Name: detection of post-replicative gaps accumulation and repair in human cells using the dna fiber assay
Uploaded: 2022-02-03T14:15:00
Description: Watch this Scientific Journal Video about Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay at JoVE.com

C.F.M.M.实验室的工作得到了圣保罗国家卫生安全基金（FAPESP，巴西圣保罗，资助#2019/19435-3，#2013/08028-1和2017/05680-0）的支持，由FAPESP和荷兰科学研究组织（NWO，荷兰）进行国际合作研究;Científico e Tecnológico （CNPq， Brasília， DF， Brazil， Grants # 308868/2018-8] 和 Coordenação de Aperfeiçoamento de Pessoal do Ensino Superior （CAPES， Brasília， DF， Brazil， Finance Code 001）.

由于该研究使用人类细胞，这项工作得到了圣保罗大学生物医学科学研究所伦理委员会（ICB-USP，批准号#48347515.3.00005467）的批准，用于人类样本的研究。
注：此处描述的协议在以前的出版物中使用，但略有修改14，16，28。这里的重点是使用紫外线C（UVC）作为DNA损伤剂。然而，其它DNA损伤剂如顺铂和羟基脲...

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标准DNA纤维测定方案的关键步骤在先前的出版物32中进行了讨论。在这里，我们描述了标准DNA纤维测定的修饰版本，以研究复制后ssDNA间隙的存在以及通过间隙填充对其进行修复，最初在14中描述。在复制后ssDNA间隙存在的背景下，在S1 Fiber方案中使用S1核酸酶很可能是在暴露于遗传毒性剂至少1小时后，以允许间隙形成和随后检测的时间。然而，必须注意的是，在...

开创性工作提供了在细菌1和人类细胞2，3中用DNA损伤剂处理后复制后单链（ssDNA）间隙积累的证据。在复制受损的DNA模板期间，DNA合成机制可以通过采用特定的转座合成DNA聚合酶或通过模板切换机制来绕过病变。或者，重复体也可以简单地跳过病变，留下一个ssDNA间隙，以便以后修复。最近，一项研究清楚地表明，利用电子显微镜可视化复制中间体的特定结构，用基因毒性剂治疗会导致真核生物中的ssDNA间隙4。这些复制后间隙区域的形成最初被认为是DNA复制2的半不连续模式的简单结果。在这种情况下，滞后链上的病变可以阻断冈崎片段的伸长，但叉子进展自然被以下冈崎片段挽救，留下ssDNA间隙。然而，进一步的研究表明，在前导链上形成间隙也是可能的，如细菌5所示。在真核生物中，PRIMPOL是一种具有primase活性的独特DNA聚合酶，被证明能够通过其重注活性6，7，8，9，10，11在复制阻断病变下游重新开始DNA合成。因此，PRIMPOL primase活性可以解释在用DNA损伤剂处理人细胞12时在前链中形成复制后ssDNA间隙。尽管如此，直到最近，这些间隙的检测以及间隙修复都需要间接或耗时的方法，例如电子显微镜4或基于质粒的测定13。使用ssDNA特异性S1核酸酶来检测人类细胞中的间隙是四十多年前早期研究的先驱，使用蔗糖梯度技术2，3。最近，我们的小组应用这种核酸酶来检测使用其他方法（如DNA纤维和彗星测定14）复制DNA（复制后间隙）中的ssDNA。这些新方法为目前关于复制后差距的研究激增铺平了道路。在这里，我们描述了一种使用S1核酸酶通过DNA纤维测定来检测复制后ssDNA间隙的策略，并解释了DNA纤维方案中的差异标记方案如何允许研究这些间隙的修复。
DNA纤维测定是一种强大的技术，已被越来越多的实验室使用，并为复制叉动力学和复制应激反应机制提供了有价值的见解。简而言之，该技术基于在复制DNA中连续掺入核苷酸类似物（例如CldU -5-氯-2'-脱氧尿苷和IdU-5-碘-2'-脱氧尿苷）。收获后，裂解细胞，DNA分子在正涂层的载玻片上扩散。然后通过特异性抗体检测CldU和IdU，这些抗体可以在荧光显微镜中可视化为双色纤维。最后，测量IdU和CldU束的长度，以识别DNA损伤诱导引起的DNA复制动力学的任何改变。该技术可用于研究不同的现象，例如叉子失速，分叉减速，新生DNA降解以及起源放电频率的变化15，16。
DNA纤维测定的一个局限性是其对几千碱基的分辨率。由于复制后ssDNA间隙可能在数百个碱基的范围内，因此不可能通过标准DNA纤维方案直接可视化这些间隙。ssDNA间隙在用基因毒性药物处理的人类细胞中复制DNA的存在以前已经间接涉及。例如，通过在S期外的细胞中募集ssDNA结合蛋白，复制蛋白A（RPA）来评估ssDNA的观察，或者通过碱性DNA解卷检测到的ssDNA的形成，以及DNA纤维测定12，17，18，19，20，21没有长时间的分叉失速 归因于ssDNA间隙的积累。此外，复制后ssDNA间隙诱导ATR依赖性G2 / M相检查点并阻止用复制毒物处理的细胞18，19，20，21，22。
S1核酸酶降解单链核酸，释放5'-磷酸基单核苷酸或寡核苷酸，与RNA相比，对ssDNA的亲和力高5倍。双链核酸（DNA：DNA、DNA：RNA 或 RNA：RNA）对 S1 核酸酶具有抗性，除非在极高浓度下使用。S1核酸酶还可以在由缺口，间隙，不匹配或环引起的单链区域切割双链DNA。因此，S1核酸酶是一种ssDNA特异性内切酶，能够切割ssDNA间隙，最终产生双链断裂23，24。因此，将S1核酸酶消化步骤添加到DNA纤维方案中间接能够检测复制后ssDNA间隙。在存在间隙的情况下，在DNA扩散之前用S1核酸酶处理暴露的细胞核将产生较短的束，这是由于ssDNA的S1裂解固有地存在于间隙14中。因此，束缩短是使用这种方法的ssDNA间隙的读出。与标准DNA纤维方案相比，具有S1核酸酶的DNA纤维只需要两个额外的步骤：细胞核暴露（细胞通透化）和用S1核酸酶处理。重要的是要注意，适当的对照是强制性的，例如用遗传毒性剂处理但没有S1核酸酶的样品和用S1核酸酶处理的没有遗传毒性剂的样品。方案本身，包括类似物的掺入，S1处理和扩散，可以在一天内完成，不需要特殊的材料。它只需要胸苷类似物，纯化的S1核酸酶，适当的一抗和二抗以及荧光显微镜。总体而言，采用S1核酸酶的DNA纤维使用相对简单的方法检测正在进行的复制分叉上的ssDNA间隙。
复制应激反应机制产生的复制后ssDNA间隙可以通过不同的机制进行修复（或填充），包括转座DNA合成或模板切换，该过程称为间隙填充或复制后修复（PRR）25。这些过程发生在前进的分叉后面，涉及复制无关的DNA合成14，26，27。基于这些发现，可以执行与标准DNA纤维测定不同的标记方案，以直接可视化G2相14，16，26，28中的间隙填充事件。具体而言，一个胸苷类似物可用于在遗传毒性治疗和复制后ssDNA间隙形成时标记复制叉，而另一个胸苷类似物可用于标记间隙填充事件。在该方案中，在基因毒性治疗1小时后立即或同时用第一胸苷类似物（例如IdU）标记细胞，以便在间隙形成时标记新生的DNA。在处理12-24小时之间的任何地方添加诺考达唑以阻止G2 / M中的细胞，防止以下S期。对于诺考达唑治疗的最后4小时，将第二个胸苷类似物（例如CldU）添加到要在间隙填充期间掺入的培养基中。重要的是，该测定只能用于检测G2中的间隙填充事件，因为来自CldU的间隙填充信号无法与由于CldU掺入S期复制DNA的信号而与信号区分开来。因此，为了最大限度地减少背景信号，损伤诱导后加入CldU的时间应与大多数细胞群进入G2相14的时间相吻合。因此，这个时间会根据细胞系和治疗条件而有所不同。建议在采用该测定之前优化细胞周期进展。这些胸苷类似物的共染色允许在基因毒性治疗期间合成的新生DNA（IdU束）上间隙填充（PRR）束（CldU贴片）的可视化，当ssDNA间隙产生时。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetic acid, Glacial</td>
			<td>Synth</td>
			<td>64-19-7</td>
			<td>Alternatively, BSA - Biosera - REF PM-T1725/100</td>
		</tr>
		<tr>
			<td>Ammonium hydroxide</td>
			<td>Synth</td>
			<td>1336-21-6</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Antibody anti-mouse IgG1 Alexa Fluor 594</td>
			<td>Invitrogen</td>
			<td>A11005</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Antibody anti-rat Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A21470</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Antibody Mouse anti-BrdU</td>
			<td>Becton Disckson</td>
			<td>347580</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Antibody Rat anti-BrdU</td>
			<td>Abcam</td>
			<td>&#160;Ab6326</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Biological security hood</td>
			<td>Pachane</td>
			<td>PA 410</td>
			<td>Use hood present in the laboratory</td>
		</tr>
		<tr>
			<td>BSA (Bovine Serum Albumin)</td>
			<td>Sigma-Aldrich</td>
			<td>A3294</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Thermo Scientific</td>
			<td>179693</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>CldU</td>
			<td>Millipore-Sigma</td>
			<td>C6891</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Cloridric acid</td>
			<td>Synth</td>
			<td>7647-01-0</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Confocal Zeiss LSM Series (7, 8 or 9)</td>
			<td>Zeiss</td>
			<td>-</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Cover glass (or coverslips)</td>
			<td>Thermo Scientific</td>
			<td>152460</td>
			<td>Alternatively, Olen - Kasvi Cover Glass (24 x 60 mm) - K5-2460</td>
		</tr>
		<tr>
			<td>DMEM - High Glucose</td>
			<td>LGC/Gibco</td>
			<td>BR30211-05/12100046</td>
			<td>Use culture media specific for the cell line used.</td>
		</tr>
		<tr>
			<td>EDTA (Ethylenediaminetetraacetic acid disodium salt dihydrate)</td>
			<td>Sigma-Aldrich</td>
			<td>E5314</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Epifluorescence Microscope Axiovert 200</td>
			<td>Zeiss</td>
			<td>-</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>FBS (Fetal Bovine Serum)</td>
			<td>Gibco</td>
			<td>12657-029</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Forma Series II Water Jacketed CO2 Incubator</td>
			<td>Thermo Scientific 3110</td>
			<td>13-998-074</td>
			<td>Use cell incubator present in the laboratory</td>
		</tr>
		<tr>
			<td>Glass slide jar</td>
			<td>Sigma-Aldrich</td>
			<td>S5516</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>56-81-5</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Idu</td>
			<td>Millipore-Sigma</td>
			<td>I7125</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Synth</td>
			<td>7791-18-6</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Merck</td>
			<td>67-56-1</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Denville</td>
			<td>M1021</td>
			<td>Alternatively, Olen - Kasvi Microscope Slides - K5-7105 OR Precision Glass Line - 7105-1</td>
		</tr>
		<tr>
			<td>MOPS (&#193;cido 3-morfolinopropano 1-sulf&#244;nico)</td>
			<td>Synth</td>
			<td>1132-61-2</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Nocodazole</td>
			<td>Sigma-Aldrich</td>
			<td>31430-18-9</td>
			<td>-</td>
		</tr>
		<tr>
			<td>PBS (Phosphate Buffer Saline)</td>
			<td>Life Thechnologies</td>
			<td>3002</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>ProLong Gold AntiFade Mountant</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td>Any antifade moutant solution for immunofluorescence could be used</td>
		</tr>
		<tr>
			<td>S1 nuclease purified from Aspergillus oryzae</td>
			<td>Invitrogen</td>
			<td>18001-016</td>
			<td>Pre-dilute the S1 nuclease (1/100 - 1/200) in S1 nuclease dilution buffer provided by the manufacturer, aliquote and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>SDS (Sodium Dodecyl Sulfate)</td>
			<td>BioRad</td>
			<td>161-0302</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Sodium Acetate Trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>6131-90-4</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Synth</td>
			<td>&#160;7647-14-5</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>57-50-1</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>West Lab Research</td>
			<td>BP152-1</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Synth</td>
			<td>9002-93-1</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>25200072</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>UVC Lamp</td>
			<td>Non Specific</td>
			<td>-</td>
			<td>Essential: emission lenght of 254 nm</td>
		</tr>
		<tr>
			<td>VLX-3W UV Radiometer</td>
			<td>Vilber Loumart</td>
			<td>-</td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Zinc Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>557-34-6</td>
			<td>Or similar</td>
		</tr>
	</tbody>
</table>

detection of post-replicative gaps accumulation and repair in human cells using the dna fiber assay

DNA纤维测定是一种简单而稳健的复制叉动力学分析方法，基于在人类细胞DNA合成过程中掺入的核苷酸类似物的免疫检测。然而，这种技术的分辨率有限，只有几千个千基。因此，标准测定无法检测到小至几百个碱基的复制后单链DNA（ssDNA）间隙。在这里，我们描述了利用S1核酸酶的DNA纤维测定的修饰版本，S1核酸酶是一种特异性切割ssDNA的酶。在存在复制后ssDNA间隙的情况下，S1核酸酶将靶向并切割间隙，产生较短的束，可用作正在进行的分叉上ssDNA间隙的读数。这些复制后ssDNA间隙是在不连续复制受损DNA时形成的。它们可以通过与基因组复制分离 的机制进行 修复，该过程称为间隙填充或复制后修复。由于间隙填充机制涉及独立于S期的DNA合成，因此DNA纤维标记方案的改变也可用于监测间隙填充事件。总而言之，DNA纤维测定的这些修饰是了解复制后间隙如何在人类细胞基因组中形成和填充的强大策略。

在S1纤维测定中，如果用基因毒性剂处理导致复制后ssDNA间隙，则与未处理的样品以及用遗传毒性剂处理但未提交S1裂解的样品相比，S1处理过的细胞核的DNA纤维的总长度在处理DNA损伤时将更短（图1）。
或者，如果与未处理的细胞相比，用S1核酸酶治疗不会显着影响DNA纤维的长度，则可能有不同的解释：（1）使用所使用的遗传毒性剂治疗时，和/或在研究?...

Watch this Scientific Journal Video about Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay at JoVE.com

Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay

在这里，我们描述了DNA纤维测定的两种修饰，以研究病变诱导后复制DNA的单链DNA间隙。S1纤维测定能够使用ssDNA特异性S1内切酶检测复制后间隙，而间隙填充测定允许间隙修复的可视化和定量。

使用DNA纤维测定检测人细胞中复制后间隙的积累和修复

The DNA fiber assay is a simple and robust method for the analysis of replication fork dynamics, based on the immunodetection of nucleotide analogs that ...

我们的实验方案为研究复制如何在受损DNA上发生开辟了新的可能性。这些技术可以检测与DNA病变相反的单链DNA间隙的形成和修复。S1核酸酶切割单链DNA并允许检测复制后间隙。这种方法最初由Meneghini博士和Cordiero-Stone博士于20世纪70年代在巴西使用，最近由我们应用于DNA纤维测定。使用这里描述的技术来研究复制后间隙的形成和修复，可以为DNA修复和复制应激反应的机制提供见解，从而保持基因组完整性。对于此处描述的两种方案，S1光纤和间隙填充测定，包括所有对照以确保准确解释数据至关重要。该程序将由我们实验室的博士生Davi J.Martins和Annabel Quinet博士演示，后者之前在我们小组工作时开发了这些技术。首先，吸出培养基，并立即将具有20微摩尔IDU培养基的培养基加入细胞中。将细胞在37摄氏度和5%二氧化碳下孵育20分钟。然后，用预热的PBS快速洗涤细胞两次，并用每平方米20焦耳的UV-C照射它们。使用未经处理的细胞作为对照。立即将含有200微摩尔CldU的培养基加入细胞中，并在37摄氏度和5%二氧化碳下孵育60分钟。之后，用预热的PBS洗涤细胞两次，并根据室温下的细胞系用CSK 100缓冲液透化它们2至10分钟。CSK通透后，与通透之前相比，几乎看不到细胞质的细胞核。小心地将PBS倒入每个孔的侧面，用PBS洗涤细胞核。然后，用S1缓冲液洗涤细胞核。将细胞核在S1缓冲液中用每毫升20个单位的S1核酸酶孵育，并且没有S1核酸酶，在37摄氏度下孵育30分钟。接下来，吸出S1缓冲液并在PBS中加入0.1%BSA。刮取细胞核并将其转移到大约注释的1.5毫升管中。始终将管子放在冰上。将细胞核以约4， 600倍G在4摄氏度下离心5分钟。以每微升约1， 500个细胞的浓度重悬于PBS中的沉淀。将试管放在冰上，并通过铺展方案立即进行DNA纤维制备。用碳铅笔注释显微镜载玻片，并将其平放在托盘上。点击管的底部以确保均质化，并在相应的载玻片顶部添加两微升细胞。加入六微升裂解缓冲液，并通过上下移液约五次来裂解细胞。使用移液器尖端将液滴稍微拉向载玻片底部，并在室温下孵育五分钟以裂解细胞核。将滑块倾斜约20至40度，并允许液滴以恒定的低速扩散到滑块的底部。让载玻片在室温下在黑暗中干燥10至15分钟。然后，将它们浸入装有新鲜制备的固定溶液的罐中，并在室温下孵育五分钟，以将DNA固定在载玻片上。用PBS洗涤载玻片两次，持续5分钟，并在室温下用新鲜制备的2.5臼齿氯酸使DNA变性40至60分钟。用PBS洗涤载玻片三次，持续五分钟，并用5%BSA阻止先前在37摄氏度下加热30至60分钟。轻轻拍打纸张以从载玻片中取出多余的液体。向载玻片滴加30微升一抗，并在顶部放置盖玻片。在室温下在黑暗潮湿的室中孵育1至1.5小时。将载玻片放入装有PBS的罐子中一到两分钟，然后小心地取下盖玻片。用PBST在罐子中洗涤三次，持续五分钟，然后将载玻片放入PBS中。轻轻敲击纸张以除去多余的液体，并向载玻片滴加30微升二抗。在上面放一个盖玻片。在室温下在黑暗潮湿的室中孵育45至60分钟。重复洗涤载玻片的步骤，再次去除多余的PBS，并向载玻片滴加20微升的安装试剂。在上面放一个盖玻片，避免气泡。在滑轨顶部附近放置一滴浸油，其中细胞被裂解。使用绿色通道并搜索背景中的绿色点以查找焦点。打开徕卡应用套件软件，单击“获取”，然后选择绿色通道。在分析之前调整绿色通道的设置。然后，更改为红色通道并调整设置。找到纤维的主要浓度，然后移动到边缘，以找到均匀分布的，不重叠的单个纤维。仅使用一个通道来选择图片的区域，并避免潜在的偏差。单击“获取叠加层”图标，然后调整软件以获得正确的显微镜放大倍率。每张幻灯片在绿色通道处拍摄大约15到20张照片，同时在整张幻灯片旁边拍摄，然后在红色通道上拍摄。现在，两个通道已合并，创建的叠加层将移动到唯一的文件夹中。可以看到带有红色和绿色的纤维。打开 ImageJ 软件，左键单击第五个图标，选择第二个选项，然后选择分割线工具。单击鼠标左键可绘制红色或绿色区域的确切路径，单击鼠标右键可停止绘制。按下键盘上的 M 按钮以测量长度。对于 S1 光纤，请跟踪每根光纤的红色和绿色区域测量值，并从不同图片中测量至少 150 根光纤的红色和绿色区域长度。S1光纤显示在这些代表性图像中。这里显示了没有间隙且不受S1核酸酶切割影响的对照新生DNA束。DNA束阳性的间隙被S1核酸酶切割，导致CldU束长度较短。可以执行文本手稿中广泛描述的差异标记方案来标记间隙填充事件。在该测定中，称为间隙填充测定，具有至少一个CldU贴片或更少CldU贴片的较长IDU轨迹对应于低间隙填充密度。较短的 IDU 束（至少具有一个 CldU 贴片）和较长的 IDU 束（具有较多的 CldU 贴片）对应于高间隙填充密度。该方案的透化步骤对于S1核酸酶作用于复制受损DNA期间形成的间隙至关重要。S1核酸酶也可用于彗星测定方案，以检测在复制治疗后仍然存在的受损DNA期间形成的间隙。S1纤维测定为复制后间隙可能导致癌症脆弱性的新范式铺平了道路，特别是在携带BRCA1和2基因突变的乳腺癌和卵巢癌中。

Research

JoVE Journal

Genetics

Detection of Copy Number Alterations Using Single Cell Sequencing

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

科研

遗传学

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

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 ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

DNA 修饰的免疫: 共焦图像的计算分析

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

CRISPR/Cas9 对小鼠和人造血祖细胞的高效基因破坏

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

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

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 ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

基于纠错 DNA 和 RNA 测序的稀有事件检测方法

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

线粒体 DNA 甲基化的精确检测方法

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

ATAC-seq Assay with Low Mitochondrial DNA Contamination from Primary Human CD4+ T Lymphocytes

原发性人类 CD4+ T 淋巴细胞低线粒体 DNA 污染的 ATAC-seq 检测

ATAC-seq has become a widely used methodology in the study of epigenetics due to its rapid and simple approach to mapping genome-wide accessible ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

使用哺乳动物细胞中的微球菌核酸酶进行染色质免疫沉淀测定

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

在彗星分析中使用冷冻组织评估DNA损伤

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...