Name: dna-magnetic particle binding analysis by dynamic and electrophoretic light scattering
Uploaded: 2017-11-09T15:00:00.000+00:00
Duration: 10 min 35 s
Description: Watch this Scientific Journal Video about DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering at JoVE.com

捷克科学基金会 (项目 GA CR 17-12816S) 和 CEITEC 2020 (LQ1601) 的财政支持得到了极大的认可。

1. 磁性纳米粒子合成 <ol> <li> 用柠檬酸稳定的磁性纳米粒子合成 (MNPs) <ol> <li> 添加20摩尔 FeCl 3 和 #8729; 6H 2 O (5.406 g) 和10摩尔 FeCl 2 和 #8729; 4H 2 O (1.988 g) 在20毫升的氧双蒸馏水 (ddd 水)。使用机械搅拌器在烧杯中的 N 2 气氛中大力搅拌获得的溶液, 直至获得透明溶液为止. </li>
		<li> 在快速机械搅拌下, 添加150毫升的 1 M NH 4 OH, 使用下降漏斗下降 (如果需要, 采取1小时或以上)。在所有 NH 4 之后, 将解决方案转移到 screwable 容器中。在75和 #176 上拧紧容器和热溶液; C 为 30 min. 
		警告: NH 4 OH 被归类为对环境具有腐蚀性和危险性. 
		注: 准备 nh 4 oh 解决方案从高品位的 28% nh 4 oh 在水中使用氧水. </li>
		<li> 用磁铁收集黑色沉淀物, 用100毫升的 ddd 水冲洗。重复3次。加入50毫升的0.5 米柠檬酸三钠二水合物沉淀。把船拧紧. 
		注: 使用柠檬酸三钠二水合物的氧溶液. </li>
		<li> 将沉淀通过超声 (35 赫、振幅 100%) 和热容器重新分散到80和 #176; C 为 1 h. 
		注: 需要 sonicator 足够的功率. </li>
		<li> 让溶液冷却到室温。用磁铁收集沉淀物并除去上清液。用 sonicator (35 赫, 振幅 100%) 重新分散50毫升的 ddd 水中的黑泥. </li>
		<li> 将等分 (25 毫升) 转换为50毫升离心管, 并加入20毫升丙酮。离心机在 2400 x g 为10分钟, 并删除清. </li>
		<li> 用低分子量切断透析装置 (500-1000 Da) 对水进行再分散沉淀和透析溶液 (24 h. 
		注: 根据制造商和 #39 的要求准备透析装置. </li>
	</ol> </li> <li> 用分枝亚 (MNPs_PEI) <ol> <li> 添加2毫升的 MNPs 溶液 (~ 0.1 克/毫升) 加入玻璃烧杯的磁性纳米微粒的合成。加入38毫升的 ddd 水和几种 (35 赫, 振幅 100%) 暂停 (10 分钟). </li>
		<li> 在快速机械搅拌下, 加10毫升的10% 分枝亚 (平均 25 kDa)。随后 3 h. </li> 的搅拌解决方案
		<li> 收集沉淀使用磁铁和清除上清液。在20毫升的 ddd 水中再分散沉淀。重复5次然后几种最后的悬浮 (35 赫, 振幅 100%). </li>
	</ol> </li> <li> 合成用二氧化硅 (MNPs_TEOS) <ol> <li> 添加10毫升获得的 MNPs 溶液 (0.1 克/毫升) 到玻璃烧杯中的磁性纳米粒子。加入60毫升的水和130毫升的乙醇。几种解决方案, 并添加6毫升 28% NH 4 哦. </li>
		<li> 在快速机械搅拌下, 加入1毫升的四乙基硅酸。随后 12 h. </li> 的搅拌解决方案
		<li> 用磁铁收集黑色沉淀物, 用50毫升乙醇 (3 次) 和50毫升水 (3 次) 冲洗。在10毫升的 ddd 水中再分散颗粒, 几种悬浮液 (35 赫, 振幅为 100%). </li>
	</ol> </li> <li> 合成含氨基的磁性纳米粒子 (MNPs_APTES) <ol> <li> 将获得的 MNPs_TEOS 溶液 (~ 0.1 克/毫升) 添加到一个玻璃烧杯中5毫升。加入45毫升的水和50毫升的乙醇。几种溶液 (35 赫, 振幅为 100%). </li>
		<li> 在快速机械搅拌下, 加入0.5 毫升 (3-氨基丙基) 烷。随后 3 h. </li> 的搅拌解决方案
		<li> 用磁铁收集黑色沉淀物, 用50毫升乙醇 (3 次) 和50毫升水 (3 次) 冲洗。在5毫升的 ddd 水中再分散颗粒, 几种悬浮液 (35 赫, 振幅为 100%). </li>
	</ol> </li> </ol> 2。磁性微粒合成 <ol> <li> 与柠檬酸盐 (基质金属蛋白酶) 的合成磁性微粒, <ol> <li> 加25毫升2米, 12.5 毫升的1米和205.875 毫升的 ddd 水进入 screwable 的容器。在磁力搅拌期间添加6.675 毫升的1米 FeSO 4 。立即将容器转移到预热的水浴 (90 和 #176; C) 为 2 h. 
		注意: 在 ddd 水中准备 3 和 KOH 解决方案. </li>
		<li> 让溶液冷却到室温。用磁铁收集黑色沉淀物, 用50毫升的 ddd 水冲洗 (5 次)。加入50毫升的0.5 米柠檬酸三钠二水合物沉淀。把船拧紧. </li>
		<li> Redisperse 通过超声 (35 赫, 振幅 100%) 和热容器在80和 #176; C 为 1 h. 收集黑色沉淀使用磁铁和洗涤它使用50毫升的 ddd 水 (10 倍). </li>
		<li> 修改与 MNPs 协议类似的分支亚 (MMPs_PEI)、二氧化硅 (MMPs_TEOS) 和氨基组 (MMPs_APTES) 的基质金属蛋白酶。有关详细信息, 请分别参阅2.2、2.3 和2.4 节. </li>
	</ol> </li> <li> 通过分支亚 (MMPs_PEI) <ol> <li> 将5毫升的基质金属蛋白酶溶液 (0.1 克/毫升) 添加到玻璃烧杯中的磁性微粒的合成。添加38毫升的 ddd 水和几种的解决方案 (35 赫, 振幅 100%, 10 分钟)。在快速机械搅拌下加入10毫升的10% 分枝亚 (平均 25 kDa)。随后 3 h. </li> 的搅拌解决方案
		<li> 用磁铁收集沉淀物并除去上清液。Redisperse 沉淀在20毫升的双蒸馏水中。重复5次然后几种最后的解决方案 (35 赫, 振幅 100%). </li>
	</ol> </li> <li> 用硅胶修饰的磁性微粒的合成 (MMPs_TEOS) <ol> <li> 将5毫升的基质金属蛋白酶溶液 (~ 0.1 克/毫升) 加入玻璃烧杯中。加入60毫升的水和130毫升的乙醇。几种悬挂 (35 赫, 振幅 100%), 并添加6毫升 28% NH 4 哦. </li>
		<li> 在快速机械搅拌下, 加入1毫升的四乙基硅酸。随后 12 h. </li> 的搅拌解决方案
		<li> 使用磁铁收集黑色沉淀物, 用50毫升乙醇 (3 次) 和50毫升水 (3 次) 冲洗。Redisperse 粒子在5毫升的 ddd 水和几种悬浮 (35 赫, 振幅 100%). </li>
	</ol> </li> <li> 合成含氨基的磁性微粒 (MMPs_APTES) <ol> <li> 将3毫升的 MMPs_TEOS 溶液 (~ 0.1 克/毫升) 添加到一个玻璃烧杯中。加入45毫升的水和50毫升的乙醇。几种悬浮 (35 赫, 振幅 100%). </li>
		<li> 在快速机械搅拌下, 加入0.5 毫升 (3-氨基丙基) 烷。随后 3 h. </li> 的搅拌解决方案
		<li> 用磁铁收集黑色沉淀物, 用50毫升乙醇 (3 次) 和50毫升水 (3 次) 洗涤。Redisperse 粒子在3毫升的 ddd 水和几种悬浮 (35 赫, 振幅 100%). </li>
	</ol> </li> </ol> 3。磁粉粒度分析使用 dl 的 <ol> <li> 粒子大小在水中 <ol> <li> 使用一个 dl 仪器, 应用后向散射探测器角度 (173 和 #176;) 和 633 nm 的光束波长。
		注: 反散射 dl 适用于集中样品, 可以避免多次散射。侧面散射 (探测器角度90和 #176;) 和前向散射 (探测器角度15和 #176;) dl 分别适用于较小的粒子和混合粒子. </li>
		<li> 在水中测量之前, 几种粒子为3分钟. 
		注: 使用稀释的微粒溶液。为此, 混合9.6 和 #181; l 的颗粒与96和 #181; l 的水. </li>
		<li> 小心吸管50和 #181; 粒子溶液的 L试管, 避免气泡的形成. 
		注: 对于每个样品, 使用一次性试管 (见资料表). </li>
		<li> 注意试管的清晰度和光束的方向。在样品夹中插入单元格并关闭腔室. </li>
		<li> 使用下列参数进行测量: 温度:25 和 #176; C; 色散相 (铁) 折射率: 2.344; 分散环境折射率 (水): 1.333 
		注: 使用折射仪测量折射率以得到更准确的结果, 特别是在开发新颖或混合材料时. </li>
	</ol> </li> </ol> 4。准备 dna 储存, 磁性微粒和捆绑缓冲 <ol> <li> dna 储存 <ol> <li> 使用 10 mM 的三盐酸缓冲 (pH = 8) 来稀释和储存 dna。使用一个标准的 DNA 样本, 可以在实验室中通过直接的 PCR 方法进行放大 (参考5.2 节的反应设置和条件)。要从噬菌体和 #955 中放大 xis 基因的498碱基对 (bp); 控制, 请使用以下引物。前引底漆: 5 和 #39;-CCTGCTCTGCCGCTTCACGC-3 和 #39;反向底漆: 5 和 #39;-TCCGGATAAAAACGTCGATGACATTTGC-3 和 #39; </li> <li> 使用商用 DNA 纯化试剂盒净化 PCR 产品. 
		注: 采用硅胶列程序的商用套件. 100 ng/和 #181; 我纯化了 DNA </li>
		<li> 用分光光度法测定 DNA 浓度 (260 nm)。稀释 10 11 拷贝/和 #181 的 DNA 存量; 按照上述指令, 可以使用 10 10 、10 9 、10 8 和 10 7 副本/和 #181 的工作解决方案; l. 
		注: PCR 产品的拷贝数可以根据以下公式计算, 假设平均分子量是 650 Da 为每个 bp: 脱氧核糖核酸拷贝/和 #181; l = (数量在 ng/和 #181; l * 6.022x10 23 拷贝/摩尔)/(498 bp * 650 克/摩尔 bp* 1x10 9 ng/g) </li> </ol> </li> <li> 磁性微粒 <ol> <li> 允许粒子在没有磁铁的情况下沉积, 并清除上清液。混合 1:20 v/v 磁性微粒与 ddd 水的比值 (颗粒: 水). 
		注: 基于方法用于描述磁性微粒的数量, 因为相等的大量 MNPs 和基质金属蛋白酶容易比较困难. </li>
	</ol> </li> <li> 绑定缓冲区 <ol> <li> 用于快速制备0.75 米醋酸钠-HCL 溶液 (pH 值 = 6), 将1毫升的3米醋酸钠与3毫升的1毫米盐酸盐混合。
		注: 所有在装订中使用的解决方案可以存储在4和 #176; c. 注意低温明显增加了结合, 特别是在乙醇溶液的情况下。仔细控制温度的解决方案, 以更好的重现性和准确性的筛选. </li>
	</ol> </li> </ol> 5。定量 PCR 技术检测 dna 分离效率的研究 <ol> <li> 由修饰的基质金属蛋白酶或 MNPs 的 dna 分离 <ol> <li> 使用96井板和为 96-井板定制的磁铁, 用于小体积实验. </li>
		<li> 使用以下绑定解决方案混合: 8、#181; l 修饰基质金属蛋白酶或 MNPs、10和 #181; 醋酸钠盐酸盐 (0.75 米, pH = 6), 60 和 #181; 85% 乙醇, 10 和 #181; l DNA (10 10 复制解决方案). </li>
		<li> 混合移和放置在磁铁上的板3分钟。当板上的磁铁, 删除解决方案, 并添加100和 #181; L 85% 乙醇洗涤. </li>
		<li> 移除乙醇. 
		注: 允许乙醇干燥几秒钟, 但不要让颗粒 over-dry 或出现裂纹。如果微粒 over-dry, 他们将很难溶解与洗脱液. </li>
		<li> 从磁体中取出板, 并添加40和 #181; 洗脱液的 L。混合移。把盘子放回磁铁上, 收集洗的 DNA (~ 37 和 #181; L)。存储淋洗在-20 和 #176; C 直到进一步分析. </li>
	</ol> </li> <li> 定量 PCR <ol> <li> 使用用于在步骤4.1.1 中准备库存 DNA 的底漆, 以量化 xis 基因的副本, 根据哈达德 et al 的协议. 10 </li> <li> 根据要测试的样本量准备主混合解决方案, 然后将混合物分发给 PCR 管或白色背景 96-井板. </li>
		<li> 对20和 #181 的完整卷使用以下数量; PCR 混合物:10 和 #181; l 聚合酶/插染料混合物 (见材料表), 2 和 #181; l DNA 样本, 2 和 #181; l 向前底漆 (10 和 #181; m), 2 和 #181; l 反转底漆 (10 和 #181; m) </li>
		<li> 根据以下程序设置 thermocycler:95、#176; c 为 120s, 40 周期 (95 和 #176; c 为十五年代, 64 和 #176; c 为十五年代, 68 和 #176; c 为四十五年代), 68 和 #176; c 为 5 min. </li>
		<li> 使用 triplicates 用于标准和样本, 计算周期阈值并构造标准曲线来估计每个样本中检索到的 DNA 的拷贝数。此处使用的工具将自动执行此功能. </li>
		<li> 在这里, 脱氧核糖核酸的拷贝的数量估计为总洗脱容量 (~ 40 和 #181; L)。与添加到绑定解决方案的原始数量 (共 10 11 副本) 相对应的 DNA 所占的百分比可能会在0到100的间隔范围内变化. </li>
	</ol> </li> </ol> 6。dna 磁性粒子结合分析使用 dl <ol> <li> 绑定 dna <ol> 进行大容量实验 (在1.5 毫升管中), 使用以下结合溶液混合物:96 和 #181; l 修饰的基质金属蛋白酶或 MNPs, 120 和 #181; 醋酸钠-盐酸盐(0.75 米, ph 值 = 6), 720 和 #181; l 85% 乙醇, 120 和 #181; l 脱氧核糖核酸 (10 10 拷贝解答) 或120和 #181; l 三 HCL 缓冲 (10 毫米, ph = 8) 为控制. </li>
	</ol> </li> <li> 齐塔人潜在分析 <ol> <li> 准备两个绑定解决方案, 一个带有 DNA, 另一个带有三 HCL 缓冲区, 以便按照步骤6.1.1 中的指示进行控制。为了避免气泡的形成, 小心吸管800和 #181; L 的粘合混合成一个一次性的细胞. 
		注: 使用一次性试管测量表面泽塔电位 (见材料表). </li>
	</ol> </li> <li> 在 dl 仪器上使用以下参数进行测量: Smoluchowski 模型具有 F (ka) 1.5、温度:25 和 #176; C、色散相 (铁) 折射率: 2.344、色散环境折射率 (水): 1.333. 
	注: DNA-磁性粒子相互作用的速率直接影响到复制的测量。一个单一测量由仪器采取 60-70 s。在这里, 仪器被指示采取10测量以1分钟延迟间隔时间。为简单起见, 结果将显示在时间 = 0, 2, 4, 6, 8, 10, 12, 14, 16 和18分钟从一读. </li>
</ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+nanoparticles+characterization:+Sizing+and+zeta+potential+measurement.">Progress in nanoparticles characterization: Sizing and zeta potential measurement.</a> Particuol. 6 (2), 112-115 (2008).</li><li>Krickl, S., Touraud, D., Kunz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+ethanolamine+stabilized+natural+rubber+latex+from+Taraxacum+kok-saghyz+and+from+Hevea+brasiliensis+using+zeta-potential+and+dynamic+light+scattering+measurements.">Investigation of ethanolamine stabilized natural rubber latex from Taraxacum kok-saghyz and from Hevea brasiliensis using zeta-potential and dynamic light scattering measurements.</a> Ind Crops Prod. 103, 169-174 (2017).</li><li>Haddad, Y., 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+Isolation+of+DNA+by+Polycharged+Magnetic+Particles:+An+Analysis+of+the+Interaction+by+Zeta+Potential+and+Particle+Size.">The Isolation of DNA by Polycharged Magnetic Particles: An Analysis of the Interaction by Zeta Potential and Particle Size.</a> Int J Mol Sci. 17 (4), (2016).</li><li>Tenorio-Neto, E. T., 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=Submicron+magnetic+core+conducting+polypyrrole+polymer+shell:+Preparation+and+characterization.">Submicron magnetic core conducting polypyrrole polymer shell: Preparation and characterization.</a> Mater Sci Eng C Mater Biol Appl. 61, 688-694 (2016).</li><li>Baharvand, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+of+ferromagnetic+iron+oxide+particles+by+polyester+resin.">Encapsulation of ferromagnetic iron oxide particles by polyester resin.</a> e-Polym. 8 (1), 1-9 (2008).</li><li>Ghorbani, Z., Baharvand, H., Nezhati, M. N., Panahi, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+polymer+particles+modified+with+beta-cyclodextrin.">Magnetic polymer particles modified with beta-cyclodextrin.</a> J Polym Res. 20 (7), (2013).</li><li>Heger, Z., 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=Paramagnetic+Nanoparticles+as+a+Platform+for+FRET-Based+Sarcosine+Picomolar+Detection.">Paramagnetic Nanoparticles as a Platform for FRET-Based Sarcosine Picomolar Detection.</a> Sci Rep. 5, (2015).</li><li>Navarro, E., Serrano-Heras, G., Casta&#241;o, M. J., Solera, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+PCR+detection+chemistry.">Real-time PCR detection chemistry.</a> Clin Chim Acta. 439, 231-250 (2015).</li></ol>

作者没有什么可透露的。

在这个协议中, 解释 DNA 与磁性粒子通过齐塔人的电位结合的理论原理是有问题的。该协议描述了磁性纳米微粒和微粒的合成和修饰。文中还介绍了制备 DNA 控制和结合液的方法。这里显示了两种筛选 DNA-粒子相互作用的策略: 定量 PCR 和 dl 方法。dl 提供了三指标的物理化学变化的粒子: 颗粒大小, 多指数和泽塔电位。颗粒大小的变化直接表明聚合或解体。多指数值描述了从分散 (指数和 #60; 0.05) 到高...

DNA 分离是分子生物学中最重要的步骤之一。核酸提取方法的发展对基因组学、基因、遗传学和转录等新兴领域产生了巨大的影响。有广泛的生物技术应用的 DNA 隔离, 包括医学 (法医/诊断工具和预后的生物标志), 和环境应用 (基因多样性, 病原体的流行, 和监测)。从不同的材料和不同的尺度, 如血液、尿液、土壤、木材和其他种类的样品, 对 DNA 进行纯化和分离的需求越来越大。1,2,3,4
纳米和微米粒子适用于 DNA 的分离, 因为它们的表面积很高, 特别是当它们可以被磁场固定时。颗粒的物理化学性质, 如大小或电荷, 会极大地影响它们对靶生物分子的束缚能力。5为了进一步增强生物分子的结合力和稳定粒子, 可以利用不同的化学修饰 (表面涂层)。根据共价键和价的相互作用, 对多种不同的结合策略进行分类。6粒子的大小直接影响其磁化特性, 而颗粒成分可以通过加入金属、合金或其他能影响其密度、孔隙度和表面的材料来定制。7没有可靠的方法来测量小颗粒的表面电荷。相反, 在滑移面上的电势 (离纳米颗粒表面的距离) 可以测量。8此值称为 "泽塔电位", 它是一种有效的工具, 通常用于通过 dl 评估纳米和微粒的稳定性。9因为它的值不仅高度依赖于色散环境的 pH 和离子强度, 而且还依赖于粒子的表面特性, 它还可以证明这一表面的变化引起的相互作用粒子和分子的兴趣。10
另一方面, 在脱水条件下的 dna 结构 (A-dna 形式) 表现出致密的构象, 以利于其沉淀 (聚集) 与一般发生的 B dna 形式相比。静电 (离子和 H 键) 是控制 DNA 与其他物质结合的主要力量, 这是由于它们阻的磷酸盐和氮碱 (特别是鸟嘌呤)。7,10
在这项工作中, 对磁性纳米微粒和微粒的三有代表性的化学修饰进行了分析 (图 1A)。介绍了纳米微粒和微粒的合成和化学改性方法。结合 dna 沉淀 (pH 值、离子强度和脱水) 的理论原理, 用于评价 dna 结合和洗脱的方法。采用定量 PCR 技术对具有代表性的纳米微粒和微粒 (图 1B) DNA 的洗脱效率进行了评价。粒子大小、多指数和泽塔电位是用于可视化粒子表面物理化学变化 (图 1C) 的重要参数。强调磁性粒子表面的化学特性是很重要的。虽然这一步骤超出了本议定书的范围, 但可以应用一些现代技术来调查化学修饰的效率。11,12,13,14傅里叶变换红外光谱 (FTIR) 可用于评价粒子表面的红外光谱, 并与自由化学修饰剂的光谱进行比较。x 射线光电子能谱 (XPS) 是另一种可用于识别材料表面元素组成的技术。其他的电化学、显微和光谱方法可以用来揭示粒子合成的质量。这项工作突出了一个新的角度来分析 DNA 磁性粒子的相互作用通过 dl。

<table><tbody><tr><td>Iron(III) chloride hexahydrate</td><td>Sigma-Aldrich</td><td>207926</td><td>Magnetic particle synthesis</td></tr><tr><td>Iron(II) chloride tetrahydrate</td><td>Sigma-Aldrich</td><td>380024</td><td>Magnetic particle synthesis</td></tr><tr><td>Iron(II) sulfate heptahydrate</td><td>Sigma-Aldrich</td><td>F8263</td><td>Magnetic particle synthesis</td></tr><tr><td>Acetone</td><td>Penta</td><td>10060-11000</td><td>Magnetic particle synthesis</td></tr><tr><td>Sodium citrate dihydrate</td><td>Sigma-Aldrich</td><td>W302600</td><td>Magnetic particle synthesis</td></tr><tr><td>Tetraethyl orthosilicate</td><td>Sigma-Aldrich</td><td>131903</td><td>Magnetic particle synthesis</td></tr><tr><td>(3-Aminopropyl)triethoxysilane</td><td>Sigma-Aldrich</td><td>440140</td><td>Magnetic particle synthesis</td></tr><tr><td>Polyethylenimine, branched, average Mw ~25,000</td><td>Sigma-Aldrich</td><td>408727</td><td>Magnetic particle synthesis</td></tr><tr><td>Ammonium hydroxide solution</td><td>Sigma-Aldrich</td><td>221228-M&amp;nbsp;</td><td>Magnetic particle synthesis</td></tr><tr><td>Ethanol</td><td>Penta</td><td>71250-11000</td><td>Magnetic particle synthesis</td></tr><tr><td>Potassium nitrate</td><td>Sigma-Aldrich</td><td>P6083</td><td>Magnetic particle synthesis</td></tr><tr><td>Potassium hydroxide</td><td>Sigma-Aldrich</td><td>1.05012</td><td>Magnetic particle synthesis</td></tr><tr><td>ow-molecular-weight cut-off membrane (Mw=1 kDa)</td><td>Spectrum labs</td><td>G235063</td><td>Magnetic particle synthesis</td></tr><tr><td>Overhead Stirrer</td><td>witeg Labortechnik GmbH</td><td>DH.WOS01035</td><td>Magnetic particle synthesis</td></tr><tr><td>Waterbath</td><td>Memmert GmbH + Co.</td><td>84198998</td><td>Magnetic particle synthesis</td></tr><tr><td>Sonicator</td><td>Bandelin</td><td>795</td><td>Magnetic particle synthesis</td></tr><tr><td>BRAND UV cuvette micro</td><td>Sigma-Aldrich</td><td>BR759200-100EA</td><td>Cuvette for size measurement</td></tr><tr><td>BRAND cap for UV-cuvette micro</td><td>Sigma-Aldrich</td><td>BR759240-100EA</td><td>Cuvette caps for size measurement</td></tr><tr><td>Folded Capillary Zeta Cell</td><td>Malvern</td><td>DTS1070</td><td>Cuvette for zeta potential measurement</td></tr><tr><td>Zetasizer Nano ZS</td><td>Malvern</td><td>ZEN3600</td><td>Device for measurement of size and zeta potential</td></tr><tr><td>Infinite 200 PRO&lt;br/&gt; NanoQuant instrument</td><td>Tecan</td><td>396 227 V1.0, 04-2010</td><td>device for measurement of DNA concentration</td></tr><tr><td>SYBR Green Quantitative RT-PCR Kit</td><td>Sigma-Aldrich</td><td>QR0100</td><td>PCR kit</td></tr><tr><td>Mastercycler pro S instrument</td><td>Eppendorf</td><td>6325 000.013</td><td>Thermocycler</td></tr><tr><td>MinElute kit</td><td>Qiagen</td><td>28004</td><td>DNA purification kit</td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>S7670</td><td>DNA binding</td></tr></tbody></table>

dna-magnetic particle binding analysis by dynamic and electrophoretic light scattering

利用磁性微粒分离 DNA 是生物技术和分子生物学研究的一个重要领域。本协议描述了通过动态光散射 (dl) 和电泳光散射 () 对 DNA 磁粒子结合的评价。dl 的分析提供了关于粒子的物理化学性质的有价值的信息, 包括颗粒大小, 多, 和泽塔电位。后者描述了粒子的表面电荷, 它在 DNA 等材料的静电结合中起主要作用。在这里, 比较分析利用三化学修饰纳米微粒和微粒及其对 DNA 结合和洗脱的影响。研究了分枝亚、四乙基硅酸和 (3-氨基丙氨) 烷的化学修饰。由于 dna 呈负电荷, 因此预期粒子表面的泽塔电位会随着 dna 的结合而减少。簇的形成也会影响粒子的大小。为了研究这些微粒在 dna 分离和洗脱中的效率, 在低 pH 值 (约 6)、高离子强度和脱水环境中, 颗粒与 dna 混合。粒子被用磁铁洗涤, 然后 DNA 被洗的三盐酸缓冲 (pH 值 = 8)。用定量聚合酶链反应 (PCR) 估计 DNA 拷贝数。对其电位、粒径、多和定量 PCR 数据进行了评价和比较。dl 是一种有洞察力和支持性的分析方法, 为 DNA 分离的微粒筛选过程增添了新的视角。

利用本文所述的协议对磁性微粒进行化学合成和改性, 合成并分析了六磁性颗粒的 DNA 结合。分析的摘要显示在表 1中。通过比较水中的颗粒大小和结合溶液, 很清楚, 所有的粒子在结合溶液中聚集 2-22 倍的褶皱。在 DNA 存在下, 一些粒子进一步聚集到更多的褶皱;然而, 这与定量 PCR 检测到的 DNA 检索没有直接关联 (表 1)。对照与 DNA 中的粒子在三粒子?...

Watch this Scientific Journal Video about DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering at JoVE.com

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

该协议描述了磁性粒子的合成, 并通过动态和电泳光散射对其 DNA 结合特性进行了评价。该方法着重于监测粒子大小的变化, 它们的多和粒子表面的泽塔电位, 它们在 DNA 等材料的结合中起着重要作用。

动态和电泳光散射的 DNA-磁性颗粒结合分析

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

dna-magnetic-particle-binding-analysis-dynamic-electrophoretic-light

Universidad San Sebastian

Research

JoVE Journal

Chemistry

12.0K 次观看.  Mendel University in Brno. 该协议描述了磁性粒子的合成, 并通过动态和电泳光散射对其 DNA 结合特性进行了评价。该方法着重于监测粒子大小的变化, 它们的多和粒子表面的泽塔电位, 它们在 DNA 等材料的结合中起着重要作用。

视频: DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

一种优化的协议电泳迁移分析使用红外荧光染料标记的寡核苷酸

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

科研

生物化学

Frequency Mixing Magnetic Detection Scanner for Imaging Magnetic Particles in Planar Samples

The setup of a planar Frequency Mixing Magnetic Detection (p-FMMD) scanner for performing Magnetic Particles Imaging (MPI) of flat samples is presented. It consists of two magnetic measurement heads on both sides of the sample mounted on the legs of a u-shaped support. The sample is locally exposed to a magnetic excitation field consisting of two distinct frequencies, a stronger component at about 77 kHz and a weaker field at 61 Hz. The nonlinear magnetization characteristics of superparamagnetic particles give rise to the generation of intermodulation products. A selected sum-frequency component of the high and low frequency magnetic field incident on the magnetically nonlinear particles is recorded by a demodulation electronics. In contrast to a conventional MPI scanner, p-FMMD does not require the application of a strong magnetic field to the whole sample because mixing of the two frequencies occurs locally. Thus, the lateral dimensions of the sample are just limited by the scanning range and the supports. However, the sample height determines the spatial resolution. In the current setup it is limited to 2 mm. As examples, we present two 20 mm &#215; 25 mm p-FMMD images acquired from samples with 1 &#181;m diameter maghemite particles in silanol matrix and with 50 nm magnetite particles in aminosilane matrix. The results show that the novel MPI scanner can be applied for analysis of thin biological samples and for medical diagnostic purposes.

A scanner for imaging magnetic particles in planar samples was developed using the planar frequency mixing magnetic detection technique. The magnetic intermodulation product response from the nonlinear nonhysteretic magnetization of the particles is recorded upon a two-frequency excitation. It can be used to take 2D images of thin biological samples.

混频磁探测仪在平面样品成像磁性微粒

The setup of a planar Frequency Mixing Magnetic Detection (p-FMMD) scanner for performing Magnetic Particles Imaging (MPI) of flat samples is presented. ...

工程学

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

用质谱和电镜分析 Biolayer 干涉传感器上的动态蛋白复合物的组装和释放

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a technique used to measure the size distribution of polymer solutions or colloidal particles. Although this technique is widely used for the assessment of polymer solutions, it is difficult to measure the particle size in concentrated solutions due to the multiple scattering effect or strong light absorption. Therefore, the concentrated solutions should be diluted before measurement. Implementation of the confocal optical component in a dynamic light scattering microscope1 helps to overcome this barrier. Using such a microscopic system, both transparent and turbid systems can be analyzed under the same experimental setup without a dilution. As a representative example, a size distribution measurement of a temperature-responsive polymer solution was performed. The sizes of the polymer chains in an aqueous solution were several tens of nanometers at a temperature below the lower critical solution temperature (LCST). In contrast, the sizes increased to more than 1.0 &#181;m when above the LCST. This result is consistent with the observation that the solution turned turbid above the LCST.

A protocol for the direct measurement of particle size distribution in concentrated solutions using dynamic light scattering microscopy is presented.

通过动态光散射显微镜混浊解决方案粒度分布的测定

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a ...

化学

Naked-Eye Detection of Rare Point Mutations in DNA

The protocol describes a naked-eye colorimetric test for the detection of somatic point mutations in an excess of wild type DNA. The future foreseen application of the method is the identification of rare mutations in circulating cell-free DNA from liquid biopsies, with a relevance in cancer diagnostics and stratification of oncological patients for personalized therapy. As a proof of concept, the test has been designed to detect the BRAFV600E mutation in the BRAF gene, which is important to identify the sub-group of melanoma patients that can benefit from targeted therapies with BRAF inhibitors. However, this colorimetric test can be easily generalized to other somatic mutations of clinical relevance due to the use of universal detection probes, thus providing strong potential in oncological diagnostics.
The test detects 0.5% of BRAFV600E in an excess of BRAFWT DNA, which matches the sensitivity of some commercial instrumental assays. Such sensitivity is clinically relevant for diagnostic purposes, allowing the early identification of drug-sensitive patients. In contrast to commercial assays based on real-time PCR, this test requires minimal instrumentation and processing, as it can be performed on DNA amplified with a standard PCR (or isothermal techniques) and provides a naked-eye readout with a one-tube reaction of a few steps in only one hour. At present, the test has been used only on synthetic DNA samples. However, the latter have been designed to mimic a real sample amplified from circulating cell-free DNA, to favor the translation of the test to clinical diagnostics.

This protocol permits the naked-eye identification of point mutated DNA in a 200-fold excess of wild type DNA molecules, by exploiting gold nanoparticles and paramagnetic microparticles.

The protocol describes a naked-eye colorimetric test for the detection of somatic point mutations in an excess of wild type DNA. The future foreseen ...

Magnetic Tweezers in a Microplate Format

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these approaches are limited single-molecule methods, which restricts their availability. To address this need, a microplate assay was developed that enables mechanical manipulation while performing standard biochemical assays. This is achieved using magnets incorporated into a microplate lid to create multiple magnetic tweezers. In this format, force is exerted across biomolecules connected to paramagnetic beads, equivalent to a typical magnetic tweezer. The study demonstrates the application of this tool with FRET-based assays to monitor protein conformations. However, this approach is widely applicable to different biological systems ranging from measuring enzymatic activity through to the activation of signaling pathways in live cells.

Here, we describe the use of a novel microplate assay to enable mechanical manipulation of biomolecules while performing ensemble biochemical assays. This is achieved using a microplate lid modified with magnets to create multiple static magnetic tweezers across the microplate.

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these ...

High-Speed Magnetic Tweezers for Nanomechanical Measurements on Force-Sensitive Elements

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are therefore poised to be useful in the field of mechanobiology. Since the method commonly relies on image-based tracking of magnetic beads, the speed limit in recording and analyzing images, as well as the thermal fluctuations of the beads, has long hampered its application in observing small and fast structural changes in target molecules. This article describes detailed methods for the construction and operation of a high-resolution MT setup that can resolve nanoscale, millisecond dynamics of biomolecules and their complexes. As application examples, experiments with DNA hairpins and SNARE complexes (membrane-fusion machinery) are demonstrated, focusing on how their transient states and transitions can be detected in the presence of piconewton-scale forces. We expect that high-speed MTs will continue to enable high-precision nanomechanical measurements on molecules that sense, transmit, and generate forces in cells, and thereby deepen our molecular-level understanding of mechanobiology.

Here, we describe a high-speed magnetic tweezer setup that performs nanomechanical measurements on force-sensitive biomolecules at the maximum rate of 1.2 kHz. We introduce its application to DNA hairpins and SNARE complexes as model systems, but it will be also applicable to other molecules involved in mechanobiological events.

高速磁镊，用于力敏元件的纳米力学测量

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are ...

对完全重构的 Cdc45/Mcm2-7/GINS （CMG） 的运动进行成像

Hybrid Ensemble and Single-molecule Assay to Image the Motion of Fully Reconstituted CMG

Eukaryotes have one replicative helicase known as CMG, which centrally organizes and drives the replisome, and leads the way at the front of replication forks. Obtaining a deep mechanistic understanding of the dynamics of CMG is critical to elucidating how cells achieve the enormous task of efficiently and accurately replicating their entire genome once per cell cycle. Single-molecule techniques are uniquely suited to quantify the dynamics of CMG due to their unparalleled temporal and spatial resolution. Nevertheless, single-molecule studies of CMG motion have thus far relied on pre-formed CMG purified from cells as a complex, which precludes the study of the steps leading up to its activation. Here, we describe a hybrid ensemble and single-molecule assay that allowed imaging at the single-molecule level of the motion of fluorescently labeled CMG after fully reconstituting its assembly and activation from 36 different purified S. cerevisiae polypeptides. This assay relies on the double functionalization of the ends of a linear DNA substrate with two orthogonal attachment moieties, and can be adapted to study similarly complex DNA-processing mechanisms at the single-molecule level.

作者聚焦:在单分子水平上研究真核复制体组分的运动动力学

We report a hybrid ensemble and single-molecule assay to directly image and quantify the motion of fluorescently labeled, fully reconstituted Cdc45/Mcm2-7/GINS (CMG) helicase on linear DNA molecules held in place in an optical trap.

杂交集合和单分子检测对完全重构的 CMG 的运动进行成像

Eukaryotes have one replicative helicase known as CMG, which centrally organizes and drives the replisome, and leads the way at the front of replication ...

动态和电泳光散射的 DNA-磁性颗粒结合分析

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高速磁镊，用于力敏元件的纳米力学测量

杂交集合和单分子检测对完全重构的 CMG 的运动进行成像

杂交集合和单分子检测对完全重构的 CMG 的运动进行成像

杂交集合和单分子检测对完全重构的 CMG 的运动进行成像