Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients
Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients
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DOI：
10.1046/j.1462-2920.2003.00536.x
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发表时间：
2004-01-01
影响因子：
5.1
通讯作者：
Friedrich, MW
中科院分区：
文献类型：
--
作者：
Lueders, T;Manefield, M;Friedrich, MW
利用核酸的稳定同位素探测技术，可以检测和鉴定参与同化同位素标记化合物为核酸的天然微生物种群的活跃成员。Sip是基于等密度梯度离心法分离同位素标记的DNA或rRNA。我们已经开发了一种高灵敏度的检测方法，用于检测离心梯度部分中的“轻”和“重”核酸。它包括总DNA或rRNA的荧光定量，以及用结构域特异的引物实时荧光PCR定量16S rRNA基因或梯度组分的16S rRNA。利用这种方法，我们发现分别用氯化铯和三氟乙酸铯密度梯度离心法可以从未标记的巴氏甲烷链霉菌的DNA或rRNA中定量地分离出全C-13标记的Extorquens DNA或rRNA。然而，在所有DNA或rRNA梯度组分中都检测到了持续较低的非特异性核酸本底，这对于解释环境SIP结果是重要的。因此，与使用溴化乙锭或梯度分馏结合指纹分析相比，对梯度组分的定量分析为提取同位素丰富的核酸提供了更高的精确度和更好的分辨率。这是对自然生态系统中代谢C-13标记化合物的微生物种群进行精细追踪的先决条件。
Stable isotope probing (SIP) of nucleic acids allows the detection and identification of active members of natural microbial populations that are involved in the assimilation of an isotopically labelled compound into nucleic acids. SIP is based on the separation of isotopically labelled DNA or rRNA by isopycnic density gradient centrifugation. We have developed a highly sensitive protocol for the detection of 'light' and 'heavy' nucleic acids in fractions of centrifugation gradients. It involves the fluorometric quantification of total DNA or rRNA, and the quantification of either 16S rRNA genes or 16S rRNA in gradient fractions by real-time PCR with domain-specific primers. Using this approach, we found that fully C-13-labelled DNA or rRNA of Methylobacterium extorquens was quantitatively resolved from unlabelled DNA or rRNA of Methanosarcina barkeri by cesium chloride or cesium trifluoroacetate density gradient centrifugation respectively. However, a constant low background of unspecific nucleic acids was detected in all DNA or rRNA gradient fractions, which is important for the interpretation of environmental SIP results. Consequently, quantitative analysis of gradient fractions provides a higher precision and finer resolution for retrieval of isotopically enriched nucleic acids than possible using ethidium bromide or gradient fractionation combined with fingerprinting analyses. This is a prerequisite for the fine-scale tracing of microbial populations metabolizing C-13-labelled compounds in natural ecosystems.