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>

<ol>
	<li>Teague, B., 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-resolution+human+genome+structure+by+single-molecule+analysis.">High-resolution human genome structure by single-molecule analysis.</a> Proc Natl Acad Sci U S A. 107 (24), 10848-10853 (2010).</li><li>Ananiev, G. E., 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=Optical+mapping+discerns+genome+wide+DNA+methylation+profiles.">Optical mapping discerns genome wide DNA methylation profiles.</a> BMC Mol Biol. 9, 68 (2008).</li><li>Zhou, 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=Validation+of+rice+genome+sequence+by+optical+mapping.">Validation of rice genome sequence by optical mapping.</a> BMC Genomics. 8, 278 (2007).</li><li>Reslewic, 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=Whole-genome+shotgun+optical+mapping+of+Rhodospirillum+rubrum.">Whole-genome shotgun optical mapping of Rhodospirillum rubrum.</a> Appl Environ Microbiol. 71 (9), 5511-5522 (2005).</li><li>Zhou, 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=Whole-genome+shotgun+optical+mapping+of+Rhodobacter+sphaeroides+strain+2.4.1+and+its+use+for+whole-genome+shotgun+sequence+assembly.">Whole-genome shotgun optical mapping of Rhodobacter sphaeroides strain 2.4.1 and its use for whole-genome shotgun sequence assembly.</a> Genome Res. 13 (9), 2142-2151 (2003).</li><li>Gupta, A., Kounovsky-Shafer, K. L., Ravindran, P., Schwartz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+mapping+and+nanocoding+approaches+to+whole-genome+analysis.">Optical mapping and nanocoding approaches to whole-genome analysis.</a> Microfluidics and Nanofluidics. 20 (44), 1-14 (2016).</li><li>Kounovsky-Shafer, K. 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=Presentation+of+large+DNA+molecules+for+analysis+as+nanoconfined+dumbbells.">Presentation of large DNA molecules for analysis as nanoconfined dumbbells.</a> Macromolecules. 46 (20), 8356-8368 (2013).</li><li>Kim, 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=Nanochannel+confinement:+DNA+stretch+approaching+full+contour+length.">Nanochannel confinement: DNA stretch approaching full contour length.</a> Lab Chip. 11 (10), 1721-1729 (2011).</li><li>Yu, H., Jo, K., Kounovsky, K. L., de Pablo, J. J., Schwartz, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+propulsion:+chemical+sensing+and+chemotaxis+of+DNA+driven+by+RNA+polymerase.">Molecular propulsion: chemical sensing and chemotaxis of DNA driven by RNA polymerase.</a> J Am Chem Soc. 131 (16), 5722-5723 (2009).</li><li>Lam, 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=Genome+mapping+on+nanochannel+arrays+for+structural+variation+analysis+and+sequence+assembly.">Genome mapping on nanochannel arrays for structural variation analysis and sequence assembly.</a> Nat Biotechnol. 30 (8), 771-776 (2012).</li><li>Cao, H., Tegenfeldt, J. O., Austin, R. H., Chou, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradient+nanostructures+for+interfacing+microfluidics+and+nanofluidics.">Gradient nanostructures for interfacing microfluidics and nanofluidics.</a> Applied Physics Letters. 81 (16), 3058-3060 (2002).</li><li>Gupta, 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=Single-molecule+analysis+reveals+widespread+structural+variation+in+multiple+myeloma.">Single-molecule analysis reveals widespread structural variation in multiple myeloma.</a> Proc Natl Acad Sci U S A. 112 (25), 7689-7694 (2015).</li><li>Wu, C. W., Schramm, T. M., Zhou, S., Schwartz, D. C., Talaat, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+mapping+of+the+Mycobacterium+avium+subspecies+paratuberculosis+genome.">Optical mapping of the Mycobacterium avium subspecies paratuberculosis genome.</a> BMC Genomics. 10, 25 (2009).</li><li>Jo, K., Schramm, T. M., Schwartz, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single-molecule+barcoding+system+using+nanoslits+for+DNA+analysis+:+nanocoding.">A single-molecule barcoding system using nanoslits for DNA analysis : nanocoding.</a> Methods Mol Biol. 544, 29-42 (2009).</li><li>Jo, K., Chen, Y. L., de Pablo, J. J., Schwartz, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elongation+and+migration+of+single+DNA+molecules+in+microchannels+using+oscillatory+shear+flows.">Elongation and migration of single DNA molecules in microchannels using oscillatory shear flows.</a> Lab Chip. 9 (16), 2348-2355 (2009).</li><li>Jo, 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=A+single-molecule+barcoding+system+using+nanoslits+for+DNA+analysis.">A single-molecule barcoding system using nanoslits for DNA analysis.</a> Proc Natl Acad Sci U S A. 104 (8), 2673-2678 (2007).</li><li>Reccius, C. H., Mannion, J. T., Cross, J. D., Craighead, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compression+and+free+expansion+of+single+DNA+molecules+in+nanochannels.">Compression and free expansion of single DNA molecules in nanochannels.</a> Phys Rev Lett. 95 (26), 268101 (2005).</li><li>Staerk, D., Hamed, A. A., Pedersen, E. B., Jacobsen, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bisintercalation+of+homodimeric+thiazole+orange+dyes+in+DNA:+effect+of+modifying+the+linker.">Bisintercalation of homodimeric thiazole orange dyes in DNA: effect of modifying the linker.</a> Bioconjug Chem. 8 (6), 869-877 (1997).</li><li>Spielmann, H. P., Wemmer, D. E., Jacobsen, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+structure+of+a+DNA+complex+with+the+fluorescent+bis-intercalator+TOTO+determined+by+NMR+spectroscopy.">Solution structure of a DNA complex with the fluorescent bis-intercalator TOTO determined by NMR spectroscopy.</a> Biochemistry. 34, 8542-8553 (1995).</li><li>Carlsson, C., Jonsson, M., Akerman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+bands+in+DNA+gel+electrophoresis+caused+by+bis-intercalating+dyes.">Double bands in DNA gel electrophoresis caused by bis-intercalating dyes.</a> Nucleic Acids Res. 23 (13), 2413-2420 (1995).</li><li>Kundukad, B., Yan, J., Doyle, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+YOYO-1+on+the+mechanical+properties+of+DNA.">Effect of YOYO-1 on the mechanical properties of DNA.</a> Soft Matter. 10 (48), 9721-9728 (2014).</li><li>Technologies, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Molecular Probes Handbook: A guide to Fluorescent Probes and Labeling Technologies. , (2010).</li><li>Riehn, R., 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=Restriction+mapping+in+nanofluidic+devices.">Restriction mapping in nanofluidic devices.</a> Proc Natl Acad Sci U S A. 102 (29), 10012-10016 (2005).</li><li>Meng, X., Cai, W., Schwartz, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+restriction+endonuclease+activity+by+DNA+binding+fluorochromes.">Inhibition of restriction endonuclease activity by DNA binding fluorochromes.</a> J Biomol Struct Dyn. 13 (6), 945-951 (1996).</li><li>Maschmann, A., Kounovsky-Shafer, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+restriction+enzyme+activity+when+cutting+DNA+labeled+with+the+TOTO+Dye+family.">Determination of restriction enzyme activity when cutting DNA labeled with the TOTO Dye family.</a> Nucleosides Nucleotides Nucleic Acids. , 1-12 (2017).</li><li>Biebricker, A. 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=The+impact+of+DNA+intercalators+on+DNA+and+DNA-processing+enzymes+elucidated+through+force-dependent+binding+kinetics.">The impact of DNA intercalators on DNA and DNA-processing enzymes elucidated through force-dependent binding kinetics.</a> Nature Communications. 6, 7304 (2015).</li><li>Gunther, K., Mertig, M., Seidel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+and+structural+properties+of+YOYO-1+complexed+DNA.">Mechanical and structural properties of YOYO-1 complexed DNA.</a> Nucleic Acids Res. 38 (19), 6526-6532 (2010).</li><li>Zhou, 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=A+clone-free,+single+molecule+map+of+the+domestic+cow+(Bos+taurus)+genome.">A clone-free, single molecule map of the domestic cow (Bos taurus) genome.</a> BMC Genomics. 16, 644 (2015).</li><li>Ray, 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=Discovery+of+structural+alterations+in+solid+tumor+oligodendroglioma+by+single+molecule+analysis.">Discovery of structural alterations in solid tumor oligodendroglioma by single molecule analysis.</a> BMC Genomics. 14, 505 (2013).</li><li>Chen, 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=Genome+sequence+of+the+model+medicinal+mushroom+Ganoderma+lucidum.">Genome sequence of the model medicinal mushroom Ganoderma lucidum.</a> Nat Commun. 3, 913 (2012).</li><li>Zhou, 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=A+single+molecule+scaffold+for+the+maize+genome.">A single molecule scaffold for the maize genome.</a> PLoS Genet. 5 (11), e1000711 (2009).</li><li>Sischka, 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=Molecular+mechanisms+and+kinetics+between+DNA+and+DNA+binding+ligands.">Molecular mechanisms and kinetics between DNA and DNA binding ligands.</a> Biophys J. 88 (1), 404-411 (2005).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> J Vis Exp. (62), (2012).</li><li>Zipper, H., Brunner, H., Bernhagen, J., Vitzthum, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigations+on+DNA+intercalation+and+surface+binding+by+SYBR+Green+I,+its+structure+determination+and+methodological+implications.">Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications.</a> Nucleic Acids Res. 32 (12), e103 (2004).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="605">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Unmethylated lambda DNA</td>
			<td style="width:111px;">NEB</td>
			<td style="width:155px;">N3013S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">YOYO-1</td>
			<td style="width:111px;">ThermoFisher Scientific</td>
			<td>Y3601</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">TOTO-1</td>
			<td style="width:111px;">ThermoFisher Scientific</td>
			<td>T3600</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">POPO-1</td>
			<td style="width:111px;">ThermoFisher Scientific</td>
			<td>P3580</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">BOBO-1</td>
			<td style="width:111px;">ThermoFisher Scientific</td>
			<td>B3582</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">YOYO-3</td>
			<td style="width:111px;">ThermoFisher Scientific</td>
			<td>Y3606</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">TOTO-3</td>
			<td style="width:111px;">ThermoFisher Scientific</td>
			<td>T3604</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">POPO-3</td>
			<td style="width:111px;">ThermoFisher Scientific</td>
			<td>P3584</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">BamHI</td>
			<td style="width:111px;">NEB</td>
			<td>R0136S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">PmlI</td>
			<td style="width:111px;">NEB</td>
			<td>R0532S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">ScaI</td>
			<td style="width:111px;">NEB</td>
			<td>R3122S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">EcoRI&#160;</td>
			<td style="width:111px;">NEB</td>
			<td>R0101S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">HindIII</td>
			<td style="width:111px;">NEB</td>
			<td>R0104S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">SmaI</td>
			<td style="width:111px;">NEB</td>
			<td>R0141S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Tris</td>
			<td style="width:111px;">Fisher</td>
			<td>BP152-1</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">EDTA</td>
			<td style="width:111px;">Fisher</td>
			<td style="width:155px;">S316-212</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">HCl</td>
			<td style="width:111px;">Fisher</td>
			<td>S25358</td>
			<td>Corrosive and irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Buffer 1</td>
			<td style="width:111px;">NEB</td>
			<td>B7201S</td>
			<td>NEB 1.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Buffer 2</td>
			<td style="width:111px;">NEB</td>
			<td>B7202S</td>
			<td>NEB 2.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Buffer 3</td>
			<td style="width:111px;">NEB</td>
			<td>B7204S</td>
			<td>NEB Cutsmart</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">High gelling temperature agarose gel</td>
			<td style="width:111px;">Lonza</td>
			<td>50041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Acetic Acid</td>
			<td style="width:111px;">Fisher</td>
			<td>S25118</td>
			<td>Hazardous</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Blue light transilluminator</td>
			<td style="width:111px;">Dark Reader</td>
			<td>DR196</td>
			<td>Wear correct eyewear&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:173px;">Ethidium bromide</td>
			<td style="width:111px;">Fisher</td>
			<td>15585011</td>
			<td style="width:167px;">Carcinogen; other alternatives are Nancy 520, Gel red, etc.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Cannon EOS Rebel T3I camera</td>
			<td style="width:111px;">Cannon</td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Apogee Model H4</td>
			<td style="width:111px;">Biorad</td>
			<td>1704510</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Power Pac Basic Power Supply</td>
			<td style="width:111px;">Biorad</td>
			<td>1645050&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Ficoll</td>
			<td style="width:111px;">Fisher Scientific</td>
			<td style="width:155px;">BP525-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Bromophenol blue</td>
			<td style="width:111px;">Fisher Scientific</td>
			<td style="width:155px;">B-392</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Xylene Cyanol</td>
			<td style="width:111px;">Eastmen</td>
			<td style="width:155px;">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

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

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and amino acids. However, the innate weak signal intensity of FRET sensors is a major challenge that prevents their application in various fields and makes the use of expensive, high-end fluorometers necessary. Previously, we built a cost-effective, high-performance FRET analyzer that can specifically measure the ratio of two emission wavelength bands (530 and 480 nm) to achieve high detection sensitivity. More recently, it was discovered that FRET sensors with bacterial periplasmic binding proteins detect ligands with maximum sensitivity in the critical temperature range of 50 - 55 &#176;C. This report describes a protocol for assessing sugar content in commercially-available beverage samples using our portable FRET analyzer with a temperature-specific FRET sensor. Our results showed that the additional preheating process of the FRET sensor significantly increases the FRET ratio signal, to enable more accurate measurement of sugar content. The custom-made FRET analyzer and sensor were successfully applied to quantify the sugar content in three types of commercial beverages. We anticipate that further size reduction and performance enhancement of the equipment will facilitate the use of hand-held analyzers in environments where high-end equipment is not available.

This protocol describes the rapid and highly sensitive quantification of F&#246;rster resonance energy transfer (FRET) sensor data using a custom-made portable FRET analyzer. The device was used to detect maltose within a critical temperature range that maximizes detection sensitivity, enabling practical and efficient assessment of sugar content.

高度敏感和快速荧光检测用便携式分析仪FRET

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and ...

科研

生物化学

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

鞘毛细管电泳 - 质谱联用生物样品的代谢谱

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer&#39;s and Parkinson&#39;s disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive &#946;-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Amyloid deposition is a hallmark of different diseases and afflicts many different organs. This paper describes the application of luminescent conjugated oligothiophene fluorescence staining in combination with fluorescence microscopy techniques. This staining method represents a powerful tool for detection and exploration of protein aggregates in both clinical and scientific setups.

用高光谱共聚焦显微镜和荧光寿命成像对发光共轭吩染色的淀粉样组织的成像

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

Dual DNA Rulers to Study the Mechanism of Ribosome Translocation with Single-Nucleotide Resolution

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. To investigate its mechanism, there are two essential technical requirements. First is single-nt resolution that can resolve normal translocation from frameshifting, during which the ribosome moves by other than 3 nt. The second is the capability to probe both the entrance and exit sides of mRNA in order to elucidate the whole picture of translocation. We report the dual DNA ruler assay that is based on the critical dissociation forces of DNA-mRNA duplexes, obtained by force-induced remnant magnetization spectroscopy (FIRMS).&#160; With 2-4 pN force resolution, the dual ruler assay is sufficient to distinguish different translocation steps. By implementing a long linker on the probing DNAs, they can reach the mRNA on the opposite side of the ribosome, so that the mRNA position can be determined for both sides. Therefore, the dual ruler assay is uniquely suited to investigate the ribosome translocation, and nucleic acid motion in general. We show representative results which indicated a looped mRNA conformation and resolved normal translocation from frameshifting.

Dual DNA ruler assay is developed to determine the mRNA position during ribosome translocation, which relies on the dissociation forces of the formed DNA-mRNA duplexes. With single-nucleotide resolution and capability of reaching both ends of mRNA, it can provide mechanistic insights for ribosome translocation and probe other nucleic acid displacements.

双DNA标尺研究单核苷酸分辨率的核糖体易位机制

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. ...

Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. FPOP uses a 248 nm excimer laser to photolyze hydrogen peroxide producing hydroxyl radicals. These radicals oxidatively modify solvent exposed side chains of 19 of the 20 amino acids. Recently, this method has been used in live cells (IC-FPOP) to study protein interactions in their native environment. The study of proteins in cells accounts for intermolecular crowding and various protein interactions that are disrupted for in vitro studies. A custom single cell flow system was designed to reduce cell aggregation and clogging during IC-FPOP. This flow system focuses the cells past the excimer laser individually, thus ensuring consistent irradiation. By comparing the extent of oxidation produced from FPOP to the protein&#39;s solvent accessibility calculated from a crystal structure, IC-FPOP can accurately probe the solvent accessible side chains of proteins.

Here, we characterize protein structure and interaction sites in living cells using a protein footprinting technique termed in-cell fast photochemical oxidation of proteins (IC-FPOP).

用细胞内快速光化学氧化蛋白质对细胞内蛋白质进行特征化

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. ...

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 ...

Cryo-EM and Single-Particle Analysis with Scipion

Cryo-electron microscopy has become one of the most important tools in biological research to reveal the structural information of macromolecules at near-atomic resolution. In single-particle analysis, the vitrified sample is imaged by an electron beam and the detectors at the end of the microscope column produce movies of that sample. These movies contain thousands of images of identical particles in random orientations. The data need to go through an image processing workflow with multiple steps to obtain the final 3D reconstructed volume. The goal of the image processing workflow is to identify the acquisition parameters to be able to reconstruct the specimen under study. Scipion provides all the tools to create this workflow using several image processing packages in an integrative framework, also allowing the traceability of the results. In this article the whole image processing workflow in Scipion is presented and discussed with data coming from a real test case, giving all the details necessary to go from the movies obtained by the microscope to a high resolution final 3D reconstruction. Also, the power of using consensus tools that allow combining methods, and confirming results along every step of the workflow, improving the accuracy of the obtained results, is discussed.

Single-particle analysis in cryo-electron microscopy is one of the main techniques used to determine the structure of biological ensembles at high resolution. Scipion provides the tools to create the whole pipeline to process the information acquired by the microscope and achieve a 3D reconstruction of the biological specimen.

使用沉降机进行冷冻电镜和单颗粒分析

Cryo-electron microscopy has become one of the most important tools in biological research to reveal the structural information of macromolecules at ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

基于荧光的连续核酸内切酶偶联DNA甲基化测定，用于筛选DNA甲基转移酶抑制剂

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

Preparation of Nucleosome Core Particles Complexed with DNA Repair Factors for Cryo-Electron Microscopy Structural Determination

DNA repair in the context of chromatin is poorly understood. Biochemical studies using nucleosome core particles, the fundamental repeating unit of chromatin, show most DNA repair enzymes remove DNA damage at reduced rates as compared to free DNA. The molecular details on how base excision repair (BER) enzymes recognize and remove DNA damage in nucleosomes have not been elucidated. However, biochemical BER data of nucleosomal substrates suggest the nucleosome presents different structural barriers dependent on the location of the DNA lesion and the enzyme. This indicates the mechanisms employed by these enzymes to remove DNA damage in free DNA may be different than those employed in nucleosomes. Given that the majority of genomic DNA is assembled into nucleosomes, structural information of these complexes is needed. To date, the scientific community lacks detailed protocols to perform technically feasible structural studies of these complexes. Here, we provide two methods to prepare a complex of two genetically fused BER enzymes (Polymerase &#946; and AP Endonuclease1) bound to a single-nucleotide gap near the entry-exit of the nucleosome for cryo-electron microscopy (cryo-EM) structural determination. Both methods of sample preparation are compatible for vitrifying quality grids via plunge freezing. This protocol can be used as a starting point to prepare other nucleosomal complexes with different BER factors, pioneer transcription factors, and chromatin-modifying enzymes.

This protocol outlines in detail the preparation of nucleosomal complexes using two methods of sample preparation for freezing TEM grids.

制备与DNA修复因子络合的核小体核心颗粒用于冷冻电镜结构测定

DNA repair in the context of chromatin is poorly understood. Biochemical studies using nucleosome core particles, the fundamental repeating unit of ...

Determining the Thermodynamic and Kinetic Association of a DNA Aptamer and Tetracycline Using Isothermal Titration Calorimetry

The determination of binding affinity and behavior between an aptamer and its target is the most crucial step in selecting and using an aptamer for application. Due to the drastic differences between the aptamer and small molecules, scientists need to put much effort into characterizing their binding properties. Isothermal Titration Calorimetry (ITC) is a powerful approach for this purpose. ITC goes beyond determining disassociation constants (Kd) and can provide the enthalpy changes and binding stoichiometry of the interaction between two molecules in the solution phase. This approach conducts continuous titration using label-free molecules and records released heat over time upon the binding events produced by each titration, so the process can sensitively measure the binding between macromolecules and their small targets. Herein, the article introduces a step-by-step procedure of the ITC measurement of a selected aptamer with a small target, tetracycline. This example proves the versatility of the technique and its potential for other applications.

The present protocol describes the use of Isothermal Titration Calorimetry (ITC) to analyze the association and dissociation kinetics of the binding between a DNA aptamer and tetracycline, including sample preparation, running standards and samples, and interpreting the resulting data.

使用等温滴定量热法测定DNA适配体和四环素的热力学和动力学关联

The determination of binding affinity and behavior between an aptamer and its target is the most crucial step in selecting and using an aptamer for ...