Metagenomic analysis of isotopically enriched DNA.
Metagenomic analysis of isotopically enriched DNA.
复制标题
同位素富集 DNA 的宏基因组分析。
DOI：
10.1007/978-1-60761-823-2_4
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
Chen Y
中科院分区：
文献类型：
--
作者：
Chen Y
这个详细的协议描述了一种方法，结合DNA稳定同位素探测为基础的富集，多重置换扩增（MDA）和宏基因组学。总之，这三种方法能够选择性地获得使用同位素标记的碳源和氮源活跃生长的未培养生物体的基因组。与稳定同位素标记的底物一起孵育可以从功能相关的微生物中富集同位素标记的DNA;这可以作为过滤器来降低宏基因组的复杂性。MDA步骤从标记的核酸产生足够的DNA用于宏基因组文库构建。随后，基因组片段可以经受与活跃的群落成员相关的系统发育或功能基因的各种筛选。MDA产生的DNA还可以作为直接高通量测序的模板，以帮助重建这些活性生物体的代谢途径。最近的概念验证研究表明，这种新的分子方法组合可以大大提高基因检测频率，并可能在未来发现新的基因，酶和代谢途径方面具有巨大的潜力。
This detailed protocol describes an approach for combining DNA stable-isotope probing-based enrichment, multiple displacement amplification (MDA), and metagenomics. Together, these three methodologies enable selective access to the genomes of uncultivated organisms that actively grow using isotopically labelled carbon and nitrogen sources. Incubations with stable-isotope-labelled substrates enrich isotopically labelled DNA from functionally relevant micro-organisms; this serves as a filter to reduce the complexity of the metagenome. The MDA step generates sufficient DNA from labelled nucleic acid for metagenomic library construction. Subsequently, genome fragments can be subjected to a variety of screens for phylogenetic or functional genes relevant to active community members. The MDA-generated DNA can also serve as template for direct high-throughput sequencing to aid reconstruction of metabolic pathways of those active organisms. Recent proof-of-concept studies have demonstrated that this novel combination of molecular methods can offer substantial enhancements to gene detection frequencies and may have great future potential for the discovery of novel genes, enzymes, and metabolic pathways.
影响因子：
2.2
作者：
Hrvatin, Sinisa;Piel, Joern
通讯作者：
Piel, Joern