Name: proofreading and dna repair assay using single nucleotide extension and maldi-tof mass spectrometry analysis
Uploaded: 2018-06-19T14:45:00.000+00:00
Duration: 11 min 8 s
Description: Watch this Scientific Journal Video about Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis at JoVE.com

我们感谢 NCFPB 综合核心设施的功能基因组学 (台北, 台湾) 和 NRPB 药物实验室 (台北, 台湾) 的技术支持。这项工作得到了台湾卫生基金会 (白介) 和科技部的研究补助金的支持, 台北, 台湾, 中华民国 [最 105-2320-b-002-047] 为伟瑶胡, [最 105-2628-b-002-051-MY3] 为康怡苏, 和 [MOST-105-2320-B-002-051-MY3] 为梁在林。

1. 底漆/模板准备
<ol><li>设计引物/模板与平衡的 G + C 内容之间的40% 和60% 之间的排序或 PCR 底漆设计。使用18到 21 nt 的引物进行适当的退火和更好的 MS 信号。</li>
	<li>设计模板, 设置50°c 为双工区域的最小熔化温度, 至少 7 nt 5 '-悬置, 以分离的信号之间的底漆和模板。 注: 例如, 对于表 1中的基板 P21/T28, 21-nt 底漆与 28 nt 模板配对。选项: 使用替代核酸 (如核酸酶抗硫代核苷酸键) 替换模板 3 ' 端上的最后4磷酸二酯键, 可以防止模板的非特定 3 ' 端退化可能造成的干扰 (例如,表 1中的 T28S4), 虽然这将增加模板准备的一些成本。</li>
	<li>采用标准淡化寡核苷酸无进一步纯化, 对本研究是满意的。使用寡核苷酸底漆/模板集中 100 pmol/µL 在 H2O 作为一个股票, 并存储在-20 摄氏度。检查底漆和模板的质量和纯度, 通过运行 MALDI 的寡核苷酸 (步骤 4-7), 以确保独特的峰值信号和良好的信噪比。</li>
	<li>确定校准和浓度规范化反应中相关序列的相等摩尔引物的相对 MALDI 的 MS 信号强度 (步骤 7) (图 2)。</li>
</ol>2. 校对反应
注: 相同的校对反应条件和协议适用于 DNA 修复 i、G i、C i 和 T i。
<ol><li>用 P21/T28 基板的 T G 错配作为表 1中校对的一个例子, 稀释底漆 P21 和模板 T28 到 12.5 pmol/µL 与 H2O;然后将稀释底漆的12µL 和12µL 稀释后的模板转移到1.5 毫升的灭菌离心管上。 注: 模板/底漆组合量足以应付2反应;相应地增加试剂的容量为多反应。</li>
	<li>紧紧地关上管子。在65°c 的水浴中孵化30分钟, 然后在37°c 的水浴中再加30分钟, 最后在冰上确保适当的退火。</li>
	<li>对1.5 毫升灭菌离心管, 添加8µL 的底漆和模板组合 (50 pmol), 2 µL 10x 校对反应缓冲器, 4 µL 的 4 ddNTPs 混合 (2 毫米的每 4 dNTPs), 和 H2O 高达18µL. 轻弹管子混合。 注: 1x 校对反应缓冲器包含50毫米氯化钠, 10 毫米氯化镁2, 1 毫米 dithiothreitol, 10 毫米三盐酸 (pH 7.9 在25°c)。参见材料表为商业来源的10x 校对反应缓冲器。反应中每个 ddNTP 的最终浓度为0.1 毫米。</li>
	<li>稀释的 DNA 聚合酶在冰冷的1x 校对反应缓冲到期望的浓度 [例如, 到一个1.5 毫升的离心管, 添加4µL 1x 校对反应缓冲器和1µL 5 U 克莱诺聚合酶从商业来源 (表材料)]。随时将稀释后的酶储存在冰上。 注: 稀释后的 DNA 聚合酶的用量足以应对2反应;相应地增加酵素的容量为多反应。体积为2.0 µL, 含2.0 克莱诺聚合酶, 可以校对超过 50 pmol 的 T G 终端不匹配 P21/T28 在5分钟。</li>
	<li>Prewarm 离心管与基质混合从步骤2.3 到预期的反应温度 (例如, 通常为37°c 为克莱诺聚合酶和25°c T4 DNA 聚合酶)。然后将稀释后的 DNA 聚合酶的2.0 µL 从步骤2.4 转移到 prewarmed 基质混合物中, 然后轻轻地将管子混合在一起。</li>
	<li>在室温下, 用酶和基片离心出几秒钟, 在 3200 x g 处旋转反应的成分。立即转移反应到加热块或水浴在期望的孵化温度和在步骤2.5。</li>
	<li>
终止反应
	<ol>
<li>添加等量的缓冲苯酚, 将其混合涡流几秒钟, 并将其离心在离心 3200 x g 的环境温度5分钟。<ol>
<li>将水相转移到干净的离心管, 加入等量的氯仿, 将其混合涡流几秒钟, 并将其离心在离心 3200 x g 处, 室温下3分钟. 将水相转移到干净的离心管中, 并在cubate 它在95°c 为5分钟;然后把它放在冰上。 注意: 苯酚和氯仿是危险化学品。处理和废物处置应遵循制度规定。</li>
		</ol>
</li>
		<li>或者, 使用酸性淬火协议。为此, 添加2µL 1 米 HCl 停止在冰上的反应6分钟, 然后添加0.64 µL 1 M 二乙醇胺 (DEA), 以中和的 pH 值的反应混合物和孵化它在95°c 为5分钟;然后把它放在冰上。</li>
	</ol>
</li>
</ol>3. 树脂除盐污染
<ol><li>使用的树脂铲从商业脱盐套件 (见材料表), 采取足够的树脂覆盖384酒窝, 6 毫克树脂酒窝板的酒窝。</li>
	<li>通过刮过盘子的顶部, 均匀地 (6 毫克/酒窝) 将树脂分散在所有的酒窝上。回收任何多余的树脂, 并将其替换在瓶子中。</li>
	<li>将2.7 步的最终反应产物从离心管转移到384井板上。分配16µL 的 H2O 稀释最终反应产物在每个井, 然后密封板和离心机, 以消除任何泡沫。</li>
	<li>取下密封, 将384井板与反应产物倒置, 盖上树脂填充的酒窝板。</li>
	<li>翻转好的酒窝板三明治, 并小心地让树脂滴入384井板 (确保384井板是冲对金属钉在树脂填充的酒窝板)。</li>
	<li>除去顶部的酒窝板, 封住含有反应产物和树脂混合物的384井板, 简单地将其离心在 3200 x g 上, 以除去任何气泡, 并将其放在转子上, 使其在室温下进行脱盐, 以淡化其含量。</li>
	<li>树脂清理后, 离心384井板 3200 x 克5分钟, 将树脂压缩到井底。</li>
</ol>4. 将反应产物转移到矩阵芯片上
<ol><li>在 nanoliter 分配器 (nanodispenser, 见材料表) 中设置样品板。</li>
	<li>把矩阵芯片 (见材料表) 和384井板从3.7 节在相应的托盘。</li>
	<li>操作 nanodispenser, 以发现反应产物到芯片;按 nanodispenser 的触摸屏上的 "运行" 按钮开始。</li>
	<li>检查在屏幕上包含饱和信息的样本点的自动捕获图像, 以确保芯片上的总斑点体积约为 5-10 nL。如果音量不足, 请重复取样。</li>
</ol>5. 建立质谱检测信息
<ol><li>准备包含导入的预期信号信息的. xls 格式的文件。例如, 在步骤2、3和4中使用 "P21_T28" (表 2) 中的 P21/T28 校对反应的设置。</li>
	<li>通过在设计器格式中右键单击导入检测组, 并从步骤5.1 中选择. xls 文件 (例如, "P21_T28"), 在应用程序程序中创建并定义新的检测方法 (参见材料表)。</li>
	<li>通过右键单击树顶并给它一个文件名 (例如, "CTT20171201" 表示检测代码和日期),通过屏幕左侧的Customer:Project:Plate选项树建立目标检测板。</li>
	<li>选择384井板类型, 按OK;将出现一个空白的盘子。</li>
	<li>在屏幕左侧选择适当的化验 (如P21_T28)。</li>
	<li>通过突出显示井和右键单击以选择添加丛, 为芯片上的每个样本斑点样本位置分配选定的检测 (例如, P21_T28)。</li>
	<li>在 xlsx 格式的芯片上编写所有测试的工作列表, 没有页眉 (例如表 3的 1201.xlsx)。通过"添加新示例项目" 选项导入工作列表。</li>
	<li>在工作列表中分配测试 (例如, 从 1201.xlsx) 到 P21_T28 检测的每个位置, 然后通过右键单击选择。</li>
</ol>6. MALDI MS 手术
<ol><li>使用应用程序将质谱仪与芯片连接 (见材料表)。</li>
	<li>选择屏幕右侧的默认设置。</li>
	<li>从步骤 5.3 (例如, CTT20171201) 中填充检测名称, 在芯片的拐角处输入芯片 ID, 并保存设置。</li>
	<li>启动 MS 控制程序 (参见材料表)。</li>
	<li>按进/出按钮, 取出侦察盘。将斑点芯片从步骤4.4 放置到侦察板上, 然后按进/出按钮, 使斑点芯片进入质谱仪。</li>
	<li>单击应用程序的 "获取" 按钮 (参见材料表) 以启动质谱并获取数据。</li>
</ol>7. 数据分析
<ol><li>打开数据分析程序 (见材料表)。</li>
	<li>浏览左上角数据库的树, 然后从步骤6.3 中选择芯片 ID。打开屏幕右侧的数据文件。</li>
	<li>单击频谱图标以显示质量频谱。若要裁剪特定范围的 m/z, 请右键单击以选择"自定义" 对话框, 然后在 "新建" 窗口中单击X 轴并设置所需的上限和下限, 然后按"确定" 以显示指定范围的频谱。</li>
	<li>通过右键单击导出, 导出用于记录保留的频谱。 注: 使用 1600 width/1,200 单位的刻度是计算机屏幕上数据检查的合理尺寸。<ol>
<li>选择 JPEG 文件类型, 单击目标, 选择浏览光盘(例如, 闪存盘 e:), 键入文件名 (例如, 1201-1. jpg), 然后单击 "导出"。</li>
	</ol>
</li>
	<li>对于数据定量, 请使用光标并单击峰值以显示左上角的峰值高度。<ol>
<li>或者, 打印导出的 JPEG 文件, 并手动使用标尺测量峰值高度。</li>
	</ol>
</li>
</ol>

<ol>
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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+residue+in+DNA+polymerases+of+the+Escherichia+coli+DNA+polymerase+I+family+is+critical+for+distinguishing+between+deoxy-+and+dideoxyribonucleotides.">A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides.</a> Proceedings of the National Academy of Sciences of the United States of America. 92 (14), 6339-6343 (1995).</li><li>Weiss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+deoxyinosine+from+the+Escherichia+coli+chromosome+as+studied+by+oligonucleotide+transformation.">Removal of deoxyinosine from the Escherichia coli chromosome as studied by oligonucleotide transformation.</a> DNA Repair. 7 (2), 205-212 (2008).</li><li>Cao, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endonuclease+V:+an+unusual+enzyme+for+repair+of+DNA+deamination.">Endonuclease V: an unusual enzyme for repair of DNA deamination.</a> Cellular and Molecular Life Sciences. 70 (17), 3145-3156 (2013).</li><li>Su, K. 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=Application+of+single+nucleotide+extension+and+MALDI-TOF+mass+spectrometry+in+proofreading+and+DNA+repair+assay.">Application of single nucleotide extension and MALDI-TOF mass spectrometry in proofreading and DNA repair assay.</a> DNA Repair. 61, 63-75 (2018).</li><li>Carver, T. E., Hochstrasser, R. A., Millar, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proofreading+DNA:+recognition+of+aberrant+DNA+termini+by+the+Klenow+fragment+of+DNA+polymerase+I.">Proofreading DNA: recognition of aberrant DNA termini by the Klenow fragment of DNA polymerase I.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (22), 10670-10674 (1994).</li><li>Santa Lucia, J., Hicks, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thermodynamics+of+DNA+structural+motifs.">The thermodynamics of DNA structural motifs.</a> Annual Review of Biophysics and Biomolecular Structure. 33, 415-440 (2004).</li><li>Su, K. 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=DNA+polymerase+I+proofreading+exonuclease+activity+is+required+for+endonuclease+V+repair+pathway+both+in+vitro+and+in+vivo.">DNA polymerase I proofreading exonuclease activity is required for endonuclease V repair pathway both in vitro and in vivo.</a> DNA Repair. 64, 59-67 (2018).</li></ol>

作者没有什么可透露的。

本研究描述了由选择的商用仪器 (见材料表) 分析的一步一步校对活动, 使用 MALDI MS。主要优点包括: 底漆和模板是免费的标签和易于执行, 允许更大的灵活性设计实验。30校对测试的流石灰完整处理需要4小时, 包括3小时手动执行校对反应和清理, 而 MALDI 的分析使用上述协议需要1小时完成。这种方法的主要缺点是化验所需的基底量。为了获得强烈的信号和一致的结果, 我们使用了 50 pmol ?...

dna 聚合酶在 dna 复制过程中的校对功能对于确保需要转移到后代1、2、3、4的遗传信息的高保真度至关重要, 5,6,7。能够评估聚合酶校对 exonucleases 的贡献将澄清保护遗传稳定性的机制。
放射性同位素标记和凝胶检测结合密度分析的 autoradiograms 或荧光粉成像8,9,10历来被用来检测 DNA 校对活动聚合 酶。虽然功能, 这些化验是费力的, 昂贵的, 不适合高通量格式。此外, 放射性同位素还遭受安全问题, 包括废物处理。另外, 通过荧光技术对校对活动进行了分析。例如, 2-aminopurine (2-AP) 可纳入延伸产品在体外聚合酶校对化验, 以产生荧光信号11,12。不幸的是, 这些方法受到低特异性, 因为 2 AP 可以对嘧啶和胞嘧啶。最近的方法包括一个敏感的基于 G 四的荧光开关探针, 用于聚合酶 3 '-5 ' exonuclease 检测13以及一个单一标记的荧光探针, 用于聚合酶校对试验, 克服了一些上述缺点14。由于需要对 DNA 基质进行特异标记, 因此对这些荧光方法的热情减弱。
与此相反, MALDI 的 DNA 分析的 MS 已经被应用于精确检测中, 在那里, 用未标记的 4 ddNTPs 的底漆延伸反应可以用来识别给定轨迹15、16、17和已广泛应用于临床应用的突变检测和癌症诊断18。利用这些基本原理, 我们建立了一个无标签的检测 DNA 聚合酶校对活动利用高分辨率, 高特异性和高通量潜力的 MALDI. 使用E。大肠杆菌DNA 聚合酶 I 克莱诺片段作为一种模型酶, dideoxyribonucleotide triphosphates (ddNTPs) 作为基质可以采取 "快照" 的校对产品后, 单核苷酸延长通过MALDI-飞行 MS (图 1)。
同样, 这种方法也被开发用于 DNA 修复化验, 其中含有 3 ' 倒数二 dI 病变的底漆受到一项模拟中 V 型修复中间体的一项检测。虽然不完全理解, 内 V 修复通路是唯一的修复系统, 已知使用的 exonuclease 的病灶切除19,20。使用 MALDI 的 MS, 我们显示一个明确定义的修复补丁, 其中 dI 可以被切除的第一个 2 nt 的底漆, 然后添加正确的补充核苷酸。
对于校对和 DNA 修复的研究, 该方法比以往的方法更快、更费力, 为机制和功能提供了补充信息。

<table><tbody><tr><td>Oligonucleotides</td><td>Mission Biotech (Taiwan)</td><td></td><td></td></tr><tr><td>phosphorothioate modified oligonucleotides</td><td>Integrated DNA Technologies (Taiwan)</td><td></td><td></td></tr><tr><td>DNA polymerase I, Large (Klenow) Fragment</td><td>New England Biolabs, MA</td><td>M0210L</td><td></td></tr><tr><td>Klenow fragment (3&#039;&amp;rarr;5&#039; exo-)</td><td>New England Biolabs, MA</td><td>M0212L</td><td></td></tr><tr><td>NEBuffer 2.1 (10X)</td><td>New England Biolabs, MA</td><td>B7202S</td><td></td></tr><tr><td>2&#039;, 3&#039; ddATP</td><td>Trilink Biotechnologies, CA</td><td>N4001</td><td></td></tr><tr><td>2&#039;, 3&#039; ddGTP</td><td>Trilink Biotechnologies, CA</td><td>N4002</td><td></td></tr><tr><td>2&#039;, 3&#039; ddTTP</td><td>Trilink Biotechnologies, CA</td><td>N4004</td><td></td></tr><tr><td>2&#039;, 3&#039; ddCTP</td><td>Trilink Biotechnologies, CA</td><td>N4005</td><td></td></tr><tr><td>dATP, dGTP, dCTP, dTTP set</td><td>Clubio, Taiwan</td><td>CB-R0315</td><td></td></tr><tr><td>SpectroCHIP array</td><td>Agena Bioscience, CA</td><td>#01509</td><td></td></tr><tr><td>MassARRAY</td><td>Agena Bioscience, CA</td><td></td><td></td></tr><tr><td>Typer 4.0 software</td><td>Agena Bioscience, CA</td><td>#10145</td><td></td></tr><tr><td>Clean Resin Tool Kit</td><td>Agena Bioscience, CA</td><td>#08040</td><td></td></tr></tbody></table>

proofreading and dna repair assay using single nucleotide extension and maldi-tof mass spectrometry analysis

维持基因组及其忠实的复制对保存基因信息至关重要。为了评估高保真度复制, 我们开发了一种简单的无标记和非无线电同位素方法, 利用矩阵辅助激光解吸电离与飞行时间 (MALDI) 质谱 (MS) 分析进行校对研究。在这里, 一个 DNA 聚合酶 [例如,克莱诺片段 (KF) 大肠杆菌 DNA 聚合酶 i () 在本研究中] 在所有四 dideoxyribonucleotide triphosphates 的存在, 用于处理不匹配的底漆模板双工。不匹配的底漆, 然后校对/延长和受 MALDI 的 MS。产品由底漆的质量变化区分到单核苷酸变异。重要的是, 校对也可以确定内部单一的不匹配, 虽然在不同的效率。2-4-核苷酸 (nt) 从 3 ' 年底的不匹配是有效地校对了由我, 和不匹配的 5 nt 从底漆总站只显示了部分更正。在 6-9 nt 从底漆 3 ' 末端没有校对发生内部不匹配。这种方法也可应用于 DNA 修复化验 (例如,评估内 V 修复通路基底损伤修复)。含 3 ' 倒数 deoxyinosine (dI) 病灶的底漆可由 i. 型矫正。事实上, 倒数第二 T i、G i 和 i 基板在添加正确的 ddN 5 '-单磷酸 (ddNMP) 之前, 有他们最后的 2 dI 含核苷酸, 而倒数第二个 C i 的不匹配是由我来容忍的, 允许引伸不修复, 显示的敏感性和分辨率的 MS 化验, 以测量 DNA 修复。

模板和底漆:
使用此处介绍的程序, 检查了从商业来源获得的等摩尔合成寡核苷酸模板和相关序列的引物, 以确定其纯度和质量 (图 3A; 注意匹配指定质量的信号和低背景) 以及峰值强度与分析物质量之间的关系 (图 2A)。测量和计算了峰值高度 (图 2B),...

Watch this Scientific Journal Video about Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis at JoVE.com

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

单核苷酸延长和 MALDI 质谱分析校对及 DNA 修复方法

采用高分辨率 MALDI 质谱和单核苷酸扩展策略, 建立了 dna 聚合酶校对和 dna 修复实验的非标记非射电同位素方法。试验证明是非常具体, 简单, 快速, 并易于执行校对和修复补丁短于 9-核苷酸。

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

proofreading-dna-repair-assay-using-single-nucleotide-extension-maldi

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Unit 1

DNA Replication

DNA Replication and Repair

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9.5K 次观看.  National Taiwan University. 采用高分辨率 MALDI 质谱和单核苷酸扩展策略, 建立了 dna 聚合酶校对和 dna 修复实验的非标记非射电同位素方法。试验证明是非常具体, 简单, 快速, 并易于执行校对和修复补丁短于 9-核苷酸。

视频: Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair

AFM imaging is a powerful technique for the study of protein-DNA interactions. This single molecule method allows the simultaneous resolution of different molecules and molecular assemblies in a heterogeneous sample. In the particular context of DNA interacting protein systems, different protein complex forms and their corresponding binding positions on target sites containing DNA fragments can thus be distinguished. Here, an application of AFM to the study of DNA lesion recognition in the prokaryotic and eukaryotic nucleotide excision DNA repair (NER) systems is presented. The procedures of DNA and protein sample preparations are described and experimental as well as analytical details of the experiments are provided. The data allow important conclusions on the strategies by which target site verification may be achieved by the NER proteins. Interestingly, they indicate different approaches of lesion recognition and identification for the eukaryotic NER system, depending on the type of lesion. Furthermore, distinct structural properties of the two different helicases involved in prokaryotic and eukaryotic NER result in and explain the different strategies observed for these two systems. Importantly, these experimental and analytical approaches can be applied not only to the study of DNA repair but also very similarly to other DNA interacting protein systems such as those involved in replication or transcription processes.

Here, the study of different DNA lesion recognition approaches via single molecule AFM imaging is demonstrated with the nucleotide excision repair system as an example. The procedures of DNA and protein sample preparations and experimental as well as analytical details for the AFM experiments are described.

原子力显微镜检查DNA损伤识别核苷酸切除修复

AFM imaging is a powerful technique for the study of protein-DNA interactions. This single molecule method allows the simultaneous resolution of different ...

科研

遗传学

A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in mammalian cell lines. Rather than globally assaying removal of xenobiotic-induced or spontaneous chromosomal DPC removal, this assay examines the repair of a homogeneous, chemically defined lesion specifically introduced at one site within a plasmid DNA substrate. Importantly, this approach avoids the use of radioactive materials and is not dependent on expensive or highly-specialized technology. Instead, it relies on standard recombinant DNA procedures and widely available real-time, quantitative polymerase chain reaction (qPCR) instrumentation. Given the inherent flexibility of the strategy utilized, the size of the crosslinked protein, as well as the nature of the chemical linkage and the precise DNA sequence context of the attachment site can be varied to address the respective contributions of these parameters to the overall efficiency of DPC repair. Using this method, plasmids containing a site-specific DPC were transfected into cells and low molecular weight DNA recovered at various times post-transfection. Recovered DNA is then subjected to strand-specific primer extension (SSPE) using a primer complementary to the damaged strand of the plasmid. Since the DPC lesion blocks Taq DNA polymerase, the ratio of repaired to un-repaired DNA can be quantitatively assessed using qPCR. Cycle threshold (CT) values are used to calculate percent repair at various time points in the respective cell lines. This SSPE-qPCR method can also be used to quantitatively assess the repair kinetics of any DNA adduct that blocks Taq polymerase.

The goal of this protocol is to quantify the repair of defined DNA-protein crosslinks on plasmid DNA. Lesioned plasmids are transfected into recipient mammalian cell lines and low-molecular weight harvested at multiple time points post-transfection. DNA repair kinetics are quantified using strand-specific primer extension followed by qPCR.

一种简便、快速、定量的测定 DNA 蛋白 Crosslinks 在转染哺乳动物细胞中的研究

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in ...

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. 
The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

基于免疫荧光显微镜的瞬时和持久双链 dna 损伤 dna 修复过程的表征

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

癌症研究

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter

Double strand breaks (DSBs) in DNA are the most cytotoxic type of DNA damage. Because a myriad of insults can result in these lesions (e.g., replication stress, ionizing radiation, unrepaired UV damage), DSBs occur in most cells each day. In addition to cell death, unrepaired DSBs reduce genome integrity and the resulting mutations can drive tumorigenesis. These risks and the prevalence of DSBs motivate investigations into the mechanisms by which cells repair these lesions. Next generation sequencing can be paired with the induction of DSBs by ionizing radiation to provide a powerful tool to precisely define the mutations associated with DSB repair defects. However, this approach requires computationally challenging and cost prohibitive whole genome sequencing to detect the repair of the randomly occurring DSBs associated with ionizing radiation. Rare cutting endonucleases, such as I-Sce1, provide the ability to generate a single DSB, but their recognition sites must be inserted into the genome of interest. As a result, the site of repair is inherently artificial. Recent advances allow guide RNA (sgRNA) to direct a Cas9 endonuclease to any genome locus of interest. This could be applied to the study of DSB repair making next generation sequencing more cost effective by allowing it to be focused on the DNA flanking the Cas9-induced DSB. The goal of the manuscript is to demonstrate the feasibility of this approach by presenting a protocol that can define mutations that stem from the repair of a DSB upstream of the CD4 gene. The protocol can be adapted to determine changes in the mutagenic potential of DSB associated with exogenous factors, such as repair inhibitors, viral protein expression, mutations, and environmental exposures with relatively limited computation requirements. Once an organism's genome has been sequenced, this method can be theoretically employed at any genomic locus and in any cell culture model of that organism that can be transfected. Similar adaptations of the approach could allow comparisons of repair fidelity between different loci in the same genetic background.

Presented here is sgRNA/CAS9 endonuclease and next-generation sequencing protocol that can be used to identify the mutations associated with double strand break repair near the CD4 promoter.

使用下一代测序来鉴定与CD4启动子附近CAS9诱导的双链断裂修复相关的突变

Double strand breaks (DSBs) in DNA are the most cytotoxic type of DNA damage. Because a myriad of insults can result in these lesions (e.g., replication ...

Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of cytosine. Thus, it is crucial for genome integrity maintenance. A highly specific, non-labeled, non-radio-isotopic method was developed to measure UDG activity. A synthetic DNA duplex containing a site-specific uracil was cleaved by UDG and then subjected to Matrix-assisted Laser Desorption/Ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. A protocol was established to preserve the apurinic/apyrimidinic site (AP) product in DNA without strand break. The change in the m/z value from the substrate to the product was used to evaluate uracil hydrolysis by UDG. A G:U substrate was used for UDG kinetic analysis yielding the Km = 50 nM, Vmax = 0.98 nM/s, and Kcat = 9.31 s-1. Application of this method to a uracil glycosylase inhibitor (UGI) assay yielded an IC50 value of 7.6 pM. The UDG specificity using uracil at various positions within single-stranded and double-stranded DNA substrates demonstrated different cleavage efficiencies. Thus, this simple, rapid, and versatile MALDI-TOF MS method could be an excellent reference method for various monofunctional DNA glycosylases. It also has the potential as a tool for DNA glycosylase inhibitor screening.

A non-labeled, non-radio-isotopic method to assay uracil-DNA glycosylase activity was developed using MALDI-TOF mass spectrometry for direct apurinic/apyrimidinic site-containing product analysis. The assay proved to be quite simple, specific, rapid, and easy to use for DNA glycosylase measurement.

尿嘧啶-DNA糖基化酶测定，采用基质辅助激光解吸/电离飞行时间质谱分析

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of ...

生物化学

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

在哺乳动物细胞中的DNA双链断裂（DSB）的维修分析

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

生物学

使用纳米荧光素酶报告基因检测定量 DNA 双链断裂修复

Assessment of DNA Double Strand Break Repair Activity Using High-throughput and Quantitative Luminescence-Based Reporter Assays

The repair of DNA double strand breaks (DSBs) is crucial for the maintenance of genome stability and cell viability. DSB repair (DSBR) in cells is mediated through several mechanisms: homologous recombination (HR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and single strand annealing (SSA). Cellular assays are essential to measure the proficiency and modulation of these pathways in response to various stimuli.
Here, we present a suite of extrachromosomal reporter assays that each measure the reconstitution of a nanoluciferase reporter gene by one of the four major DSBR pathways in cells. Upon transient transfection into cells of interest, repair of pathway-specific reporter substrates can be measured in under 24 h by the detection of Nanoluciferase (NanoLuc) luminescence.
These robust assays are quantitative, sensitive, titratable, and amenable to a high-throughput screening format. These properties provide broad applications in DNA repair research and drug discovery, complementing the currently available toolkit of cellular DSBR assays.

作者聚焦：开发针对 DNA 损伤反应的新型抗癌疗法

We present protocols for the assessment of double strand break repair pathway proficiency in cells using a suite of luminescence-based extrachromosomal reporter substrates.

使用基于高通量和定量发光的报告基因分析评估 DNA 双链断裂修复活性

The repair of DNA double strand breaks (DSBs) is crucial for the maintenance of genome stability and cell viability. DSB repair (DSBR) in cells is ...

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem1,2. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via F&ouml;rster Resonance Energy Transfer (FRET)3,4. The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors.

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.

定量，实时分析，利用分子信标病​​变特定的细胞裂解液中的碱基切除修复活动

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base ...

单核苷酸延长和 MALDI 质谱分析校对及 DNA 修复方法

摘要

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此视频中的章节

原子力显微镜检查DNA损伤识别核苷酸切除修复

一种简便、快速、定量的测定 DNA 蛋白 Crosslinks 在转染哺乳动物细胞中的研究

基于免疫荧光显微镜的瞬时和持久双链 dna 损伤 dna 修复过程的表征

使用下一代测序来鉴定与CD4启动子附近CAS9诱导的双链断裂修复相关的突变

尿嘧啶-DNA糖基化酶测定，采用基质辅助激光解吸/电离飞行时间质谱分析

在哺乳动物细胞中的DNA双链断裂（DSB）的维修分析

使用基于高通量和定量发光的报告基因分析评估 DNA 双链断裂修复活性

使用基于高通量和定量发光的报告基因分析评估 DNA 双链断裂修复活性

使用基于高通量和定量发光的报告基因分析评估 DNA 双链断裂修复活性

定量，实时分析，利用分子信标病变特定的细胞裂解液中的碱基切除修复活动

单核苷酸延长和 MALDI 质谱分析校对及 DNA 修复方法

摘要

探索更多视频

此视频中的章节

原子力显微镜检查DNA损伤识别核苷酸切除修复

一种简便、快速、定量的测定 DNA 蛋白 Crosslinks 在转染哺乳动物细胞中的研究

基于免疫荧光显微镜的瞬时和持久双链 dna 损伤 dna 修复过程的表征

使用下一代测序来鉴定与CD4启动子附近CAS9诱导的双链断裂修复相关的突变

尿嘧啶-DNA糖基化酶测定，采用基质辅助激光解吸/电离飞行时间质谱分析

在哺乳动物细胞中的DNA双链断裂（DSB）的维修分析

使用基于高通量和定量发光的报告基因分析评估 DNA 双链断裂修复活性

使用基于高通量和定量发光的报告基因分析评估 DNA 双链断裂修复活性

使用基于高通量和定量发光的报告基因分析评估 DNA 双链断裂修复活性

定量，实时分析，利用分子信标病​​变特定的细胞裂解液中的碱基切除修复活动

定量，实时分析，利用分子信标病变特定的细胞裂解液中的碱基切除修复活动