Name: a magnetic-bead-based mosquito dna extraction protocol for next-generation sequencing
Uploaded: 2021-04-15T14:00:00
Description: Watch this Scientific Journal Video about A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing at JoVE.com

我们感谢由美国疾病控制和预防中心资助的太平洋西南地区病媒-伯恩疾病卓越中心（合作协议 1U01CK000516）、CDC 赠款 NU50CK000420-04-04、 美国农业部国家粮食和农业研究所（哈奇项目1025565）、UF/IFAS佛罗里达医学昆虫学实验室研究金给陈泽宇、NSF CAMTech IUCRC第二期赠款（AWD05009_MOD0030）和佛罗里达州卫生部（合同CODQJ）。本文的结论和结论是作者的，不一定代表美国鱼类和野生动物管理局的观点。

1. DNA提取前的一般样品储存和制剂
<ol><li>如果样品储存在 > 70% 酒精中以软化组织，则将样品在 100 μL PCR 级水中水合 1 小时（或过夜），达到 4 °C。</li>
</ol>2. 样品中断
<ol><li>将孵化器或摇动热块设置在 56 °C 下。</li>
	<li>制作蛋白酶K（PK）缓冲/酶混合物。每只蚊子提取需要 2 微升蛋白酶 K （100 毫克/兆升） 和 98 μL 蛋白酶 K 缓冲器 （总计 100 微升） 。要为多个标本准备主组合...

<ol>
	<li>Nieman, C. C., Yamasaki, Y., Collier, T. C., Lee, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+DNA+extraction+protocol+for+improved+DNA+yield+from+individual+mosquitoes.">A DNA extraction protocol for improved DNA yield from individual mosquitoes.</a> F1000Research. 4, 1314 (2015).</li><li>Lee, 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=Genome-wide+divergence+among+invasive+populations+of+Aedes+aegypti+in+California.">Genome-wide divergence among invasive populations of Aedes aegypti in California.</a> BMC Genomics. 20 (1), 204 (2019).</li><li>Schmidt, H., 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=Abundance+of+conserved+CRISPR-Cas9+target+sites+within+the+highly+polymorphic+genomes+of+Anopheles+and+Aedes+mosquitoes.">Abundance of conserved CRISPR-Cas9 target sites within the highly polymorphic genomes of Anopheles and Aedes mosquitoes.</a> Nature Communications. 11 (1), 1425 (2020).</li><li>Schmidt, H., 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=Transcontinental+dispersal+of+Anopheles+gambiae+occurred+from+West+African+origin+via+serial+founder+events.">Transcontinental dispersal of Anopheles gambiae occurred from West African origin via serial founder events.</a> Communications Biology. 2, 473 (2019).</li><li>Norris, L. C., 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=Adaptive+introgression+in+an+African+malaria+mosquito+coincident+with+the+increased+usage+of+insecticide-treated+bed+nets.">Adaptive introgression in an African malaria mosquito coincident with the increased usage of insecticide-treated bed nets.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (3), 815-820 (2015).</li><li>Main, B. J., 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+genetic+basis+of+host+preference+and+resting+behavior+in+the+major+african+malaria+vector,+Anopheles+arabiensis.">The genetic basis of host preference and resting behavior in the major african malaria vector, Anopheles arabiensis.</a> Plos Genetics. 12 (9), 1006303 (2016).</li><li>Yamasaki, Y. 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=Improved+tools+for+genomic+DNA+library+construction+of+small+insects.">Improved tools for genomic DNA library construction of small insects.</a> F1000Research. 5, 211 (2016).</li><li>Tabuloc, C. 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=Sequencing+of+Tuta+absoluta+genome+to+develop+SNP+genotyping+assays+for+species+identification.">Sequencing of Tuta absoluta genome to develop SNP genotyping assays for species identification.</a> Journal of Pest Science. 92, 1397-1407 (2019).</li><li>Campos, 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=Complete+mitogenome+sequence+of+Anopheles+coustani+from+S&#227;o+Tom&#233;+island.">Complete mitogenome sequence of Anopheles coustani from S&#227;o Tom&#233; island.</a> Mitochondrial DNA. Part B, Resources. 5 (3), 3376-3378 (2020).</li><li>Cornel, A. J., 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=Complete+mitogenome+sequences+of+Aedes+(Howardina)+busckii+and+Aedes+(Ochlerotatus)+taeniorhynchus+from+the+Caribbean+Island+of+Saba.">Complete mitogenome sequences of Aedes (Howardina) busckii and Aedes (Ochlerotatus) taeniorhynchus from the Caribbean Island of Saba.</a> Mitochondrial DNA. Part B, Resources. 5 (2), 1163-1164 (2020).</li><li>Lucena-Aguilar, G., 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+source+selection+for+downstream+applications+based+on+dna+quality+indicators+analysis.">DNA source selection for downstream applications based on dna quality indicators analysis.</a> Biopreservation and Biobanking. 14 (4), 264-270 (2016).</li></ol>

这里描述的协议可以适应其他昆虫物种。尼曼等人介绍的协议的原始版本已经在多个物种上进行了测试，包括伊蚊、伊蚊、伊·布希、埃·塔尼奥尔欣丘斯、阿诺菲莱斯·阿拉比安西斯、安·科卢齐、安·库斯塔尼、 安达利， 安· 预计此协议将适用于这些以及其他节肢动?...

最近开发的改进的DNA提取协议1允许许多高影响下游研究涉及全基因组测序2，3，4，5，6。这种基于磁珠的DNA提取协议提供了来自单个蚊子样本的可靠DNA产量，这反过来又降低了从现场采集中获取足够数量的样本的成本和时间。
人口和景观基因组学的最新进展与全基因组测序成本的降低直接相关。虽然以前的DNA提取协议1 提高了与高通量测序相关的效率，但没有资金的小型实验室/研究可能会选择不使用这些新的强大景观和人口基因组学工具，因为实施协议的成本（例如，专用仪器的成本）。
在这里，提出了一个修改的DNA提取协议，使用与Neiman等人类似的磁珠提取步骤来获得 高纯度DNA，但不依赖于高成本的仪器进行组织裂解和DNA提取。该协议适用于需要>10ng高质量DNA的实验。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AE Buffer</td>
			<td>Qiagen</td>
			<td>19077</td>
			<td>Elution buffer</td>
		</tr>
		<tr>
			<td>AL Buffer</td>
			<td>Qiagen</td>
			<td>19075</td>
			<td>Lysis buffer</td>
		</tr>
		<tr>
			<td>AW1 Buffer</td>
			<td>Qiagen</td>
			<td>19081</td>
			<td>Washing buffer 1</td>
		</tr>
		<tr>
			<td>AW2 Buffer</td>
			<td>Qiagen</td>
			<td>19072</td>
			<td>Washing buffer 2</td>
		</tr>
		<tr>
			<td>MagAttract Suspension G</td>
			<td>Qiagen</td>
			<td>1026901</td>
			<td>magnetic bead</td>
		</tr>
		<tr>
			<td>Magnetic bead separator</td>
			<td>Epigentek</td>
			<td>Q10002-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop</td>
			<td>ThermoFisher</td>
			<td>ND-2000</td>
			<td>microvolume spectrophotometer</td>
		</tr>
		<tr>
			<td>PK Buffer</td>
			<td>ThermoFisher</td>
			<td>4489111</td>
			<td>Proteinase K buffer</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>ThermoFisher</td>
			<td>A25561</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit</td>
			<td>Invitrogen</td>
			<td>Q33238</td>
			<td>fluorometer</td>
		</tr>
	</tbody>
</table>

a magnetic-bead-based mosquito dna extraction protocol for next-generation sequencing

最近公布的一项使用磁珠和自动DNA提取仪器的DNA提取协议表明，有可能从保存完好的单个蚊子身上提取出高质量和数量的DNA，足以进行下游全基因组测序。然而，对于许多实验室来说，依赖昂贵的自动DNA提取仪器可能令人望而却步。在这里，该研究提供了一个经济实惠的磁珠为基础的DNA提取协议，这是适合中低吞吐量。这里描述的协议是使用个别伊 蚊 样本成功测试的。与高质量DNA提取相关的成本降低将增加高吞吐量测序在资源有限的实验室和研究中的应用。

使用 100 μL 的脱毛缓冲器使用荧光仪测量每个蚊子头/胸组织的平均 DNA 产量为 4.121 ng/μL （N = 92，标准偏差 3.513）。这足以满足全基因组库建设所需的10-30ng基因组DNA输入要求。DNA 的数量可能因蚊子体型和保存条件而异，在 0.3-29.7 ng/μL 之间。一些高变异性是由于研究中使用的磁珠的特性。不同品牌的磁珠可能会产生更一致的浓度，如上一份报告<sup class=...

Watch this Scientific Journal Video about A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing at JoVE.com

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

这里描述的是一个DNA提取协议，使用磁珠从蚊子中产生高质量的DNA提取。这些提取适合下游的下一代测序方法。

用于下一代测序的基于磁珠的蚊子DNA提取协议

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high ...

这种基于预算的磁珠DNA提取协议使资源有限的实验室和研究能够访问高通量测序。该协议不需要昂贵的设备进行高质量的DNA提取，它可以帮助某些诊断或研究的情况下，获得足够数量的DNA与高通量测序的质量是具有挑战性的。此协议很容易通过一次性演示进行复制。应首先用少量样本进行尝试，以熟悉工作流程。首先，通过加入100微升PCR级水，在4摄氏度下孵育一小时，软化组织，使储存在70%以上酒精中的蚊子组织样本得到水合。孵化后，丢弃水，加入100微升蛋白酶K缓冲酶混合物。然后使用微中心管虫子使组织同质化。同质化后，离心组织在56摄氏度下解压并孵育两到三个小时。为了提取DNA，移液器100微升的组织裂解成一个新的清洁微中心管。然后添加磁珠主组合的215微升。使用移液器，将解酯和磁珠混合物混合 10 到 20 秒，然后让它在室温下站立 10 分钟。在孵化过程中，偶尔轻轻摇动管子，以最大限度地将DNA与磁珠结合。接下来，将管子放在磁珠分离器上，直到溶液变得清晰。然后使用移液器，轻轻吸气管中的液体，而不会干扰持有DNA的磁珠。将管子从磁珠分离器移开，通过添加 325 微升洗涤缓冲器来清洗珠子。在室温下通过管道彻底混合并孵育一分钟。孵化后，将管子放在磁珠分离器上，取出先前演示的超高纳特，然后将管子从磁珠分离器移开，用洗涤缓冲器洗涤珠子一次。第二次用缓冲器清洗后，用250微升的洗涤缓冲器两次以同样的方式清洗珠子。最后一次洗涤后，将管子从磁珠分离器移开，并添加 100 微升的洗脱缓冲器。通过管道彻底混合，然后在室温下孵育管子两分钟，然后放回磁分离器上。当溶液变得清晰时，将超高纳特转移到一个新的，干净的0.5毫升微中心管，并适当地存储。这里显示为典型的微卷荧光仪读数蚊子DNA在一个包含0.5毫摩尔EDTA的椭芽缓冲器。平均260至280纳米吸收比为2.3。此表显示了不同提取方法在一个工作日内处理的成本和样本数量。本协议提取的典型试剂和消耗成本约为每个样品 9.50。此成本相当于任何典型的磁珠提取方法。本协议的主要成本效益来自不需要自动DNA提取仪器。在处理多个样品时，请务必更改样品之间的移液器提示，以防止 DNA 交叉污染。我们使用这种方法提取的高质量DNA进行全基因组测序。我们目前正在使用测序数据来识别公共卫生和疾病控制中关注的杀虫剂耐药性或免疫基因的新突变。拥有经济实惠的高吞吐量测序解决方案为旨在了解影响许多新基因控制策略成功的蚊子分散和基因流动的人口基因组学和景观基因组学打开了令人兴奋的大门。

Research

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蚊子养育的议定书“（冈比亚按蚊）

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科研

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有针对性的DNA甲基化分析通过新一代测序

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

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Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to ...

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