Name: isolation and genome analysis of single virions using 'single virus genomics'
Uploaded: 2013-05-26T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics' at JoVE.com

愿我们，感谢肯Nealson的整个手稿的准备过程中他的见解和建议。

1。病毒悬浮液的制备通过流式细胞仪分离出单个病毒颗粒之前，准备不固定的病毒悬液。 <ol><li>使用先前建立的协议，确保最终的病毒悬浮液中分离病毒颗粒被包含在0.1微米过滤TE液（Tris-EDTA，pH8）中。我们建议您使用其他方法虽然已开发出采用化学絮凝7切向流过滤（TFF）接近6。 </li><li>枚举使用既定的协议， 例如 ，使用萤光8-10或流式细胞...

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	<li>Raghunathan, 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=Genomic+DNA+amplification+from+a+single+bacterium.">Genomic DNA amplification from a single bacterium.</a> Appl. Environ. Microbiol. 71, 3342-337 (2005).</li><li>Lasken, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+genomic+sequencing+using+Multiple+Displacement+Amplification.">Single-cell genomic sequencing using Multiple Displacement Amplification.</a> Curr. Opin. Microbiol. 10, 510-516 (2007).</li><li>Ishoey, T., Woyke, T., Stepanauskas, R., Novotny, M., Lasken, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+sequencing+of+single+microbial+cells+from+environmental+samples.">Genomic sequencing of single microbial cells from environmental samples.</a> Curr. Opin. Microbiol. 11, 198-204 (2008).</li><li>Edwards, R. A., Rohwer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+metagenomics.">Viral metagenomics.</a> Nature Reviews Microbiology. 3, 504-510 (2005).</li><li>Rohwer, F., Prangishvili, D., Lindell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+viruses+in+the+environment.">Roles of viruses in the environment.</a> Environ. Microbiol. 11, 2771-2774 (2009).</li><li>Williamson, S. 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应用SVG方法时，必须考虑一些重要因素。基因分型，在概念证明型实验13，是不是保守引物的环境或未知的菌株可在所有病毒组的有效选项。此外，背景DNA合成或非特异性扩增常见的扩增过程中使用MDA反应14。非特异性扩增已被归因于新兴的反应试剂和/或通过一种机制，允许从随机六聚体在反应混合物中的扩增的DNA污染。当使用病毒的组合，有可能是一个更高的可能性由于作为粒?...

<table border="1">
	<tbody>
		<tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>1X TE buffer</td>
			<td>Invitrogen</td>
			<td>12090-015</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Buffer-saturated Phenol</td>
			<td>Invitrogen</td>
			<td>15513-039</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GenomiPhi HY kit</td>
			<td>GE Healthcare</td>
			<td>25-6600-22</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glycoblue</td>
			<td>Invitrogen</td>
			<td>AM9516</td>
			<td>15 mg/ml</td>
		</tr>
		<tr>
			<td>LMP agarose</td>
			<td>Invitrogen</td>
			<td>16520100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PTFE microscope slide</td>
			<td>Electron Microscopy Sciences</td>
			<td>63430-04</td>
			<td>24 well, 4 mm Diameter</td>
		</tr>
		<tr>
			<td>SybrGreen</td>
			<td>Invitrogen</td>
			<td>S7585</td>
			<td>10,000X</td>
		</tr>
		<tr>
			<td>&beta;-agarase</td>
			<td>New England Biolabs</td>
			<td>M0392S</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

isolation and genome analysis of single virions using 'single virus genomics'

全基因组扩增和测序的单一微生物细胞使基因组特性，而不需要栽培1-3。病毒，是无处不在的，我们星球上的4最众多的实体和重要的在各种环境中5，通过类似的方法尚未透露。在这里，我们描述了一种方法，用于分离和表征单个病毒颗粒的基因组被称为“单个病毒的基因组学”（SVG）。 SVG利用流式细胞仪分离单个病毒和全基因组扩增，得到高分子量的基因组DNA（基因组DNA），可以用于在随后的测序反应。

Watch this Scientific Journal Video about Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics' at JoVE.com

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

单一的病毒基因组（SVG）是一种方法来隔离和放大的基因组单virons。病毒悬浮液的混合组合，用流式细胞仪检测到载玻片离散井含琼脂糖，从而捕获病毒粒子和减少下游加工过程中的基因组剪切排序。全基因组扩增是通过使用多重置换扩增（MDA）的产生，是适合用于测序的基因组物质中。

使用“单个病毒基因组的单病毒颗粒的分离和基因组分析&#39;

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

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