DNA buoyant density shifts during 15N-DNA stable isotope probing.
DNA-based stable isotope probing (SIP) is a novel technique for the identification of organisms actively assimilating isotopically labeled compounds. Herein, we define the limitations to using 15N-labeled substrates for SIP and propose modifications to compensate for these shortcomings. Changes in DNA buoyant density (BD) resulting from 15N incorporation were determined using cultures of disparate GC content (Escherichia coli and Micrococcus luteus). Incorporation of 15N into DNA increased BD by 0.015+/-0.002 g mL(-1) for E. coli and 0.013+/-0.002 g mL(-1) for M. luteus. The DNA BD shift was greatly increased (0.045 g mL(-1)) when dual isotope (13C plus 15N) labeling was employed. Despite the limited DNA BD shift following 15N enrichment, we found the use of gradient fractionation, followed by a comparison of T-RFLP profiles from fractions of labeled and control treatments, facilitated detection of enrichment in DNA samples from either cultures or soil.
United States Department of Agriculture-Agricultural Research Service, University of Illinois at Urbana-Champaign, S-306 Turner Hall, 1102 S. Goodwin Ave., Urbana, IL 61801, USA. cupplesa@msu.edu
Alison M
AM
Cupples
Elizabeth A
EA
Shaffer
Joanne C
JC
Chee-Sanford
Gerald K
GK
Sims
D002247:Carbon Isotopes / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D004247:DNA / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods*; D016982:Micrococcus luteus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009587:Nitrogen Isotopes / Q000378:metabolism*; D012150:Polymorphism, Restriction Fragment Length
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D004247:DNA