粒体DNA，核糖​​体RNA基因对十二染色体的读取，和三个内部对照质粒pBR322中，pUC19和pUG72该被掺入样品之前柱纯化（ 图3）。 视频显示连续的例子读取映射到第四号染色体的HXT7 _ARS432_ HXT6轨迹。以前，[HXT6 / 7 圈 ]通过圆-SEQ十S288C群（每1×10 10个细胞）和环状DNA结构检测通过反向PCR分析20证实。在[HXT6 / 7 CIrcle]也被记录在每三个CEN.PK种群（ 图4A）的。此外，大多数CEN.PK的重复样本中共同eccDNA基因重叠从S288C数据集（ 图4B）eccDNA基因。 为了测试圈-SEQ协议的特异性为环状DNA纯化，两个样本，每个30微克基因组DNA，进行了测试。一个样品补充有来自两个样品由圆-SEQ协议进行纯化100ng的质粒DNA和eccDNA。柱分离后，将DNA产率的样品1.27％（380毫微克）无质粒（GD）和1.60％（480毫微克），用于将样品与质粒（GD + P）。核酸外切酶处理的效率是为后29小时，并使用PCR对ACT1 72小时线性DNA含量进行测试。未有样品含有扩增ACT1（数据未示出）。每个核酸外切酶处理的样品的分数为进一步由O29 mplified 聚合酶和酶反应的产物通过碘化丙啶染色（ 图5A-F）和琼脂糖凝胶电泳（ 图5G）分析。核酸外切酶处理后，样品显示出最小的碘化丙啶染色（ 图5A-B）。的O29 -只用基因组DNA扩增样品揭示螺纹状结构（ 图5C）与对照样品（ 图5E）。该O29 -这又增加了质粒扩增产物透露灶（ 图5D）类似质粒控制（ 图5F）。这些图像表明，O29聚合酶富集了线性DNA环状DNA。大多数线性染色体DNA是由29小时核酸外切酶处理（ 图5A-B，G）后的样品取出。然而，大量的核酸外切酶治疗时间超过100小时，使用100多个单位是<html>需要除去所有染色体的线性DNA，如O29 -扩增样品仍然显示螺纹状结构的一个背景72小时外切核酸酶处理（ 图5C-D）的后。 <img alt="图1" src="/files/ftp_upload/54239/54239fig1.jpg" /> 图1.圈-SEQ方法的概要的协议有5个步骤：1）细胞培养，2）纯化和eccDNA的富集通过柱色谱法，3）中的洗脱级分其余线性染色体DNA的消化，4）的放大通过O29 DNA聚合酶，和5）高度富集eccDNA的测序和映射的DNA读取到S.酵母参考基因组。 <a href="https://www.jove.com/files/ftp_upload/54239/54239fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> p_upload / 54239 / 54239fig2.jpg“/&gt; 图2.毗连读取&gt; 1 kb的作为序列深度的函数。EccDNA从1×10 10细胞增加，因为序列深度的函数（百万映射阅读）。图：从单倍体CEN.PK S.生物一式三份10 10的细胞分裂分离酵母种群（C1，C2，C4）。 <a href="https://www.jove.com/files/ftp_upload/54239/54239fig2large.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/54239/54239fig3.jpg" /> 图3.检测已知环状DNA元件。（AB）撒在％的读取范围的曲线图（读密度），用于在CEN.PK生物质粒复制C1，C2和C4。映射（A）读取到内源性酵母质粒是：2μ; [rDNA的 圈子（从第十二号染色体核糖体RNA基因）;与mtDNA（线粒体DNA）。 （B）独特的读取映射到控制质粒。对照质粒柱纯化之前被掺入样品。每个细胞的质粒的比率分别为：pBR322中（加号）1：1，pUC19中（圆圈）1:50，及pUG72（三角形）1：2,500。 <img alt="图4" src="/files/ftp_upload/54239/54239fig4.jpg" /> 图4.常见eccDNA元素CEN.PK和S288C。（A）维恩图显示476之间的重叠 基因上的三个CEN.PK样品（C1，C2，C4）在294 eccDNA元素。 16共同重叠eccDNA基因/质粒注释（所有基因的名字都在数据集1）。 （B）中从三个CEN.PK样品（C1，C2，C4）推定eccDNAs所有记录，基因的维恩图，相比于上推定eccDNAs所有记录，基因10 S288C样品：S1-S2，R1〜R4，Z1-Z4（见参考文献20）。示出的是13生物学重复（S1-S2，R1〜R4，Z1-Z4，C1-C3）与基因/质粒和重叠的至少2菌株背景和3或更多的实验设置推定eccDNA区域。 C示例，CEN.PK; R和Z的样品，S288C BY4741; S个样本，S288C M3750。 <a href="https://www.jove.com/files/ftp_upload/54239/54239fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54239/54239fig5.jpg" /> 图5.核酸外切酶后，可视化的DNA样本和 O 2 9治疗。DNA的（AF）碘化丙啶染色。比例尺，10微米。 （A，C和E）与基因组DNA样本（GD）; （B和D）与GD加质粒（GD + P）个样本。 （ <str<html>翁&gt; AB）后29小时核酸外切酶处理（EXO 29 H）; （CD）72小时核酸外切酶处理后O29聚合酶放大后（EXO 72小时+ O29）。 （E）后Ë基因组DNA控制：O29聚合酶放大; （F）质粒控制（5.5 KB）后， 29聚合酶放大; （ 克邻）的琼脂糖凝胶电泳。左起：L，1 kb的标记; P，EXO 29小时后质粒控制（5.5 KB）; GD，EXO后29小时（样品为A）; GD + P，EXO后29小时（样品为B）; GD和GD + P，EXO后29小时+ O29; GD和GD + P，EXO后72小时+ O29（样品为CD）。见表S1 额外的信息。 <a href="https://www.jove.com/files/ftp_upload/54239/54239fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> ASET 1“SRC =”/文件/ ftp_upload / 54239 / 54239dataset1.jpg“/&gt; 在CEN.PK.数据集1.潜在的DNA函证地区 <a href="https://www.jove.com/files/ftp_upload/54239/DATASET1-CENPK.xlsx">请点击这里下载此文件。</a> 示出的是序列数据，并为348的区域进行分析。列AD，eccDNA映射。 A，样品（左起第一列）从假定的eccDNA被确定; B，染色体; CD，启动和假定eccDNAs结束坐标。 EH，eccDNA内容。 E，在该区域的自主复制序列（ARS）;楼在该地区完整的基因; G，包含在该区域的基因的一部分; H，BLASTN确定的基因。 IO，EccDNA覆盖和p值。我，在BP独特注解序列最长的区域; Ĵ，映射的所有的读取次数; K，映射的所有覆盖率从映射百万读取（FPKM）读取由每KB片段;推定的eccDNA L，p值相比，由Monte Carlo模拟的机会发生;男，号联合国iquely映射读取;没有;作为K和L只用唯一映射读取（UFPKM）。对于映射参数读取和描述20蒙特卡罗模拟人。

Watch this Scientific Journal Video about Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells at JoVE.com

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

从真核细胞染色体外的环状DNA基因组范围内的净化

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

Research

JoVE Journal

Biology

Purification of Mitochondria from Yeast Cells

从酵母细胞线粒体的纯化

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

科研

生物学

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

Genome-wide Analysis of Aminoacylation Charging Levels of tRNA Using Microarrays

（充电）酰化tRNA的使用微阵列技术水平的全基因组分析

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both ...

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

采用芯片识别亚型特定的靶基因的全基因组分析

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

从原核文化精简纯化的质粒DNA

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high ...

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

miRNA的目标使用的任务目标ID库的全基因组屏幕

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

定量和自动化高通量的全基因组RNAi屏幕 C。线虫</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

A Novel Bayesian Change-point Algorithm for Genome-wide Analysis of Diverse ChIPseq Data Types

一种新型的全基因组分析的贝叶斯变点算法的多元ChIPseq数据类型

ChIPseq is a widely used technique for investigating protein-DNA interactions. Read density profiles are generated by using next-sequencing of ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

全基因组基因缺失<em&gt;链球菌血统</em通过高通量PCR

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

多聚核糖体分离和哺乳动物Translatomes分析在基因组范围内

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...