Development of a SYBR safe™ technique for the sensitive detection of DNA in cesium chloride density gradients for stable isotope probing assays

ABSTRACT Stable isotope probing (SIP) of nucleic acids is a powerful tool that can identify the functional capabilities of noncultivated microorganisms as they occur in microbial communities. While it has been suggested previously that nucleic acid SIP can be performed with 15N, nearly all applications of this technique to date have used 13C. Successful application of SIP using 15N-DNA (15N-DNA-SIP) has been limited, because the maximum shift in buoyant density that can be achieved in CsCl gradients is approximately 0.016 g ml−1 for 15N-labeled DNA, relative to 0.036 g ml−1 for 13C-labeled DNA. In contrast, variation in genome G+C content between microorganisms can result in DNA samples that vary in buoyant density by as much as 0.05 g ml−1. Thus, natural variation in genome G+C content in complex communities prevents the effective separation of 15N-labeled DNA from unlabeled DNA. We describe a method which disentangles the effects of isotope incorporation and genome G+C content on DNA buoyant density and makes it possible to isolate 15N-labeled DNA from heterogeneous mixtures of DNA. This method relies on recovery of “heavy” DNA from primary CsCl density gradients followed by purification of 15N-labeled DNA from unlabeled high-G+C-content DNA in secondary CsCl density gradients containing bis-benzimide. This technique, by providing a means to enhance separation of isotopically labeled DNA from unlabeled DNA, makes it possible to use 15N-labeled compounds effectively in DNA-SIP experiments and also will be effective for removing unlabeled DNA from isotopically labeled DNA in 13C-DNA-SIP applications.

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Identification of the Functionally Active Methanotroph Population in a Peat Soil Microcosm by Stable-Isotope Probing

Applied and Environmental Microbiology ◽

10.1128/aem.68.3.1446-1453.2002 ◽

2002 ◽

Vol 68 (3) ◽

pp. 1446-1453 ◽

Cited By ~ 170

Author(s):

Samantha A. Morris ◽

Stefan Radajewski ◽

Toby W. Willison ◽

J. Colin Murrell

Keyword(s):

Stable Isotope ◽

Gradient Centrifugation ◽

Peat Soil ◽

Pcr Amplification ◽

Cesium Chloride ◽

Stable Isotope Probing ◽

Soil Microcosm ◽

16S Rrna Genes ◽

Rrna Genes ◽

Type I

ABSTRACT The active population of low-affinity methanotrophs in a peat soil microcosm was characterized by stable-isotope probing. “Heavy” 13C-labeled DNA, produced after microbial growth on 13CH4, was separated from naturally abundant 12C-DNA by cesium chloride density gradient centrifugation and used as a template for the PCR. Amplification products of 16S rRNA genes and pmoA, mxaF, and mmoX, which encode key enzymes in the CH4 oxidation pathway, were analyzed. Sequences related to extant type I and type II methanotrophs were identified, indicating that these methanotrophs were active in peat exposed to 8% (vol/vol) CH4. The 13C-DNA libraries also contained clones that were related to β-subclass Proteobacteria, suggesting that novel groups of bacteria may also be involved in CH4 cycling in this soil.

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Characterization of Growing Microorganisms in Soil by Stable Isotope Probing with H218O

Applied and Environmental Microbiology ◽

10.1128/aem.02021-06 ◽

2007 ◽

Vol 73 (8) ◽

pp. 2541-2546 ◽

Cited By ~ 65

Author(s):

Egbert Schwartz

Keyword(s):

Stable Isotope ◽

Cesium Chloride ◽

Luria Broth ◽

Stable Isotope Probing ◽

Soil Samples ◽

16S Rrna Genes ◽

Rrna Genes ◽

Soil Dna ◽

Total Fluorescence ◽

Isopycnic Centrifugation

ABSTRACT A new approach to characterize growing microorganisms in environmental samples based on labeling microbial DNA with H2 18O is described. To test if sufficient amounts of 18O could be incorporated into DNA to use water as a labeling substrate for stable isotope probing, Escherichia coli DNA was labeled by cultivating bacteria in Luria broth with H2 18O and labeled DNA was separated from [16O]DNA on a cesium chloride gradient. Soil samples were incubated with H2 18O for 6, 14, or 21 days, and isopycnic centrifugation of the soil DNA showed the formation of two bands after 6 days and three bands after 14 or 21 days, indicating that 18O can be used in the stable isotope probing of soil samples. DNA extracted from soil incubated for 21 days with H2 18O was fractionated after isopycnic centrifugation and DNA from 17 subsamples was used in terminal restriction fragment length polymorphism (TRFLP) analysis of bacterial 16S rRNA genes. The TRFLP patterns clustered into three groups that corresponded to the three DNA bands. The fraction of total fluorescence contributed by individual terminal restriction fragments (TRF) to a TRFLP pattern varied across the 17 subsamples so that a TRF was more prominent in only one of the three bands. Labeling soil DNA with H2 18O allows the identification of newly grown cells. In addition, cells that survive but do not divide during an incubation period can also be characterized with this new technique because their DNA remains without the label.

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