Stable isotope probing with 15Nachieved by disentangling the effects of genome G+C content and isotope enrichment on DNA density

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 N-15, nearly all applications of this technique to date have used C-13. Successful application of SIP using N-15-DNA (N-15-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 N-15-labeled DNA, relative to 0.036 g ml(-1) for C-13-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 N-15-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 N-15-labeled DNA from heterogeneous mixtures of DNA. This method relies on recovery of heavy DNA from primary CsCl density gradients followed by purification of N-15-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 C-13-DNA-SIP applications.