Name: extraction of diatom dna from water samples and tissues
Uploaded: 2023-11-10T13:10:00.000+00:00
Duration: 4 min 20 s
Description: Watch this Scientific Journal Video about Diatom Testing for Forensic Drowning Examination at JoVE.com

这项工作得到了国家自然科学基金（82060341,81560304）和海南省院士创新平台科研项目（YSPTZX202134）的支持。

该研究已获得海南医科大学伦理委员会的批准。本研究中使用的组织样本不被视为涉及人类受试者的研究。这些标本是出于法医病理诊断的目的而获得的，其余的用于本实验中硅藻DNA的提取。研究人员无法轻易识别个人以获得相关利益相关者的知情同意。
注：为保证本实验中报告的研究方法的普遍适用性，本实验基本遵循所用试剂盒的操作说明，仅修改了部分步骤。本实验中使用的水样是从实验室附近的池塘中随机采集的（补充图1A）。在该实验中，确认溺水尸体的肺组织为研究组织，以证明提取方案（补充图1B）。在法医实践中，有时还需要使用溺水尸体的其他器官和组织（如肝、脾、肾、骨髓等）来提取硅藻DNA，这需要对这种实验方法进行微小的相应改进，这将在实验的相应章节中解释。该实验中使用的肺组织样本来自法医案件中明显淹死的尸体。已经进行了形态学测试，以证明肺组织含有硅藻（补充图2）。
1.样品的预处理
<ol><li>水样的预处理<ol><li>将 10 mL 水样放入离心管中，并以 13,400 x g 离心 5 分钟。用移液管小心弃去 9.8 mL 上清液，并将剩余在底部的约 200 μL 富集硅藻水样品转移到 2 mL 离心管中。 注意：可以先进行预实验，如果 10 mL 水样不足以富集，可以增加初始量。</li>
	</ol></li>
	<li>组织样本的预处理<ol><li>从溺水的尸体中取出0.5克肺边缘组织，将肺组织完全切碎或研磨，直到它变得浑浊。防止外源硅藻污染是这一步的核心。用剪刀反复将组织切成小块。</li>
	</ol></li>
</ol>2. DNA提取
注意：所有离心步骤均在室温下完成。使用离心力为14,500 x g的台式离心机;在实验开始之前，需要准备预热到70°C的水浴（或金属浴）。所有步骤必须严格遵循无菌操作原则。
<ol><li>水样和组织的组装 注意：水样和组织硅藻DNA提取方法相同。<ol><li>将玻璃珠加入含有预处理水样的 2 mL 离心管中。倒置 10 倍至 15 倍以充分混合。添加的玻璃珠由大小玻璃珠以1：1的质量比混合而成。大玻璃珠的直径为 1.5-2.0 毫米，小玻璃珠的直径为 0.4-0.6 毫米。</li>
		<li>取 0.5 g 切碎的组织，如上所述将玻璃珠加入含有组织的 2 mL 离心管中。倒置 10 倍至 15 倍混合。</li>
	</ol></li>
	<li>向试管中加入 40 μL 蛋白酶 K （20 mg/mL）。在室温下放置 15 分钟，在此期间，倒置，每 3 分钟混合 10 次。 注意：如果组织没有完全消化，可以适当增加蛋白酶K的量，直到溶液变得透明。</li>
	<li>向离心管中加入200μL结合缓冲液，立即涡旋，混合4分钟（振荡混合频率：3000rpm）。 注意：在此步骤中，应严格控制振荡强度和时间，以确保足够的混合强度和时间。</li>
	<li>将离心管置于70°C水浴中10分钟，溶液变得澄清。</li>
	<li>向离心管中加入100μL异丙醇，涡旋并混合15秒，此时可能出现絮状沉淀。 注意：在上述操作步骤中，以适当的强度充分混合非常重要，但应避免剧烈摇晃，以防止DNA剪切。</li>
	<li>将上一步得到的溶液与絮凝剂沉淀物一起放入吸附柱中（吸附柱置于收集管中）。吸附柱长度为3.0厘米，直径为1.0厘米，吸附基体为硅基膜。</li>
	<li>以8,000× g 离心30秒，弃去收集管中的废液，并将吸附柱放回收集管中。</li>
	<li>向吸附柱中加入 500 μL 抑制剂去除缓冲液。以 13,400 x g 离心 30 秒，然后丢弃收集管中的废液。</li>
	<li>向吸附柱中加入700μL洗涤缓冲液。以 13,400 x g 离心 30 秒，然后丢弃收集管中的废液。 注意： 首次使用前，将指定量的无水乙醇加入洗涤缓冲液瓶中。</li>
	<li>向吸附柱中加入 500 μL 洗涤缓冲液。以 13,400 x g 离心 30 秒，然后丢弃收集管中的废液。</li>
	<li>将吸附柱放回空的收集管中。以14,500× g 离心2分钟，并尽可能除去洗涤缓冲液，以避免洗涤缓冲液中残留的乙醇抑制下游反应。</li>
	<li>取出吸附柱，放入干净的离心管中。向吸附膜的中间部分加入 100 μL 洗脱缓冲液。</li>
	<li>将吸附柱置于室温下3-5分钟，并以13,400× g 离心1分钟。</li>
	<li>将上一步得到的溶液再次加入离心吸附柱中。将离心吸附柱置于室温下2分钟，并以13,400× g 离心1分钟。 注：洗脱缓冲液应在70°C水浴中预热约10分钟。洗脱体积应不小于50μL;否则，DNA产量将降低。</li>
	<li>将提取的硅藻DNA储存在2-8°C以备将来使用。如果要长时间储存DNA溶液，请将其储存在-20°C。</li>
</ol>3. PCR检测
注意：由于法医样本中硅藻的含量通常很低，溺水尸体的组织样本提取物也可能含有不同程度的组织和器官（例如本实验中的肺）及其自身的DNA。因此，直接检测DNA提取物中的总DNA并不能反映硅藻DNA提取的情况。本实验选择硅藻特异性引物，采用PCR产物对提取物中的硅藻DNA提取情况进行评价。产物可采用琼脂糖凝胶电泳进行观察分析，也可采用实时荧光定量PCR熔解曲线进行分析，灵敏度更高。
<ol><li>加入 2 μL 从水样和组织中提取的 DNA 作为模板。选择以下两种方法之一进行检查。</li>
	<li>常规PCR检测<ol><li>使用可特异性扩增硅藻 18S rDNA 片段的引物22 。具体请参见 表1。</li>
		<li>根据引物的特性建立PCR反应体系和扩增条件。具体请参见 表2。</li>
		<li>在2%琼脂糖凝胶上运行PCR扩增产物。使用凝胶成像仪观察和分析成像。</li>
	</ol></li>
	<li>荧光定量PCR检测<ol><li>使用上述可特异性扩增硅藻18S rDNA片段的引物，制备实时荧光定量PCR反应体系。具体请参见 表3。</li>
		<li>进行PCR扩增并分析获得的扩增曲线和Ct值。同时，设置程序，通过荧光定量PCR熔融曲线技术，将扩增产物的双链逐渐熔融成单链，然后对得到的熔解曲线进行分析。</li>
	</ol></li>
</ol>

使用改良试剂盒进行经济的硅藻 DNA 提取

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硅藻细胞受到坚硬的硅质细胞壁17的保护，必须破坏这种结构才能提取硅藻DNA。普通试剂盒不易破坏硅藻的硅质外壳;因此很难成功提取硅藻DNA21。我们的实验室改进了最常用的血液DNA提取试剂盒，在硅藻提取过程中添加了不同直径和不同质量比的玻璃珠。同时进行涡旋振荡，可充分打破硅藻壳，提高硅藻DNA提取效果。如上一份报告21所示，玻璃珠的直径、振荡强度和时间将直接影响DNA提取的质量。涡旋振荡时间不宜过长（不超过10分钟）。如果时间太长，DNA会因过度振动而断裂。同时，时间不宜太短。如果时间太短（不少于2分钟），硅藻壳将无法完全去除，这将使DNA提取效率低下。在选择玻璃珠的同时，选择不同圆周直径的珠子，大的玻璃珠保证了碰撞强度，小的玻璃珠保证了玻璃珠在碰撞破碎过程中没有死角碰撞，提高了破壳效率。
要从样品中提取硅藻DNA，通常需要富集硅藻。这个过程将对硅藻DNA的提取产生很大的影响。富集的目的是提高样品中硅藻的浓度，使提取的硅藻DNA的浓度在可检测范围内;用于下一步应用，如实时荧光定量PCR和高通量测序。
由于硅藻在自然界中分布广泛4，因此防止实验室污染是重中之重28.DNA提取过程中的污染会导致与实际结果的偏差和使用价值的损失。因此，在提取硅藻DNA时，应始终注意无菌操作原则，并采取一切可能的措施来减少和避免外源硅藻的污染。从尸体中提取组织时，应采取措施防止其被外源性硅藻污染。提取组织后，应将其储存在无硅藻污染的容器或样品袋中。组织不应用甲醛处理。提取和处理组织的设备应反复用蒸馏水清洗2-3次，然后用紫外线或高温灭菌，以确保没有其他来源的硅藻污染。测试前应用无硅藻器械从浅表组织上切下组织，然后更换器械以挑出下部组织进行检查。同时，规范操作流程也可以有效避免实验室污染问题。
由于硅藻是许多水生生物的食物，它们在长期储存过程中会丢失29.硅藻也可以在光照条件下生长和繁殖。因此，取样后应尽快启动DNA提取步骤，并应进行快速的样品处理，以防止样品中硅藻的损失。如果不能立即进行DNA提取，则应将所有样品冷冻并置于黑暗中，以尽可能防止硅藻的还原。
我们的研究选择了一种基于蛋白酶 K 消化方法的常见且廉价的血液 DNA 提取试剂盒，该试剂盒经过改进，可以从水样和组织中提取硅藻 DNA，效果良好30。组织被充分切割以与蛋白酶 K 完全接触，并且组织被完全消化。成功提取的可能原理如下：在振荡步骤中，硅藻的硬壳在快速移动的玻璃珠之间反复撞击和破碎，露出细胞膜;不同尺寸玻璃珠的配置可以填补单一尺寸玻璃珠碰撞之间的间隙，从而尽可能充分地覆盖碰撞接触面;失去硅壳后，蛋白酶 K 有助于从硅藻中释放 DNA。与目前使用的土壤DNA提取试剂盒相比，改进后的血液DNA提取试剂盒可以满足硅藻DNA提取的要求，更经济，更容易获得。它不仅便于法医硅藻检查的使用，而且适用于其他硅藻研究相关学科，也可用于其他浮游生物DNA的提取。从水样中提取硅藻DNA时，需要根据水中浮游生物的数量来调整蛋白酶K的使用量。根据初步实验，可以增加或减少蛋白酶K的量。从组织中提取硅藻DNA时，不同的器官组织类型、组织污染和其他因素都会影响消化所需的蛋白酶K的量。在实验中，试剂盒中的蛋白酶K在使用后应保存在-20°C，否则，蛋白酶K的灭活会影响提取过程。 
总之，硅藻DNA提取应在取样和样品处理后尽快进行，否则应保存在-20°C下保存。样品处理、储存和整个提取过程应小心进行，以防止外源硅藻污染。否则，无法评估提取的硅藻DNA是否在样品中或外源性污染。当使用蛋白酶K消化组织时，其剂量可能是一个问题。组织消化不足可增加蛋白酶 K 的量。因此，充分剪切组织很重要。对组织的充分消化还可以防止组织碎片堵塞吸附柱内的质膜。膜被堵塞后，不能用移液器吸头挑出堵塞处或反复离心，会损坏膜。最终的DNA溶液中含有许多杂质，会影响硅藻DNA的检测。振荡强度和时间也是关键问题。第一次涡旋振荡时间控制在4-5 min，振荡频率控制在3000 rpm，否则会影响DNA提取。如果PCR后电泳条带不清晰，可能需要进一步增加DNA模板的量。本研究中提取的DNA是DNA的混合物，包括硅藻DNA和其他物种的DNA，因此无法确定硅藻DNA的浓度。因此，它不适用于需要高纯度DNA模板的应用。
研究表明，不同的硅藻种类可以被不同的引物扩增31,32。本研究仅使用硅藻18S rDNA上的硅藻特异性引物D512和D978来测试硅藻DNA是否成功提取，不能覆盖所有硅藻种类22,33。此外，由于该对引物的扩增产物片段长度为390-410 bp，因此改进的方法选择硅藻DNA的所有片段大小来测试是否对实验参数有影响，这需要进一步研究。后续研究还将使用不同区域和不同扩增长度的引物来探索提取效果。
与传统的硅藻DNA提取方法相比，该方法具有低成本和大规模硅藻DNA提取的主要优点。它可以补充硅藻形态学方法的优势，可以满足初级法医实验室的溺水诊断需求。同时，也扩大了硅藻DNA提取试剂盒的选择范围，具有良好的应用前景。将来，这种方法将不仅限于硅藻DNA的提取，还可用于从其他藻类中提取DNA。

在法医实践中，确定尸体是在水中溺水还是死后被扔进水里，对于案件的妥善解决至关重要1.这也是法医学实践中亟待解决的难题之一2.硅藻在自然环境中（尤其是在水中）含量丰富3,4。在溺水过程中，由于缺氧和应激反应，患者会出现剧烈的呼吸运动，吸入大量的溺水液。因此，水中的硅藻随溺水液进入肺部，一些硅藻可以通过肺泡-毛细血管屏障进入血液循环，并随着血流扩散到内脏器官5,6。在肺、肝和骨髓等内部组织和器官中检测到硅藻是死前溺水的有力证据 7,8。目前，法医硅藻测试主要基于形态学测试方法。在对组织进行一系列预消化后，在显微镜下对未消化的硅藻进行形态定性和定量估计。在此期间，需要使用危险且对环境不友好的试剂，例如硝酸。这个过程非常耗时，需要研究人员具有扎实的分类学专业知识和丰富的经验。这些都给法医人员带来了一定的挑战9.硅藻DNA检测技术是近年来发展起来的一种用于硅藻检测的新技术10,11,12。该技术通过分析硅藻的特定DNA序列组成来实现硅藻的物种鉴定13,14。PCR技术和测序技术是常用的技术方法，但它们的基础是从硅藻中成功提取DNA。然而，硅藻具有不同于其他生物的特殊结构，使其DNA提取技术也不同。
硅藻的细胞壁具有高度的硅化程度，其主要成分是二氧化硅15,16,17。硅质细胞壁非常坚硬，在提取DNA之前必须将其破坏。普通的DNA提取试剂盒通常难以直接用于硅藻DNA的提取，因为它们不能破坏硅藻的硅质外壳18。因此，破坏硅藻的硅质外壳是提取硅藻DNA需要解决的关键技术问题之一。
同时，由于法医研究样本中所含的硅藻数量，无论是水样还是溺水尸体的器官和组织，往往都是有限的，因此有必要富集硅藻。富集的本质是物质的分离。在尝试将硅藻聚集在一起时，尽量减少其他材料成分（干扰成分）的含量。在法医工作中，实验室经常使用离心或膜过滤富集方法来分离硅藻细胞19。然而，由于真空泵设备应用不广，膜富集法在普通的初级法医实验室中并不常用。因此，离心法仍然是法医实验室20中常用的硅藻富集法。
从硅藻中提取DNA目前主要用于法医实践，其应用存在很大的局限性。目前，市场上用于法医学的硅藻DNA提取试剂盒很少，而且价格普遍昂贵21。本文提供了一种改进的硅藻DNA提取方法，使硅藻DNA提取变得简单、方便且具有成本效益。这增加了后续硅藻分子生物学检测的应用，可以通过硅藻检测更好地解决法医溺水相关问题。该方法通过添加玻璃珠并设置适当的涡旋时间来破坏硅藻的硅质细胞壁。通过这种方式，蛋白酶 K 和结合溶液迅速裂解细胞并使细胞中的各种酶失活。基因组DNA在吸附柱的基质膜中被吸收，最后被洗脱缓冲液洗脱。这种改进的全血基因提取试剂盒提高了血液试剂盒在法医检验材料中的硅藻DNA提取效果，降低了法医实践中硅藻DNA提取的成本，可以更好地应用于基层法医研究。

<table><tbody><tr><td>Binding Buffer</td><td>BioTeke</td><td>B010006022</td><td>rapidly lysing cells</td></tr><tr><td>ChemoHS qPCR Mix</td><td>Monad</td><td>00007547-120506</td><td>qPCR Mix</td></tr><tr><td>D2000 DNA ladder</td><td>Real-Times(Beijing) Biotechnology</td><td>RTM415</td><td>Measure the position of electrophoretic bands</td></tr><tr><td>D512</td><td>Taihe Biotechnology</td><td>TW21109196</td><td>forword primer</td></tr><tr><td>D978</td><td>Taihe Biotechnology</td><td>TW21109197</td><td>reverse primer</td></tr><tr><td>Elution buffer</td><td>BioTeke</td><td>B010006022</td><td>A low-salt elution buffer washes off the DNA</td></tr><tr><td>Glass bead</td><td>Yingxu Chemical Machinery(Shanghai)&amp;nbsp;</td><td>70181000</td><td>Special glass beads for dispersing and grinding</td></tr><tr><td>Import adsorption column</td><td>BioTeke</td><td>B2008006022</td><td>Adsorption column with silica matrix membrane</td></tr><tr><td>Inhibitor Removal Buffer</td><td>BioTeke</td><td>B010006022</td><td>Removal of Inhibitors in DNA Extraction</td></tr><tr><td>Isopropanol</td><td>BioTeke</td><td>B010006022</td><td>Precipitate or isolate DNA</td></tr><tr><td>MIX-30S Mini Mixer</td><td>Miulab</td><td>MUC881206</td><td>oscillatory action</td></tr><tr><td>Proteinase K</td><td>BioTeke</td><td>B010006022</td><td>Inactivation of intracellular nucleases and other proteins</td></tr><tr><td>Rotor-Gene Q 5plex HRM</td><td>Qiagen</td><td>R1116175</td><td>real-time fluorescence quantification PCR</td></tr><tr><td>Speed Micro-Centrifuge</td><td>Scilogex</td><td>9013001121</td><td>centrifuge</td></tr><tr><td>Tanon 3500R Gel Imager</td><td>Tanon</td><td>16T5553R-455</td><td>gel imaging</td></tr><tr><td>Taq Mix Pro</td><td>Monad</td><td>00007808-140534</td><td>PCR Mix</td></tr><tr><td>Thermo Cycler</td><td>Zhuhai Hema</td><td>VRB020A</td><td>ordinary PCR</td></tr><tr><td>Wash Buffer</td><td>BioTeke</td><td>B010006022</td><td>Remove impurities such as cell metabolites</td></tr></tbody></table>

extraction of diatom dna from water samples and tissues

硅藻测试是法医实践中确定尸体是否溺水、推断溺水地点的重要辅助手段。硅藻试验也是环境和浮游生物领域的重要研究内容。以硅藻DNA为主要研究对象的硅藻分子生物学检测技术是一种新的硅藻检测方法。硅藻DNA提取是硅藻分子检测的基础。目前，常用用于硅藻DNA提取的试剂盒价格昂贵，增加了开展相关研究的成本。本实验室对通用全血基因组DNA快速提取试剂盒进行了改进，获得了满意的硅藻DNA提取效果，从而为相关研究提供了一种经济实惠的基于玻璃珠的DNA提取解决方案。使用该方案提取的硅藻DNA可以满足许多下游应用，例如PCR和测序。

由于目前使用的DNA提取方法提取的DNA溶液中含有来自样品中不同来源的所有DNA成分，因此通过该协议获得的DNA也不例外。因此，DNA溶液不仅仅是硅藻基因组DNA的溶液。通过查阅文献22,23,24选择可特异性扩增硅藻18S rDNA片段的引物。采用NCBI-Blast Primer对引物进行验证，结果表明，18S rDNA的正向引物D512和反向引物D978是藻类特异性引物，覆盖了多种硅藻。扩增结果未显示任何人类基因。它们被用作可靠的DNA生物标志物进行硅藻测试，因此选择它们来验证该实验的结果。以提取的DNA为模板进行硅藻特异性PCR扩增，琼脂糖凝胶电泳扩增产物显示，水样和组织均有硅藻DNA条带，这些电泳条带在250-500 bp之间（接近500 bp），与引物靶标扩增产物长度大小（390-410 bp）一致。这表明提取方案成功提取了硅藻DNA（图1A，B）。
实时荧光定量PCR技术是一种特异性严、灵敏度高的基因检测技术。通过在PCR反应系统中添加荧光基团，使荧光信号的积累可以使整个PCR过程得到实时监测，灵敏度高25,26。由于含量低和其他原因，有时很难在琼脂糖凝胶电泳中观察到通过常规PCR特异性扩增的产物27（图1C）。通过实时荧光定量PCR的特异性扩增，得到了具有四个特征阶段的典型扩增曲线（图2）。同时，通过实时荧光定量PCR熔解曲线技术，将扩增产物的DNA双链在高温下熔融成单链，染料从双链中游离，荧光值降低。通过检测荧光值的变化，得到熔融曲线（图3）。得到的熔解曲线具有特征峰，峰值在85.5 °C左右，证明目标DNA存在特异性扩增，表明提取的DNA溶液中存在硅藻DNA。染料也可以直接加入到常规的PCR扩增产物中，通过实时荧光定量PCR熔解曲线技术可以得到熔解曲线，以证明是否存在特异性扩增。因此，当常规PCR-琼脂糖凝胶电泳无法证明提取的DNA溶液中是否存在硅藻DNA时，可以选择灵敏度更高的实时荧光定量PCR技术进行验证和评估。在本实验中，实时荧光定量PCR熔解曲线技术仅用于定性分析。
如前21报道所示，传统的土壤DNA提取试剂盒和改良的血液DNA提取试剂盒在从水样和组织中提取硅藻DNA基本相同。硅藻DNA提取的成本差异主要是两种试剂盒的价格差异，其他耗材的成本基本相同。本研究对分子生物学实验中常用的血液DNA提取试剂盒进行简单改进，可以获得更好的实验结果，不仅增加了研究人员在硅藻DNA提取相关研究活动中选择检测试剂盒的范围，而且降低了检测成本。
该协议的改进过程已发布21.通过设计不同质量比和涡旋振荡时间的组合，将玻璃珠振荡的最佳DNA提取条件添加到常规试剂盒中，从而提高法医样品中硅藻DNA的提取效果，特别是在组织样品中。当涡旋振荡频率为3000 rpm时，得到DNA提取的最佳组合;涡旋振荡时间为4 min，直径为1.5-2.0 mm的大玻璃珠与直径为0.4-0.6 mm的小玻璃珠的质量比为1：1。对试剂盒的常规方法和改进方法进行了比较。结果表明，所用试剂盒可以满足硅藻DNA扩增的需要，改进方法在水样中扩增后的电泳条带亮度与常规方法相似，在组织样品中扩增后电泳条带更亮（图4）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65792/65792fig01.jpg"> 图1：PCR扩增后使用方案提取的DNA的电泳结果。 总共使用 2 μL DNA 提取物作为 DNA 模板进行 PCR 特异性扩增。将PCR扩增产物通过2%琼脂糖凝胶在1x TAE缓冲溶液上电泳，并在100V的恒定电压下运行25-30分钟。所有凝胶均使用D2000 DNA分子量标准。（A）PCR扩增后水样中硅藻DNA的电泳结果，泳道1为空白对照;泳道 2-7 是来自同一区域不同位置的水样。（B）组织中硅藻DNAPCR扩增后的电泳结果。泳道 1-6 是从同一肺组织的不同位置切除的组织;泳道 7 是空白控件。（C）在硅藻DNA扩增后，电泳结果未显示相应位置的电泳带，说明没有扩增产物或扩增产物太少。此时，电泳结果无法表明所提出的DNA溶液中是否存在硅藻DNA，这需要通过更灵敏的方法（如PCR熔解曲线）进行。 <a href="https://www.jove.com/files/ftp_upload/65792/65792fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65792/65792fig02.jpg"> 图2：硅藻DNA qPCR扩增曲线。 以常规PCR和琼脂糖凝胶电泳共2 μL未显示条带的DNA溶液提取物为模板，制备实时荧光定量PCR系统并设置程序。最后，得到了线性基线阶段、指数阶段开始阶段、指数阶段和平台阶段四个特征阶段的典型放大曲线。通过分析也得到了每条曲线的Ct值，表明DNA提取成功。每个数字代表相应曲线的Ct值。阈值为 5.2。y 轴的单位是 Rn. <a href="https://www.jove.com/files/ftp_upload/65792/65792fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65792/65792fig03.jpg"> 图3：硅藻DNA熔解曲线。 使用实时荧光定量PCR系统，设置了温度从60-95°C上升的程序。 这产生了熔解曲线，特异性扩增的双链DNA在高温下熔融成单链，染料中不含双链，荧光值降低至0。该图是通过取原始图的负倒数获得的。横坐标为温度，最高峰值对应的横坐标为Tm值，图中熔融曲线的Tm值为85.5 °C。 每条曲线的编号与 图1C中每条车道的编号相对应。 <a href="https://www.jove.com/files/ftp_upload/65792/65792fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65792/65792fig04.jpg"> 图4：改进工艺的电泳结果。 （A）不同玻璃珠比和涡旋振荡时间组合的电泳结果。泳道1、4、7、10、13，大小玻璃珠混合质量比为1：2，泳道2、5、8、11、14，大小玻璃珠混合质量比为1：1，泳道3、6、9、12、15，大小玻璃珠混合质量比为2：1， 1-3号车道振荡时间为2 min，4-6号车道为3 min，7-9号车道为4 min，10-12号车道振荡时间为5 min，13-15号车道振荡时间为6 min。泳道 16 是空白控件。（B）改进前后试剂盒提取产物PCR扩增后的电泳结果。泳道 1-3 是提取水样的常规方法，泳道 4-6 是提取水样的改进方法，泳道 7-9 是提取组织的常规方法，泳道 10-12 是改进的组织提取方法，泳道 13 是空白对照。结果已从21 修改。 <a href="https://www.jove.com/files/ftp_upload/65792/65792fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
补充图1：水样和组织样。 所用的水样取自实验室附近的一个池塘，所用的组织样本被确认为溺水尸体的肺组织。（A） 实验水样采集地点是实验室附近的一个池塘。（B）富集水样和切碎的肺组织样。 <a href="https://www.jove.com/files/ftp_upload/65792/Supplemental%20Figure%20S1.zip" target="_blank">请点击这里下载此文件。</a>
补充图2：硅藻的显微照片。 图像（A）和（B）是在400倍光学显微镜下拍摄的。箭头指向硅藻。图像（C）和（D）是在电子显微镜下拍摄的。图像（A）和（B）显示了溺水地点水中的硅藻。硅藻被命名为 Fragilaria 和 Navicula。图像（C）和（D）显示了肺部的硅藻，称为 Nitzschia 和 Navicula。 <a href="https://www.jove.com/files/ftp_upload/65792/Supplementary%20Figure%202.zip" target="_blank">请点击这里下载此文件。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>目标</td>
			<td>引物名称</td>
			<td>序列</td>
		</tr><tr><td rowspan="2">18S r脱氧核糖核酸</td>
			<td>forword 底漆 D512</td>
			<td>ATTCCAGCTCCAATAGCG</td>
		</tr><tr><td>反向引物D978</td>
			<td>GACTACGATGGTATCTAATC</td>
		</tr></tbody></table>表1：使用的引物列表。 下表描述了本文中使用的特定引物的序列、名称和靶区。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="4">PCR反应系统</td>
		</tr><tr><td colspan="2">2 个 Taq Mix Pro</td>
			<td colspan="2">10 微升</td>
		</tr><tr><td colspan="2">forword 底漆 D512</td>
			<td colspan="2">0.5 μL （10 μmol/L）</td>
		</tr><tr><td colspan="2">反向引物D978</td>
			<td colspan="2">0.5 μL （10 μmol/L）</td>
		</tr><tr><td colspan="2">模板 DNA</td>
			<td colspan="2">2 微升</td>
		</tr><tr><td colspan="2">无核酸酶水</td>
			<td colspan="2">至 20 μL</td>
		</tr><tr><td colspan="4">注意：空白对照模板DNA被等量的无核酸酶水取代。</td>
		</tr><tr><td colspan="4">PCR程序</td>
		</tr><tr><td>温度</td>
			<td colspan="2">时间</td>
			<td>周期</td>
		</tr><tr><td>94 摄氏度</td>
			<td colspan="2">10 分钟</td>
			<td>1</td>
		</tr><tr><td>94 摄氏度</td>
			<td colspan="2">45 秒</td>
			<td rowspan="3">40</td>
		</tr><tr><td>50 摄氏度</td>
			<td colspan="2">45 秒</td>
		</tr><tr><td>72 摄氏度</td>
			<td colspan="2">1 分钟</td>
		</tr><tr><td>72 摄氏度</td>
			<td colspan="2">10 分钟</td>
			<td>1</td>
		</tr></tbody></table>表2：PCR组分。 下表描述了常规PCR反应系统的配置以及PCR的反应温度、时间和循环次数的设置。所用反应系统的总体积为20 μL。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="4">实时荧光定量PCR反应系统</td>
		</tr><tr><td colspan="2">2x qPCR混合液</td>
			<td colspan="2">10 微升</td>
		</tr><tr><td colspan="2">forword 底漆 D512</td>
			<td colspan="2">0.45 μL （10 μmol/L）</td>
		</tr><tr><td colspan="2">反向引物D978</td>
			<td colspan="2">0.45 μL （10 μmol/L）</td>
		</tr><tr><td colspan="2">模板 DNA</td>
			<td colspan="2">2 微升</td>
		</tr><tr><td colspan="2">无核酸酶水</td>
			<td colspan="2">至 20 μL</td>
		</tr><tr><td colspan="4">注意：空白对照模板DNA被等量的无核酸酶水取代。</td>
		</tr><tr><td colspan="4">实时荧光定量PCR程序</td>
		</tr><tr><td>温度</td>
			<td colspan="2">时间</td>
			<td>周期</td>
		</tr><tr><td>94 摄氏度</td>
			<td colspan="2">10 分钟</td>
			<td>1</td>
		</tr><tr><td>94 摄氏度</td>
			<td colspan="2">45 秒</td>
			<td rowspan="3">40</td>
		</tr><tr><td>50 摄氏度</td>
			<td colspan="2">45 秒</td>
		</tr><tr><td>72 摄氏度</td>
			<td colspan="2">1 分钟</td>
		</tr></tbody></table>表3：实时荧光定量PCR组分。 下表描述了实时定量PCR反应系统的配置以及PCR的反应温度、时间和循环次数的设置。所用反应系统的总体积为20 μL。

Watch this Scientific Journal Video about Diatom Testing for Forensic Drowning Examination at JoVE.com

作者聚焦： 用于法医溺水检查的硅藻检测 

本文介绍了使用改良的通用 DNA 提取试剂盒提取硅藻 DNA 的方案。

从水样和组织中提取硅藻DNA

Diatom testing is an essential auxiliary means in forensic practice to determine whether the corpse drowned in water and to infer the drowning location. ...

我们的研究主要集中在溺水的话题上。我们想回答在水中发现的尸体是否死于溺水的问题，以及回答另一个人落入水中的另一个问题。这意味着要知道溺水的位置。与其他技术相比，我们提供了一种简单、方便、经济的硅藻DNA提取方案，我们可以更好地用于法医研究，特别是在基本的法医实践中。我们的实验室将根据水中的模式，如死亡原因、死后间隔等，进一步关注溺水相关问题。此外，我们还通过法医昆虫学和热带法医中的一些特殊现象进行研究。

extraction-of-diatom-dna-from-water-samples-and-tissues

Nanyang Technological University

Research

JoVE Journal

Medicine

Extraction of Diatom DNA from Water Samples and Tissues

1.8K 次观看.  Hainan Medical University, Hainan Provincial Academician Workstation (tropical forensic medicine). 我们的研究主要集中在溺水的话题上。我们想回答在水中发现的尸体是否死于溺水的问题，以及回答另一个人落入水中的另一个问题。这意味着要知道溺水的位置。与其他技术相比，我们提供了一种简单、方便、经济的硅藻DNA提取方案，我们可以更好地用于法医研究，特别是在基本的法医实践中。我们的实验室将根据水中的模式，如死亡原因、死后间隔等，进一步关注?...

视频: 作者聚焦： 用于法医溺水检查的硅藻检测 

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with &#39;end-point&#39; PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction.

从海岸沉积物和16S量化的rRNA基因和转录同时DNA-RNA提取采用Real-time PCR检测

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of ...

科研

环境科学

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes &#8804;4 L or &gt;4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

We describe a protocol for filtration of water samples with a filter cartridge and extraction of environmental DNA (eDNA) without having to cut open the housing to remove the filter. This protocol is developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

滤芯水过滤样品和环境DNA的提取使用

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

Capture of Environmental DNA (eDNA) from Water Samples by Flocculation

The analysis of environmental DNA (eDNA) has become a widely used approach to problem solving in species management. The detection of cryptic species including invasive and (or) species at risk is the goal, typically accomplished by testing water and sediment for the presence of characteristic DNA signatures. Reliable and efficient procedures for the capture of eDNA are required, especially those that can be performed easily in the field by personnel with limited training and citizen scientists. The capture of eDNA using membrane filtration is widely used currently. This approach has inherent issues that include the choice of filter material and porosity, filter fouling, and time required on site for the process to be performed. Flocculation offers an alternative that can be easily implemented and applied to sampling regimes that strive to cover broad territories in limited time.

<meta charset="utf8"></head><body>通过絮凝从水样品中捕获环境 DNA eDNA

Environmental DNA (eDNA) is typically captured from water samples using alcohol precipitation, filtration, or flocculation. Presented here is an alternative protocol that uses lanthanum chloride to enable collection of dissolved and particulate-bound nucleic acids from water samples containing eukaryotic cells, prokaryotic cells, and virus.

通过絮凝从水样品中捕获环境 DNA （eDNA）

The analysis of environmental DNA (eDNA) has become a widely used approach to problem solving in species management. The detection of cryptic species ...

DNA Extraction from 0.22 &mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been treated with storage/lysis buffer and archived at -80&deg;C, and to purify this DNA using a cesium chloride density gradient.  The protocol begins with two one-hour incubation steps to liberate DNA from cells and remove RNA.  Next, a series of Phenol:Chloroform and Chloroform extractions are performed followed by centrifugation to remove proteins and cell membrane components, collection of the aqueous DNA extract, and several buffer exchange steps to wash and concentrate the extract.  Part Five describes the optional purification via cesium chloride density gradient.  It is recommended to work with less than 15 samples at one time to avoid confusion and cut down protocol time. The total time required for this protocol depends on the number of samples to be extracted. For 10-15 samples and assuming the proper centrifugation equipment is available, this entire protocol should take 3 days. Make sure you have the hybridization ovens set to temperature at the outset of the process.

We describe a method for extraction of high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;m Sterivex filters, followed by cesium chloride density gradient centrifugation for purification.

从0.22微米Sterivex过滤器和氯化铯密度梯度离心法的DNA提取

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been ...

生物学

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

超高分子量基因组DNA的提取土壤和沉积物

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

Visualizing Methane-Cycling Microbial Dynamics in Coastal Wetlands

Coastal wetlands are the largest biotic source of methane, where methanogens convert organic matter into methane and methanotrophs oxidize methane, thus playing a critical role in regulating the methane cycle. The wetlands in South Texas, which are subject to frequent weather events, fluctuating salinity levels, and anthropogenic activities due to climate change, influence methane cycling. Despite the ecological importance of these processes, methane cycling in South Texas coastal wetlands remains insufficiently explored. To address this gap, we developed and optimized a method for detecting genes related to methanogens and methanotrophs, including mcrA as a biomarker for methanogens and pmoA1, pmoA2, and mmoX as biomarkers for methanotrophs. Additionally, this study aimed to visualize the spatial and temporal distribution patterns of methanogen and methanotroph abundance utilizing the geographic information system (GIS) software ArcGIS Pro. The integration of these molecular techniques with advanced geospatial visualization provided critical insights into the spatial and temporal distribution of methanogen and methanotroph communities across South Texas wetlands. Thus, the methodology established in this study offers a robust framework for mapping microbial dynamics in wetlands, enhancing our understanding of methane cycling under varying environmental conditions, and supporting broader ecological and environmental change studies.

The protocol detects key methane-cycling genes in South Texas coastal wetlands and visualizes their spatial distribution to enhance understanding of methane regulation and its environmental impacts in these dynamic ecosystems.

可视化沿海湿地中的甲烷循环微生物动力学

Coastal wetlands are the largest biotic source of methane, where methanogens convert organic matter into methane and methanotrophs oxidize methane, thus ...

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. ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

从石蜡包埋材料的遗传和表观遗传分析的DNA提取

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

超高分子量DNA提取微生物席

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

从水样和组织中提取硅藻DNA

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可视化沿海湿地中的甲烷循环微生物动力学

以DNA为基础的鱼类物种鉴定协议

从石蜡包埋材料的遗传和表观遗传分析的DNA提取

超高分子量DNA提取微生物席