Enhancement of gene detection frequencies by combining DNA-based stable-isotope probing with the construction of metagenomic DNA libraries
Enhancement of gene detection frequencies by combining DNA-based stable-isotope probing with the construction of metagenomic DNA libraries
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DOI：
10.1007/s11274-005-9042-z
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发表时间：
2006-04-01
影响因子：
4.1
通讯作者：
Daniel, Rolf
中科院分区：
文献类型：
--
作者：
Schwarz, Sebastian;Waschkowitz, Tanja;Daniel, Rolf
以C-13标记的生长底物为诱饵的DNA稳定同位素探针法是从积极参与消耗这些底物的微生物中选择性提取DNA的有力工具。为了提高新天然产物筛选中靶基因的检测频率，我们首次将基于DNA的sip与元基因组文库的构建相结合。为了分离依赖辅酶B-12的甘油脱水酶基因，以甘油-C-13(3)为唯一碳源，对瓦登海沉积物样品中的甘油发酵微生物进行了富集。随后，通过密度离心法从天然丰富的C-12-DNA中分离出C-13标记的DNA，并用于文库构建。对构建的靶基因文库的筛选表明，基于DNA的SIP用于富集含脱水酶基因的基因组的基因检测频率比传统的使用未标记的甘油进行富集法记录的基因检测频率高2.1-3.8倍。
DNA-based stable-isotope probing (SIP) using C-13-labeled growth substrates as bait is a powerful tool for the selective DNA isolation from microorganisms that are actively involved in consuming these substrates. To enhance the detection frequency of target genes in screens for new natural products, we have combined for the first time DNA-based SIP with the construction of metagenomic libraries. To isolate genes encoding coenzyme B-12-dependent glycerol dehydratases an enrichment of glycerol-fermenting microorganisms from a sediment sample of the Wadden Sea was performed by using glycerol-C-13(3) as sole carbon source. Subsequently, the C-13-labeled DNA was separated from the naturally abundant C-12-DNA by density centrifugation, and used for library generation. Screening of the constructed libraries for the target genes revealed that the gene detection frequencies employing DNA-based SIP for enrichment of genomes harboring dehydratase genes were 2.1- to 3.8-fold higher than those recorded by using a traditional step with unlabeled glycerol for enrichment.