Name: determining if dna stained with a cyanine dye can be digested with restriction enzymes
Uploaded: 2018-02-02T13:30:00.000+00:00
Duration: 6 min 58 s
Description: Watch this Scientific Journal Video about Determining if DNA Stained with a Cyanine Dye Can Be Digested with Restriction Enzymes at JoVE.com

这项研究由国家普通医学科学研究所 (研究院) (5605100122001)、国立卫生研究院 (NIH) 的一个组成部分以及内布拉斯加州大学科尔尼 (UNK) 暑期学生研究计划 (SSRP) 和 UNK本科研究奖学金 (报告)。

1. 染料、缓冲剂和琼脂糖凝胶的制备
<ol><li>
为染色 dna 或对染色 dna 的消化准备下列溶液。
	<ol>
<li>制备 1x TE (三盐酸和乙酸酸;edta) 缓冲使用10毫米三盐酸和1毫米 edta 在一个毕业的圆筒或容量烧瓶。室温贮存 (18-25 ° c)。</li>
		<li>制作每种染料的等分: TOTO-1、YOYO-1、POPO-1、BOBO-1、TOTO-3、YOYO-3、POPO-3、BOBO-3 (表 1)。吸管4µL 1 毫米染料到深琥珀色或黑色1.5 毫升管和添加36µL 的 1x TE, 混合使用吸管;这种浓度使100µM 染料溶液 (40 µL 总)。为每种染料完成此步骤。贮存在4° c。 注意: 避免将染料暴露在光中以防止染料漂白或降解。</li>
		<li>使用10毫米双三丙烷-HCl、10 mm 氯化镁2和100µg/毫升牛血清白蛋白 (BSA) 制备缓冲1。准备2使用50毫米氯化钠, 10 毫米三盐酸, 10 毫米氯化镁2, 和100µg/毫升 BSA。使用50毫米醋酸钾、20毫米三乙酸、10毫米醋酸镁和100µg/毫升 BSA 制备缓冲3。每个酶都需要这些特定的缓冲器来起作用。</li>
	</ol>
</li>
	<li>
准备以下溶液凝胶电泳。
	<ol>
<li>将0.25% 溴蓝色、0.25% 二甲苯蓝、15% 聚在 dH2o 中存储在室温下, 制作一个6x 加载染料。</li>
		<li>为了使0.7% 琼脂糖凝胶, 称0.07 克的高凝胶温度 (HGT) 琼脂糖在一个锥形瓶, 增加100毫升的1x 泰缓冲区, 并放置一个倒置烧杯的顶部的烧瓶。<ol>
<li>在热板上加热溶液, 直到气泡开始在烧瓶底部形成并浮到顶端。</li>
			<li>从热板中取出溶液, 让溶液冷却, 直到烧瓶可以徒手触碰, 然后将凝胶倒入 24.5 cm x 21.8 厘米的凝胶模中, 用凝胶梳来制造水井。</li>
			<li>在梳子或凝胶的表面上取出气泡, 用吸管顶端弹出, 让凝胶凝固至少15分钟。这可以储存在一个冰箱 (4 ° c) 包裹在塑料包装1周, 以防止干燥的凝胶或污染。有关如何运行凝胶的详细信息, 请参阅 Lee et al.33. 注: 使用30个单独的井的凝胶梳子。每井应该是4.5 毫米宽与深度2毫米。井的高度将取决于倒入盒子的凝胶量。</li>
		</ol>
</li>
		<li>用40毫米三盐酸、20毫米醋酸和1毫米 edta 制备1x 泰 (三乙酸-edta) 缓冲液。室温贮存 (18-25 ° c)。</li>
	</ol>
</li>
</ol>2. 染色 DNA 的制备
<ol><li>
使用不同的染料等分染色的λ DNA (300-800 ng)。每个染料 (表 1) 将有六不同浓度的测试, 为48的反应, 每限制酶。以下过程概述了1反应的设置 (图 1)。
	<ol>
<li>在4° c 下, 用 1x TE 稀释 100 ng/µL 的λ DNA。用300µL 的总体积来解决。</li>
		<li>染色化λ DNA对于每个预期从文摘, 估计 75-100 ng/波段。添加适量的染料, 以产生浓度1.9 µM, 3.8 µM, 19.4 µM, 32.2 µM, 48.3 µM, 63.5 µM/100 ng 的 DNA。</li>
		<li>在4° c 的夜间孵育 (15 小时-18 小时)。</li>
		<li>让一个反应不作为控件25。 注: 使用化 DNA 进行消化反应。甲基化敏感的酶不能在 DNA 中切割细胞化区域, 并且会出现不完全消化的现象。</li>
	</ol>
</li>
</ol>3. 限制性酶法的制备
<ol><li>
用限制性酶消化染色 dna, 以确定该酶是否能消化 prestained dna (图 1)。
	<ol>
<li>次日, 增加 20 U 限制性酵素, 3 µL 适当的缓冲 (表 2) 和蒸馏, 蒸压和过滤的水共计30µL。对于每种酶反应, 选择具有最高切削效率的缓冲器, 可在制造商的网站上找到。</li>
		<li>将反应放在水浴中, 在最佳酶活性的温度下, 表2列出了 Maschmann et al中使用的6种酶, 用于 2-4 h (表 2)25。</li>
		<li>停止反应, 增加2µL 0.5 米 EDTA pH 8.0。</li>
	</ol>
</li>
</ol>4. 琼脂糖凝胶 Electroeluting 染色 DNA
<ol><li>去除侧面从 24.5 cm x 21.8 cm 胶凝体模子 (步 1.2.2) 和安置胶凝体入胶凝体电泳箱。倒入2.5 升的1x 泰缓冲器到盒子里。小心地从凝胶中取出凝胶梳, 使井可访问33。</li>
	<li>将限制酶反应移到塑料薄膜上, 并将每个反应与6x 加载染料结合起来。对于每个反应溶液, 添加1µL 6x 负载染料每 5-6 µL 的 DNA 溶液 (1x)。然后吸管的反应 (载入染料, < 18 µL) 入井。</li>
	<li>混合1µL 1 kb 阶梯, 9 µL 1x TE, 2 µL 6x 负载染料。加载解决方案的井侧翼的其他解决方案。</li>
	<li>用深蓝色或黑色的纸或织物覆盖凝胶盒。将凝胶盒连接到电源, 将电源设置为30伏, 并在夜间运行 (15-20 小时)。在凝胶运行时关灯, 以防止 photocleavage 标记的 DNA。</li>
	<li>第二天早上关掉电源。采取的凝胶使用托盘和转移到一个容器与45µL 溴溴和1升的1x 泰缓冲区。在室温下将容器存放在黑暗中或用箔盖住, 以防暴露在光线下。让凝胶染色至少45分钟室温。 注意: 使用溴溴化物和手套, 戴上安全眼镜。在清理含有溴溴溶液的容器并扔掉凝胶时, 请参考您的状态处置指南, 以确定如何处理使用过的溴溴化物;此外, 还可以使用其他污渍代替溴溴化物。</li>
</ol>5. 琼脂糖凝胶的成像
<ol><li>将凝胶放在蓝色光 transilluminator 上, 在400-500 纳米之间发出最大的光输出。与连接到车站的摄像头拍照。 注意: 请务必使用照明灯的盖子, 在打开光源之前戴上防护眼镜。 注: 此步骤也可使用任何 UV 光源或标准的凝胶扫描仪完成。</li>
	<li>通过将文件拖到 ImageJ 状态栏中查看 ImageJ 中的图片, 图像会弹出。</li>
	<li>通过比较凝胶的带与预期的在硅中凝胶模式, 确定实验的消化率。为了确定消化率, 实验波段的数量除以预期的 DNA 带数24,25。如果实验消化的 DNA 带模式有预期的模式, 消化率为1。如果图案的碎片比预期的多, 则消化率大于1。如果模式的碎片少于预期, 则消化率小于1。</li>
</ol>

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作者没有什么可透露的。

为了消化荧光标记的 DNA (图 1), 需要一系列步骤。首先, DNA 在一夜之间被荧光染色。DNA 可以用青菁聚孵育一段较短的时间;但是, 卡尔松et al.发现, 由于不完整的染色20, dna 为每个 dna 大小创建了双波段。为了纠正这种现象, DNA 可以在一夜之间被染色以防止双波段的发生。这是协议中的关键步骤。如果染料没有孵育足够长的时间, 染料-DNA 可?...

托托系列 (TOTO-1、YOYO-1、POPO-1、BOBO-1、TOTO-3、YOYO-3、POPO-3 和 BOBO-3;表 1)用于多种实验, 其中 DNA 的可视化是 必需的1,234568,9,10,11,12,13,14,15,16,17. 由于其量子产率、灵敏度和对 DNA 分子的高亲和性, 使菁二聚体被广泛应用于18、19、20。菁二聚体染料对双链 DNA 有很大的选择性, 当插层有100到1000倍的荧光21。吡啶染料 (YOYO-1、TOTO-1、YOYO-3 和 TOTO-3) 的发射波长较其 quinolium 染料 (BOBO-1、POPO-1、BOBO-3 和 POPO-3) 相对较短 (表 1)22。此外, 菁聚插入 DNA 的量子产率高 (0.2-0.6)22。然而, 使用一种酶来确定 dna 分子的甲基化分布,2或在已经沾有荧光染料的 dna 分子的拉伸23中需要一种方法来确定什么酶能消化染色的 dna。任何类型的染料, intercalates 成 dna 或任何酶, 提供了一个可辨认的 dna 基质的模式可以用于这种方法。
孟et al.首先通过使用各种不同染料的凝胶电泳测定 prestained DNA 的消化率24。Maschmann 的et al.深入研究了染料的托托系列。两者都确定了染色 dna 的消化率, 看看是否可以用限制性酶25来消化带有特定染料染色的 dna。其他方法研究染料插层与 DNA 的结合效应使用光学镊子26或核磁共振27。任何一种方法都需要专用设备;然而, 这种方法允许大多数分子生物学实验室确定染料是否干扰限制性酶消化的设备。
此外, 在其他测量给定分子长度的方法中, 光学制图在表面上拉长了不 dna 分子, 并消化了 dna, 以确定片段的拉伸和大小。染料的插层显示增加了荧光染色 DNA 分子的轮廓长度, 并根据所使用的染料, 其轮廓长度是不同的21。该方法已在各种基因组中得到了应用 1,3461328293031. 然而, 如果分子是前染色的, 根据染料和酶, 该酶可能无法切割 DNA 染色与给定的染料。因此, 这种方法确定是否 DNA 染色的特定染料可以消化的酶。此外, 根据染料的浓度和染料的使用, 在凝胶中的 dna 带的迁移速度会比原生 dna 的移动慢得多, 因为 dna 骨架部分解除, 使染料空间插入基对32.
然而, 有时这些染料可以部分或完全抑制某些限制性酶的作用7,24。这被认为是由于荧光染料的附着导致 DNA 结构的改变, 这可能会阻止酶识别其特定的序列。了解这些染料如何影响限制性酶可以帮助在实验中的甲基化剖面或染色 DNA 的延伸是必需的。
在我们的方法, DNA 被染色的荧光的兴趣和消化的限制酶。然后在凝胶上电泳 DNA, 对其进行了成像, 并测定了限制性酶消化率。根据凝胶上的切口形态选择限制性酶。太多的波段导致 dna 带的重叠, 而太少的波段没有给出完整的 dna 分子的图像。有一个甜美的斑点, 能够确定的轮廓的消化 DNA 分子;因此, 它将取决于所使用的 DNA 和酶。这种方法的优点是它的简单性;它只需要在限制消化和凝胶电泳中使用的设备。

<table><tbody><tr><td>Unmethylated lambda DNA</td><td>NEB</td><td>N3013S</td><td></td></tr><tr><td>YOYO-1</td><td>ThermoFisher Scientific</td><td>Y3601</td><td></td></tr><tr><td>TOTO-1</td><td>ThermoFisher Scientific</td><td>T3600</td><td></td></tr><tr><td>POPO-1</td><td>ThermoFisher Scientific</td><td>P3580</td><td></td></tr><tr><td>BOBO-1</td><td>ThermoFisher Scientific</td><td>B3582</td><td></td></tr><tr><td>YOYO-3</td><td>ThermoFisher Scientific</td><td>Y3606</td><td></td></tr><tr><td>TOTO-3</td><td>ThermoFisher Scientific</td><td>T3604</td><td></td></tr><tr><td>POPO-3</td><td>ThermoFisher Scientific</td><td>P3584</td><td></td></tr><tr><td>BamHI</td><td>NEB</td><td>R0136S</td><td></td></tr><tr><td>PmlI</td><td>NEB</td><td>R0532S</td><td></td></tr><tr><td>ScaI</td><td>NEB</td><td>R3122S</td><td></td></tr><tr><td>EcoRI&amp;nbsp;</td><td>NEB</td><td>R0101S</td><td></td></tr><tr><td>HindIII</td><td>NEB</td><td>R0104S</td><td></td></tr><tr><td>SmaI</td><td>NEB</td><td>R0141S</td><td></td></tr><tr><td>Tris</td><td>Fisher</td><td>BP152-1</td><td>Irritant</td></tr><tr><td>EDTA</td><td>Fisher</td><td>S316-212</td><td>Toxic</td></tr><tr><td>HCl</td><td>Fisher</td><td>S25358</td><td>Corrosive and irritant</td></tr><tr><td>Buffer 1</td><td>NEB</td><td>B7201S</td><td>NEB 1.1</td></tr><tr><td>Buffer 2</td><td>NEB</td><td>B7202S</td><td>NEB 2.1</td></tr><tr><td>Buffer 3</td><td>NEB</td><td>B7204S</td><td>NEB Cutsmart</td></tr><tr><td>High gelling temperature agarose gel</td><td>Lonza</td><td>50041</td><td></td></tr><tr><td>Acetic Acid</td><td>Fisher</td><td>S25118</td><td>Hazardous</td></tr><tr><td>Blue light transilluminator</td><td>Dark Reader</td><td>DR196</td><td>Wear correct eyewear&amp;nbsp;</td></tr><tr><td>Ethidium bromide</td><td>Fisher</td><td>15585011</td><td>Carcinogen; other alternatives are Nancy 520, Gel red, etc.</td></tr><tr><td>Cannon EOS Rebel T3I camera</td><td>Cannon</td><td></td><td></td></tr><tr><td>Apogee Model H4</td><td>Biorad</td><td>1704510</td><td></td></tr><tr><td>Power Pac Basic Power Supply</td><td>Biorad</td><td>1645050&amp;nbsp;</td><td></td></tr><tr><td>Ficoll</td><td>Fisher Scientific</td><td>BP525-25</td><td></td></tr><tr><td>Bromophenol blue</td><td>Fisher Scientific</td><td>B-392</td><td></td></tr><tr><td>Xylene Cyanol</td><td>Eastmen</td><td>T1579</td><td></td></tr></tbody></table>

determining if dna stained with a cyanine dye can be digested with restriction enzymes

荧光显微镜的 DNA 可视化利用了各种染料, 如菁染料。这些染料因其对 DNA 的亲和力和敏感性而被利用。为了确定在实验完成后 dna 分子是否全长, 需要用限制性酶消化 dna 来确定染色分子是否全长。然而, 染色的 dna 可能会抑制酶, 因此需要一种方法来确定什么酶可以用于荧光染色 dna。在这种方法中, dna 在一夜之间被染有菁染料, 使染料和 dna 平衡。接下来, 染色 DNA 被一种限制性的酶消化, 装入凝胶和电泳。将实验的 DNA 消化带与在硅片中的文摘进行比较, 以确定限制性酶活性。如果有相同数量的波段如预期, 那么反应是完整的。超过预期的波段表明部分消化和较少的带表示不完全消化。这种方法的优点是它的简单性, 它使用的设备, 科学家将需要一个限制酶检测和凝胶电泳。这种方法的一个局限性是, 大多数科学家可用的酶是商业上可用的酶;但是, 任何限制酶都可以使用。

为了确定插染料是否会影响消化 DNA 的限制性酶, 必须遵循正确的步骤顺序 (图 1)。一旦 DNA 被一种特定的酶染色和消化, 可以采取凝胶的图片来确定碎片的数量及其大小 (图 2)。为了确定酶的效率, 预期可见带的总数除以可见带数。酶的效率等同于统一, 如果数量的波段预期和看到匹配。如果在凝胶上有更多的带, 然后预期, 该值?...

Watch this Scientific Journal Video about Determining if DNA Stained with a Cyanine Dye Can Be Digested with Restriction Enzymes at JoVE.com

Determining if DNA Stained with a Cyanine Dye Can Be Digested with Restriction Enzymes

染色 DNA 分子荧光显微镜允许科学家在实验中看到他们。在这里提出的方法, DNA 分子是前染色的荧光染料和消化的甲基化和非甲基化的敏感限制酶。

用限制性酶来测定染有菁染料的 DNA

Visualization of DNA for fluorescence microscopy utilizes a variety of dyes such as cyanine dyes. These dyes are utilized due to their high affinity and ...

determining-if-dna-stained-with-cyanine-dye-can-be-digested-with

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

8.7K 次观看.  University of Nebraska - Kearney. 染色 DNA 分子荧光显微镜允许科学家在实验中看到他们。在这里提出的方法, DNA 分子是前染色的荧光染料和消化的甲基化和非甲基化的敏感限制酶。

视频: Determining if DNA Stained with a Cyanine Dye Can Be Digested with Restriction Enzymes

DNA Electrophoresis Using Thiazole Orange Instead of Ethidium Bromide or Alternative Dyes

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, DNA is visualized by staining. This article demonstrates how to use thiazole orange to stain DNA. Thiazole orange compares favorably to common staining methods, in that it is sensitive, inexpensive, excitable with UV or blue light (to prevent sample damage), and safer than ethidium bromide. Labs already equipped to run DNA electrophoresis experiments using ethidium bromide can generally switch dyes with no additional changes to existing protocols, using UV light for detection. Blue-light detection to avoid sample damage can additionally be achieved with a blue-light source and emission filter. Labs already equipped for blue-light detection can simply switch dyes with no additional changes to existing protocols.

Here, we present a protocol to use thiazole orange for the detection of DNA in gel electrophoresis experiments. The use of thiazole orange allows elimination of ethidium bromide, and fluorescence detection can be achieved with either UV or blue light.

用噻唑橙代替溴代或替代染料进行 DNA 电泳

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, ...

科研

生物化学

In-Nucleus Hi-C in Drosophila Cells

The genome is organized into topologically associating domains (TADs) delimited by boundaries that isolate interactions between domains. In Drosophila, the mechanisms underlying TAD formation and boundaries are still under investigation. The application of the in-nucleus Hi-C method described here helped to dissect the function of architectural protein (AP)-binding sites at TAD boundaries isolating the Notch gene. Genetic modification of domain boundaries that cause loss of APs results in TAD fusion, transcriptional defects, and long-range topological alterations. These results provided evidence demonstrating the contribution of genetic elements to domain boundary formation and gene expression control in Drosophila. Here, the in-nucleus Hi-C method has been described in detail, which provides important checkpoints to assess the quality of the experiment along with the protocol. Also shown are the required numbers of sequencing reads and valid Hi-C pairs to analyze genomic interactions at different genomic scales. CRISPR/Cas9-mediated genetic editing of regulatory elements and high-resolution profiling of genomic interactions using this in-nucleus Hi-C protocol could be a powerful combination for the investigation of the structural function of genetic elements.

The genome is organized in the nuclear space into different structures that can be revealed through chromosome conformation capture technologies. The in-nucleus Hi-C method provides a genome-wide collection of chromatin interactions in Drosophila cell lines, which generates contact maps that can be explored at megabase resolution at restriction fragment level.

果蝇细胞中的核内高C

The genome is organized into topologically associating domains (TADs) delimited by boundaries that isolate interactions between domains. In Drosophila, ...

遗传学

用于本科研究和教学的染色体构象捕获 （3C）

Getting an A with the 3Cs: Chromosome Conformation Capture for Undergraduates

Chromosome conformation capture (3C) is a powerful tool that has spawned a family of similar techniques (e.g., Hi-C, 4C, and 5C, referred to here as 3C techniques) that provide detailed information of the three-dimensional organization of chromatin. The 3C techniques have been used in a wide range of studies, from monitoring the changes in chromatin organization in cancer cells to identifying enhancer contacts made with gene promoters. While many of the studies using these techniques are asking big genome-wide questions with intricate sample types (i.e., single-cell analysis), what is often lost is that the 3C techniques are grounded in basic molecular biology methods that are applicable to a broad range of studies. By addressing tightly focused questions of chromatin organization, this cutting-edge technique can be used to enhance the undergraduate research and teaching lab experience. This paper presents a 3C protocol and provides adaptations and points of emphasis for implementation at primarily undergraduate institutions in undergraduate research and teaching experiences.

作者聚焦：用 3C 获得 A：本科生的染色体构象捕获  

Here, we present an adaption of the chromosome conformation capture (3C) technique in detail with an emphasis on undergraduate involvement and learning.

获得 3C 的 A：本科生的染色体构象捕获

Chromosome conformation capture (3C) is a powerful tool that has spawned a family of similar techniques (e.g., Hi-C, 4C, and 5C, referred to here as 3C ...

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

限制性核酸内切酶介导的 DNA 切割的单分子停留时间分析

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

嗨 - C：一个方法研究基因组的三维架构。

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

生物学

DNA-based Fish Species Identification Protocol

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. This versatile method employs PCR amplification of genomic DNA extracted from fish samples, followed by restriction fragment length polymorphism (RFLP) analysis to generate fragment patterns that can be resolved on the Agilent 2100 Bioanalyzer and matched to the correct species using RFLP pattern matching software.
The fish identification method uses a simple, reliable, spin column- based protocol to isolate DNA from fish samples. The samples are treated with proteinase K to release the nucleic acids into solution. DNA is then isolated by suspending the sample in binding buffer and loading onto a micro- spin cup containing a silica- based fiber matrix. The nucleic acids in the sample bind to the fiber matrix. The immobilized nucleic acids are washed to remove contaminants, and total DNA is recovered in a final volume of 100 &mu;l. The isolated DNA is ready for PCR amplification with the provided primers that bind to sequences found in all fish genomes. The PCR products are then digested with three different restriction enzymes and resolved on the Agilent 2100 Bioanalyzer. The fragment lengths produced in the digestion reactions can be used to determine the species of fish from which the DNA sample was prepared, using the RFLP pattern matching software containing a database of experimentally- derived RFLP patterns from commercially relevant fish species.

This publication describes how to use the Agilent Fish Species Identification System to identify the species of a fish by extracting DNA and performing PCR and RFLP analysis.

以DNA为基础的鱼类物种鉴定协议

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. ...

<meta charset="utf8"></head><body>使用单分子测定法研究 TRF1/2 与端粒 DNA 的相互作用

Analyzing Telomeric Protein-DNA Interactions Using Single-Molecule Magnetic Tweezers

Telomeres, the protective structures at the ends of chromosomes, are crucial for maintaining cellular longevity and genome stability. Their proper function depends on tightly regulated processes of replication, elongation, and damage response. The shelterin complex, especially Telomere Repeat-binding Factor 1 (TRF1) and TRF2, plays a pivotal role in telomere protection and has emerged as a potential anti-cancer target for drug discovery. These proteins bind to the repetitive telomeric DNA motif TTAGGG, facilitating the formation of protective structures and recruitment of other telomeric proteins. Structural methods and advanced imaging techniques have provided insights into telomeric protein-DNA interactions, but probing the dynamic processes requires single-molecule approaches. Tools like magnetic tweezers, optical tweezers, and atomic force microscopy (AFM) have been employed to study telomeric protein-DNA interactions, revealing important details such as TRF2-dependent DNA distortion and telomerase catalysis. However, the preparation of single-molecule constructs with telomeric repetitive motifs continues to be a challenging task, potentially limiting the breadth of studies utilizing single-molecule mechanical methods. To address this, we developed a method to study interactions using full-length human telomeric DNA with magnetic tweezers. This protocol describes how to express and purify TRF2, prepare telomeric DNA, set up single-molecule mechanical assays, and analyze data. This detailed guide will benefit researchers in telomere biology and telomere-targeted drug discovery.

<meta charset="utf8"></head><body>作者聚焦:用于研究端粒蛋白-DNA 相互作用的先进单分子技术

This protocol demonstrates using single-molecule magnetic tweezers to study interactions between telomeric DNA-binding proteins (Telomere Repeat-binding Factor 1 [TRF1] and TRF2) and long telomeres extracted from human cells. It describes the preparatory steps for telomeres and telomeric repeat-binding factors, the execution of single-molecule experiments, and the data collection and analysis methods.

用限制性酶来测定染有菁染料的 DNA

摘要

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此视频中的章节

用噻唑橙代替溴代或替代染料进行 DNA 电泳

果蝇细胞中的核内高C

获得 3C 的 A：本科生的染色体构象捕获

获得 3C 的 A：本科生的染色体构象捕获

获得 3C 的 A：本科生的染色体构象捕获

限制性核酸内切酶介导的 DNA 切割的单分子停留时间分析

嗨 - C：一个方法研究基因组的三维架构。

以DNA为基础的鱼类物种鉴定协议

使用单分子磁镊分析端粒蛋白-DNA 相互作用

使用单分子磁镊分析端粒蛋白-DNA 相互作用