Stable isotope probing with 15N achieved 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 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.
Buckley
Daniel H
DH
Department of Crop and Soil Sciences, 705 Bradfield Hall, Cornell University, Ithaca, NY 14853, USA. dhb28@cornell.edu
Huangyutitham
Varisa
V
Hsu
Shi-Fang
SF
Nelson
Tyrrell A
TA
D001431:Bacteriological Techniques; D001482:Base Composition; D006690:Bisbenzimidazole; D002499:Centrifugation, Density Gradient; D002586:Cesium; D002712:Chlorides; D004269:DNA, Bacterial / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D007553:Isotope Labeling / Q000379:methods*; D009587:Nitrogen Isotopes; D011550:Pseudomonas aeruginosa / Q000235:genetics
D002712:Chlorides; D004269:DNA, Bacterial; D009587:Nitrogen Isotopes; D002586:Cesium; C028019:cesium chloride; D006690:Bisbenzimidazole