Name: following the dynamics of structural variants in experimentally evolved populations
Uploaded: 2023-02-03T13:35:00
Description: Watch this Scientific Journal Video about Following the Dynamics of Structural Variants in Experimentally Evolved Populations at JoVE.com

这项工作得到了ERC HGTCODONUSE（ERC-2015-CoG-682819）对S.B.的支持，这项工作中使用的数据（部分）是通过蒙彼利埃进化科学研究所的GenSeq技术设施在ANR“Investissements d'avenir”计划（ANR-10-LABX-04-01）的支持下产生的。

建立此协议需要精确了解祖先序列中的插入、删除、反转或复制点。该信息通常通过末端或中间点样本的全基因组测序（WGS）获得。在下面的协议中，给出了每个步骤插入突变情况的一般原则，以及一个代表性案例，其中遵循大肠杆菌实验进化群体中 IS10 插入 mutS 基因的频率。在该群体中，终点群体的WGS确定在位置2，463和2，471之间插入了1，329 bp IS10，导致该插入位点重复。该方法?...

<ol>
	<li>Mahmoud, M., 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=Structural+variant+calling:+the+long+and+the+short+of+it.">Structural variant calling: the long and the short of it.</a> Genome Biology. 20 (1), 246 (2019).</li><li>Bragg, D. C., 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=Disease+onset+in+X-linked+dystonia-parkinsonism+correlates+with+expansion+of+a+hexameric+repeat+within+an+SVA+retrotransposon+in+TAF1.">Disease onset in X-linked dystonia-parkinsonism correlates with expansion of a hexameric repeat within an SVA retrotransposon in TAF1.</a> Proceedings of the National Academy of Sciences. 114 (51), 11020-11028 (2017).</li><li>Stransky, N., Cerami, E., Schalm, S., Kim, J. L., Lengauer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+landscape+of+kinase+fusions+in+cancer.">The landscape of kinase fusions in cancer.</a> Nature Communications. 5, 4846 (2014).</li><li>Tenaillon, O., 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=The+molecular+diversity+of+adaptive+convergence.">The molecular diversity of adaptive convergence.</a> Science. 335 (6067), 457-461 (2012).</li><li>Vandecraen, J., Chandler, M., Aertsen, A., Van Houdt, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+insertion+sequences+on+bacterial+genome+plasticity+and+adaptability.">The impact of insertion sequences on bacterial genome plasticity and adaptability.</a> Critical Reviews in Microbiology. 43 (6), 709-730 (2017).</li><li>Andersson, D. I., Gene Hughes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=amplification+and+adaptive+evolution+in+bacteria.">amplification and adaptive evolution in bacteria.</a> Annual Review of Genetics. 43, 167-195 (2009).</li><li>Bailey, S. F., Bataillon, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+the+experimental+evolution+programme+help+us+elucidate+the+genetic+basis+of+adaptation+in+nature.">Can the experimental evolution programme help us elucidate the genetic basis of adaptation in nature.</a> Molecular Ecology. 25 (1), 203-218 (2016).</li><li>Consuegra, J., 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=Insertion-sequence-mediated+mutations+both+promote+and+constrain+evolvability+during+a+long-term+experiment+with+bacteria.">Insertion-sequence-mediated mutations both promote and constrain evolvability during a long-term experiment with bacteria.</a> Nature Communications. 12 (1), 980 (2021).</li><li>Burch, C. L., Romanchuk, A., Kelly, M., Wu, Y., Jones, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+determination+of+barriers+to+horizontal+gene+transfer.">Genome-wide determination of barriers to horizontal gene transfer.</a> bioRxiv. , (2022).</li><li>Slomka, 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=Experimental+evolution+of+Bacillus+subtilis+reveals+the+evolutionary+dynamics+of+horizontal+gene+transfer+and+suggests+adaptive+and+neutral+effects.">Experimental evolution of Bacillus subtilis reveals the evolutionary dynamics of horizontal gene transfer and suggests adaptive and neutral effects.</a> Genetics. 216 (2), 543-558 (2020).</li><li>Choudhury, D., Saini, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+Escherichia+coli+in+different+carbon+environments+for+2,000+generations.">Evolution of Escherichia coli in different carbon environments for 2,000 generations.</a> Journal of Evolutionary Biology. 32 (12), 1331-1341 (2019).</li><li>Tenaillon, O., 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=Tempo+and+mode+of+genome+evolution+in+a+50,000-generation+experiment.">Tempo and mode of genome evolution in a 50,000-generation experiment.</a> Nature. 536 (7615), 165-170 (2016).</li><li>Behringer, M. G., 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=Escherichiacoli+cultures+maintain+stable+subpopulation+structure+during+long-term+evolution.">Escherichiacoli cultures maintain stable subpopulation structure during long-term evolution.</a> Proceedings of the National Academy of Sciences. 115 (20), 4642-4650 (2018).</li><li>Voordeckers, K., 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=Adaptation+to+high+ethanol+reveals+complex+evolutionary+pathways.">Adaptation to high ethanol reveals complex evolutionary pathways.</a> PLoS Genetics. 11 (11), 1005635 (2015).</li><li>Levy, S. F., 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=Quantitative+evolutionary+dynamics+using+high-resolution+lineage+tracking.">Quantitative evolutionary dynamics using high-resolution lineage tracking.</a> Nature. 519 (7542), 181-186 (2015).</li><li>Bruger, E. L., Marx, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+decade+of+genome+sequencing+has+revolutionized+studies+of+experimental+evolution.">A decade of genome sequencing has revolutionized studies of experimental evolution.</a> Current Opinion in Microbiology. 45, 149-155 (2018).</li><li>Grubaugh, N. D., 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=An+amplicon-based+sequencing+framework+for+accurately+measuring+intrahost+virus+diversity+using+PrimalSeq+and+iVar.">An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar.</a> Genome Biology. 20 (1), 8 (2019).</li><li>Bedhomme, 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=Evolutionary+changes+after+translational+challenges+imposed+by+horizontal+gene+transfer.">Evolutionary changes after translational challenges imposed by horizontal gene transfer.</a> Genome Biology and Evolution. 11 (3), 814-831 (2019).</li><li>Tenaillon, O., Toupance, B., Le Nagard, H., Taddei, F., Godelle, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutators,+population+size,+adaptive+landscape+and+the+adaptation+of+asexual+populations+of+bacteria.">Mutators, population size, adaptive landscape and the adaptation of asexual populations of bacteria.</a> Genetics. 152 (2), 485-493 (1999).</li></ol>

在这里，我们提出了一种具有成本效益的方法，该方法可以跟踪实验进化群体中新兴适应性SV等位基因的动力学。该方法将经典PCR技术和自动平行毛细管电泳相结合，可以确定两个等位基因的相对数量。一旦建立，它允许并行定量许多样品中的等位基因比例，并且比WGS便宜得多。该方法可被视为等效于非SNP突变的扩增子测序，并可解决大SV扩增子测序的技术局限性。我们已经表明，该方法使用克服...

结构变异（SV）是基因组序列的改变，通常影响50 bp或更多。所描述的 SV 的四类是大插入、大删除、反转和重复。直到最近，就其表型效应及其作为疾病遗传决定因素的作用或其对适应的贡献而言，对单核苷酸变异（SNV）的关注比对结构变异的关注更多。这可能是因为检测SNV和预测其表型效应更容易。然而，短读长深度测序技术极大地改善了SV的检测，至少在单个个体或克隆基因组中是这样1。同时，它们的表型效应得到了更好的表征，并且已经记录了许多关于它们作为人类疾病2，3或适应新环境4的遗传决定因素的例子。
缺失和插入通常是由于移动遗传元件（MGE）插入引起的，比单核苷酸多态性（SNP）更具破坏性，并导致移码突变和蛋白质结构修饰。基因内的缺失和MGE插入几乎总是导致基因失活，当插入序列（IS）包含启动子或终止序列时，插入到非编码区域会导致相邻基因的抑制或组成性表达5。虽然必需基因的敲除对细菌适应性有明显的不利影响，但在某些情况下，非必需基因的缺失是有益的。尽管有其固有的成本，但重复也可能是有利的，并参与适应，因为它们导致基因剂量的变化;根据条件6，特定蛋白质活性的增加可能是有利的。
微生物实验进化种群通常从克隆开始。这种遗传多样性的最初缺乏，加上试管的“封闭环境”特征，导致通过水平基因转移和重组获得基因的进化潜力非常有限。在这些特定条件下，对缺失、重复和基因组内 MGE 插入的适应的贡献尤为重要;细菌通常通过功能丧失突变（主要是由于缺失或MGE插入）来适应，影响在稳定，通常营养丰富，单一栽培的人工环境中无用的基因7。在运行时间最长的大 肠杆菌 进化实验中，IS150插入在50，000代后进化的人群中特别频繁，IS元件占突变的35%，这些突变在保留其祖先点突变率的种群中达到高频率8。
进化和重新测序研究将实验进化和下一代测序（NGS）技术相结合，以研究细菌如何在表型和基因组水平上适应不同的环境条件和压力，例如不同的碳和能源，抗生素和渗透应激9，10，11.这些研究通常仅在实验终点获得进化群体或克隆的基因组信息，在某些情况下，在许多中间时间点12，13，14。这些数据提供了对适应给定环境所涉及的基因和途径的见解，但很少允许研究人员随着时间的推移跟踪从头出现和扫荡等位基因的动态。
遵循这些动态的一种方法是选择有限数量的分离感兴趣的等位基因（因为它们影响的基因的功能，因为它们在独立群体中平行扫描等），并使用扩增子测序来量化等位基因比例，在同一测序运行中汇集许多时间点15。该方法已成功用于跟踪实验16 和自然17 微生物种群中小尺寸变异（SNP或1 bp插入缺失）的动力学。然而，在较大的插入缺失或MGE插入的情况下，扩增子的大小差异会引起PCR效率差异，从而扭曲了读取和等位基因比例之间的关系。在某些情况下，两个等位基因之间的大小差异优于扩增子的经典长度。在这里，我们将三重态PCR技术与自动平行毛细管电泳相结合，以根据大小判别量化插入等位基因的相对频率。这种方法允许利用未充分利用的实验时间点来确定新兴突变等位基因的动力学，并以具有成本效益的方式跟踪其固定或丢失的频率。我们应用这种方法来跟踪通过IS10插入突变的新兴 mutS-等位基因，为突变的基因型提供超突变表型。
这种方法需要两个靶等位基因，大小差异为≥5%。首先，引物三联体被设计成产生相似大小的片段，它们共享一个共同的引物。其次，优化PCR条件，并使用野生型（WT）和突变gDNA的混合物产生校准曲线。最后，通过PCR扩增样品，并通过平行定量毛细管电泳定量每个等位基因的相对频率。

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		<tr>
			<td>96 Well Skirted PCR Plate</td>
			<td>4titude</td>
			<td>4Ti - 0740</td>
			<td>PCR</td>
		</tr>
		<tr>
			<td>Agarose molecular biology grade</td>
			<td>Eurogentec</td>
			<td>EP-0010-05</td>
			<td>Agarose gel electrophoresis&#160;</td>
		</tr>
		<tr>
			<td>Agilent DNF-474 HS NGS Fragment Kit Quick Guide for the Fragment Analyzer Systems</td>
			<td>Agilent</td>
			<td></td>
			<td>PDF instruction guide</td>
		</tr>
		<tr>
			<td>Buffer TBE</td>
			<td>Panreac appliChem</td>
			<td>A4228,5000Pc</td>
			<td>Agarose gel electrophoresis&#160;</td>
		</tr>
		<tr>
			<td>Calibrated Disposable Inoculating Loops and Needles</td>
			<td>LABELIANS</td>
			<td>8175CSR40H</td>
			<td>Bacterial culture</td>
		</tr>
		<tr>
			<td>Dneasy Blood and Tissue Kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td>DNA extraction</td>
		</tr>
		<tr>
			<td>Electrophoresis power supply</td>
			<td>Amilabo</td>
			<td>ST606T</td>
			<td>Agarose gel electrophoresis&#160;</td>
		</tr>
		<tr>
			<td>Fragment Analyzer Automated CE System</td>
			<td>Agilent</td>
			<td></td>
			<td>Parallel capillary electrophoresis</td>
		</tr>
		<tr>
			<td>Fragment DNA Ladder</td>
			<td>Agilent</td>
			<td>DNF-396, range 1-6000bp</td>
			<td>Parallel capillary electrophoresis</td>
		</tr>
		<tr>
			<td>GENTAMICIN SULFATE SALT BIOREAGENT</td>
			<td>Sigma-Aldrich</td>
			<td>G1264-1G</td>
			<td>Bacterial culture</td>
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		<tr>
			<td>High Sensitivity diluent marker</td>
			<td>Agilent</td>
			<td>DNF-373</td>
			<td>Parallel capillary electrophoresis</td>
		</tr>
		<tr>
			<td>High Sensitivity NGS quantitative analysis kit</td>
			<td>Agilent</td>
			<td>DNF-474</td>
			<td>Parallel capillary electrophoresis</td>
		</tr>
		<tr>
			<td>Ladder quick load 1 kb plus DNA ladder</td>
			<td>NEB</td>
			<td>N0469S</td>
			<td>Agarose gel electrophoresis&#160;</td>
		</tr>
		<tr>
			<td>LB Broth, VegitoneNutriSelect Plus</td>
			<td>Millipore</td>
			<td>28713</td>
			<td>Bacterial culture</td>
		</tr>
		<tr>
			<td>Master Mix PCR High Fidelity Phusion Flash</td>
			<td>Thermo Fisher Scientific</td>
			<td>F548L</td>
			<td>PCR</td>
		</tr>
		<tr>
			<td>Primers</td>
			<td>Eurogentec</td>
			<td></td>
			<td>PCR</td>
		</tr>
		<tr>
			<td>Prosize data analysis software v.4</td>
			<td>Agilent</td>
			<td>V.4</td>
			<td>Parallel capillary electrophoresis</td>
		</tr>
		<tr>
			<td>Qubit assays</td>
			<td>Invitrogen</td>
			<td>MAN0010876</td>
			<td>DNA quantification</td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS Assay Kit</td>
			<td>LIFE TECHNOLOGIES SAS</td>
			<td>Q32854</td>
			<td>DNA quantification</td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>Eppendorf</td>
			<td>Ep gradients</td>
			<td>PCR</td>
		</tr>
		<tr>
			<td>UVbox, eBOX VX5</td>
			<td>Vilber Lourmat</td>
			<td></td>
			<td>Agarose gel electrophoresis visualisation</td>
		</tr>
		<tr>
			<td>Water for injectable preparation</td>
			<td>Aguettant</td>
			<td>PROAMP</td>
			<td>PCR</td>
		</tr>
	</tbody>
</table>

following the dynamics of structural variants in experimentally evolved populations

结构变异（SV）（即缺失、插入、重复和倒置）现在已知在表型变异中起重要作用，因此在疾病确定或适应新环境等过程中起重要作用。然而，单核苷酸变异比SV受到更多的关注，可能是因为它们更容易检测，并且它们的表型效应更容易预测。短读长深度测序技术的发展极大地改善了SV的检测，但从汇集测序（poolseq）数据中量化其频率在技术上仍然复杂且昂贵。
在这里，我们提出了一种相当简单且便宜的方法，该方法允许研究人员跟踪SV等位基因频率的动态。作为应用的一个例子，我们跟踪细菌实验进化群体中插入序列（IS）的频率。该方法基于围绕结构变异边界的引物三重体设计，使得通过复制野生型（WT）和衍生等位基因产生的扩增子在大小上相差至少5%，并且它们的扩增效率相似。然后通过平行毛细管电泳确定每个扩增子的数量，并将其归一化为校准曲线。该方法可以很容易地扩展到其他结构变异（缺失、重复和倒置）的频率定量，以及自然人群（包括患者体内病原体群体）的池-序列方法。

使用从祖先克隆中提取的DNA和从第1，000代S2.11群体中分离的超突变子克隆，我们建立了 图2所示的校准曲线。实验室制备的DNA混合物和平行毛细管电泳仪器测量的实际突变比例通过斜率为1.0706的线性关系连接，R2 为0.9705。此外，生物学重复之间存在良好的一致性;标准曲线的九个点的标准差在0.61和17.74之间。
<img alt="Figure 2" class="xfigimg" src="/files/ftp...

Watch this Scientific Journal Video about Following the Dynamics of Structural Variants in Experimentally Evolved Populations at JoVE.com

Following the Dynamics of Structural Variants in Experimentally Evolved Populations

我们开发了一种具有成本效益的方法来跟踪非单核苷酸多态性等位基因动力学，该方法可以轻松适应实验进化冷冻档案。将三重PCR技术与自动平行毛细管电泳相结合，以量化实验进化过程中插入等位基因的相对频率。

跟踪实验进化人群中结构变异的动态

Structural variants (SVs) (i.e., deletions, insertions, duplications, and inversions) are now known to play an important role in phenotypic variation, and ...

插入、缺失、重复和反转等结构变异在过去更难通过实验进行跟踪，并且它们的频率无法通过扩增子测序准确测量。在这里，我们提供了一种简单的具有成本效益的技术，该技术将一式三份引物设计和平行毛细管电分离置换相结合，以随着时间的推移跟踪结构变异等位基因频率。该方法旨在补充实验进化中的终点测序，并追溯新兴新生等位基因的频率。我们希望这种方法也能使那些使用宿主内病原体数据的人受益。演示该程序的将是来自我们实验室的研究助理Jeanne Hamet。这些方法的不同步骤将显示在派生等位基因由IS10插入称为mutS的细菌基因中产生的情况下。首先设计一对引物，以扩增突变插入位点周围野生型等位基因上的短扩增子。在插入序列中设计第三个引物正向引物二，以从野生型扩增子中产生轻微的大小变量。首先从固定野生型和突变等位基因克隆的 24 小时培养物中提取 DNA。定量DNA后，用水将每个DNA提取物稀释至每微升5纳克。以 50/50 的比例固定两个样本。设置一个 20 微升反应，其中包含 10 纳克 DNA 样品、引物和 PCR 预混液。接下来，使用2%琼脂糖凝胶通过电泳分离PCR产物。野生型扩增子和突变体之间的大小差异必须在凝胶上验证。为了获得校准曲线，首先以各种比例混合野生型DNA和突变型DNA。将PCR产物稀释至每微升浓度0.1纳克。要制备分离凝胶，请将新鲜凝胶和染料混合。更换入口缓冲液。将冲洗缓冲液放在平行毛细管电泳仪的正确抽屉位置。将22微升稀释剂标记物添加到96孔板的孔中。现在向每个样品孔中加入两微升稀释的PCR产物。在一个孔中，从高灵敏度试剂盒中添加一个DNA大小的分子量标准，大小范围为1到6， 000个碱基对。将微孔板放入平行毛细管电泳仪中，在仪器软件上选择运行。使用数据分析软件分析结果。首先，根据峰的已知大小识别峰。量化每个扩增子以产生校准曲线。最后，验证是否正确测量了两个等位基因的相对数量。然后使用该校准曲线计算PCR扩增和平行毛细管电泳后结构变异的数量。这些具有代表性的结果是在mutS基因的破坏性插入之后。敲低和mutS导致超突变表型，允许细菌采样比其基本突变率更多的突变。使用所提出的方法，跟踪了1000代的mutS结构变异频率。揭示了新兴突变等位基因的非单调轨迹。突变等位基因首次在第680代被发现。然后等位基因的频率迅速增加，到713代达到67%。然后这种增长停滞不前，在接下来的53代中仅增加了10%，达到76%出乎意料的是，突变等位基因频率在13代的过程中从76%下降到49%，之后它增加到固定。这种方法将使那些从事实验进化的人受益。该协议允许用户获取冻结的档案并探索进化轨迹，否则端点测序数据无法观察到这些轨迹。

Research

JoVE Journal

Biology

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

人死后脑组织中的单细胞群获得高品质的RNA

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression ...

科研

生物学

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

Evaluation of the Spatial Distribution of &#947;H2AX following Ionizing Radiation

评价以下电离辐射的空间分布的γH2AX

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

漂白实验（FRAP和FLIP）来衡量生活胚胎干细胞的染色质的蛋白质动力学

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

在整个细胞群在单细胞水平上研究细胞周期蛋白B的蛋白水解

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

从基因组DNA选择性捕获的5  - 羟甲基

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Robust 3D DNA FISH Using Directly Labeled Probes

强大的3D直接标记的探针DNA鱼

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

切片培养法继牙胚在植文化的发展

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

隔离增效变种的Hsp104利用酵母Proteinopathy模型

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

研究核苷酸合并: 酵母基因组 Ribonucleotides 的链特异检测及核苷酸诱变的检测

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be ...