The aim of this work was to obtain pure extracellular DNA molecules so as to estimate their longevity in soil by an isotope-based approach. Extracellular DNA molecules were extracted from all horizons of a forest soil and purified by the procedure of Davis (1986) without (DNA1) or with (DNA2) a successive treatment with binding resins followed by elution. The two differently purified DNA samples were compared for their A260/A280 ratio, PCR amplification, and natural abundance of stable (13C and 15N) and radioactive (14C) isotopes. The purity index and the PCR amplification did not differentiate the efficiency of the two purification procedures. The isotopic signature of DNA was more sensitive and was strongly affected by the purification procedures. The isotopic measurements showed that the major contaminant of extracellular DNA1 was the soil organic matter (SOM), even if it is not possible to exclude that the similar δ13C, δ15N and Δ14C values of DNA and SOM could be due to the use of SOM-deriving C and N atoms for the microbial synthesis of DNA. For extracellular DNA2, extremely low values of Δ14C were obtained, and this was ascribed to the presence of fossil fuel-derived substances used during the purification, although in amounts not revealed by GC-MS analysis. The fact that it is not possible to obtain contaminant-free DNA molecules and the potential use of soil native organic compounds during the microbial synthesis of DNA make not achievable to estimate the age of soil extracellular DNA by radiocarbon dating..
Purification and isotopic signatures (d13C, d15N, D14C) of soil extracellular DNA
AGNELLI, Alberto;
2007
Abstract
The aim of this work was to obtain pure extracellular DNA molecules so as to estimate their longevity in soil by an isotope-based approach. Extracellular DNA molecules were extracted from all horizons of a forest soil and purified by the procedure of Davis (1986) without (DNA1) or with (DNA2) a successive treatment with binding resins followed by elution. The two differently purified DNA samples were compared for their A260/A280 ratio, PCR amplification, and natural abundance of stable (13C and 15N) and radioactive (14C) isotopes. The purity index and the PCR amplification did not differentiate the efficiency of the two purification procedures. The isotopic signature of DNA was more sensitive and was strongly affected by the purification procedures. The isotopic measurements showed that the major contaminant of extracellular DNA1 was the soil organic matter (SOM), even if it is not possible to exclude that the similar δ13C, δ15N and Δ14C values of DNA and SOM could be due to the use of SOM-deriving C and N atoms for the microbial synthesis of DNA. For extracellular DNA2, extremely low values of Δ14C were obtained, and this was ascribed to the presence of fossil fuel-derived substances used during the purification, although in amounts not revealed by GC-MS analysis. The fact that it is not possible to obtain contaminant-free DNA molecules and the potential use of soil native organic compounds during the microbial synthesis of DNA make not achievable to estimate the age of soil extracellular DNA by radiocarbon dating..I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
