Stable Isotope Probing with ^sup 15^N Achieved by Disentangling the Effects of Genome G+C Content and Isotope Enrichment on DNA Density[white triangle down],[dagger]

• Published in: Applied and environmental microbiology 2007-05, Vol.73 (10), p.3189
• Main Authors: Buckley, Daniel H, Varisa Huangyutitham, Shi-Fang, Hsu, Nelson, Tyrrell A
• Format: Article
• Language: English
• Subjects: Deoxyribonucleic acid; DNA; Genomics; Isotopes; Microbiology; Microorganisms
• ISSN: 0099-2240; 1098-5336

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 ^sup 15^N, nearly all applications of this technique to date have used ^sup 13^C. Successful application of SIP using ^sup 15^N-DNA (^sup 15^N-DNA-SIP) has been limited, because the maximum shift in buoyant density that can be achieved in CsC1 gradients is approximately 0.016 g ml^sup -1^ for ^sup 15^N-labeled DNA, relative to 0.036 g ml^sup -1^ for ^sup 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^sup -1^. Thus, natural variation in genome G+C content in complex communities prevents the effective separation of ^sup 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 ^sup 15^N-labeled DNA from heterogeneous mixtures of DNA. This method relies on recovery of "heavy" DNA from primary CsC1 density gradients followed by purification of ^sup 15^N-labeled DNA from unlabeled high-G+C-content DNA in secondary CsC1 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 ^sup 15^N-labeled compounds effectively in DNA-SIP experiments and also will be effective for removing unlabeled DNA from isotopically labeled DNA in ^sup 13^C-DNA-SIP applications. [PUBLICATION ABSTRACT]