Nanoliter reactors improve multiple displacement amplification of genomes from single cells.
Nanoliter reactors improve multiple displacement amplification of genomes from single cells.
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DOI：
10.1371/journal.pgen.0030155
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发表时间：
2007-09
期刊：
影响因子：
4.5
通讯作者：
Quake, Stephen R.
中科院分区：
文献类型：
--
作者：
Marcy, Yann;Ishoey, Thomas;Lasken, Roger S.;Stockwell, Timothy B.;Walenz, Brian P.;Halpern, Aaron L.;Beeson, Karen Y.;Goldberg, Susanne M. D.;Quake, Stephen R.
由于只有一小部分环境细菌适合实验室培养，因此人们对直接从单细胞进行基因组测序非常感兴趣。通过多位移扩增（Multiple Displacement Amplification， MDA）方法可以从一个细胞中获得足够的DNA进行测序，从而消除了开发培养方法的需要。在这里，我们使用微流控装置分离大肠杆菌个体，并在60-nl的反应中通过MDA扩增基因组DNA。我们的研究结果证实了一篇报道，即减少MDA反应体积降低了非特异性合成，这可能是由污染的DNA模板和引物之间不利的相互作用造成的。通过qPCR评估基因组扩增的质量，并将其与标准50 μl体积的单细胞扩增进行比较。放大偏差在纳升体积中大大减少，从而提供了所有序列的更均匀的表示。微升和纳升体积的单细胞扩增子通过高通量焦磷酸测序提供了高质量的序列数据，从而展示了从单细胞测序基因组的直接途径。操纵或分析单个细胞的遗传物质或基因组通常具有挑战性。生物化学DNA扩增技术可用于从单个细胞中复制基因组的许多拷贝，在本文中，我们研究了这种扩增作为反应体积的函数的效果。我们发现，在纳升体积中单细胞基因组扩增比在微升体积中有效得多，可以更好地代表起始基因组，减少产物中的偏差。因此，从单个细胞中获得高质量的基因组序列应该是可能的。这是有用的，因为在纯培养中可以获得的微生物很少，因此只适用于单细胞分析。
Since only a small fraction of environmental bacteria are amenable to laboratory culture, there is great interest in genomic sequencing directly from single cells. Sufficient DNA for sequencing can be obtained from one cell by the Multiple Displacement Amplification (MDA) method, thereby eliminating the need to develop culture methods. Here we used a microfluidic device to isolate individual Escherichia coli and amplify genomic DNA by MDA in 60-nl reactions. Our results confirm a report that reduced MDA reaction volume lowers nonspecific synthesis that can result from contaminant DNA templates and unfavourable interaction between primers. The quality of the genome amplification was assessed by qPCR and compared favourably to single-cell amplifications performed in standard 50-μl volumes. Amplification bias was greatly reduced in nanoliter volumes, thereby providing a more even representation of all sequences. Single-cell amplicons from both microliter and nanoliter volumes provided high-quality sequence data by high-throughput pyrosequencing, thereby demonstrating a straightforward route to sequencing genomes from single cells. It is often challenging to manipulate or analyze the genetic material or genome of an individual cell. Biochemical DNA amplification technologies can be used to make many copies of the genome from a single cell, and in this paper we investigated how well such amplification works as a function of the reaction volume. We found that single-cell genome amplification in nanoliter volumes is much more effective than in microliter volumes, providing better representation of the starting genome with less bias in the product. It should therefore be possible to obtain high-quality genome sequences from single cells. This is useful because very few microbes can be obtained in pure culture, and are therefore only amenable to single-cell analysis.
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影响因子：
56.9
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Venter, JC;Remington, K;Smith, HO
通讯作者：
Smith, HO
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作者：
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Lasken, Roger S;Stockwell, Timothy B
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Stockwell, Timothy B