Stable Isotope Probing with 15 N Achieved by Disentangling the Effects of Genome G+C Content and Isotope Enrichment on DNA Density

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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 15 N, nearly all applications of this technique to date have used 13 C. Successful application of SIP using 15 N-DNA ( 15 N-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 15 N-labeled DNA, relative to 0.036 g ml −1 for 13 C-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 15 N-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 15 N-labeled DNA from heterogeneous mixtures of DNA. This method relies on recovery of “heavy” DNA from primary CsCl density gradients followed by purification of 15 N-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 15 N-labeled compounds effectively in DNA-SIP experiments and also will be effective for removing unlabeled DNA from isotopically labeled DNA in 13 C-DNA-SIP applications.