New radioisotope-free method for measuring bacterial production using [15N5]-2′-deoxyadenosine and liquid chromatography mass spectrometry (LC–MS) in aquatic environments
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This study proposed a non-radioactive quantitative measurement of bacterial production using stable isotope nitrogen-15-labeled deoxyadenosine ([15N5]-2′-deoxyadenosine; 15N-dA) by liquid chromatography mass spectrometry (LC–MS). The method of preparing 5N-dA described in this study included incubation of seawater or lake water with 15N-dA for 5–24 h, filtration onto a membrane filter, DNA extraction, enzymatic hydrolysis of DNA to nucleosides and quantification of 15N-dA by LC–MS. In the DNA extraction, the silica beads method was examined first, but a large amount of salts and enzymatic inhibitors used in the method caused failure of subsequent procedures, such as enzymatic hydrolysis and LC–MS analysis. On the other hand, the magnetic beads method showed much better results for the extraction. The incorporation rate of 15N-dA was significantly positively correlated to that of tritium-labeled thymidine (3H-TdR) in samples of coastal seawaters and lake waters. The average 15N-dA: 3H-TdR incorporation ratio for the seawater sample was 0.55 with 2.5 and 97.5 % confidence intervals of 0.51 and 0.58, respectively; the average ratio for the lake water sample was 0.28 with 2.5 and 97.5 % confidence intervals of 0.23 and 0.34, respectively. The results suggest that the 15N-dA method can be applied to the measurement of bacterial production in aquatic ecosystems, and that this method can accurately predict the DNA synthesis rates measured by the conventional method.
KeywordsBacterial production Stable isotope Liquid chromatography mass spectrometry (LC–MS) DNA synthesis 2′-deoxyadenosine
We thank Dr. Kazuhiro Iwasaki for valuable advice on DNA extraction, Dr. Shota Takumi for valuable advice on enzymatic DNA hydrolysis and Dr. Kazuaki Shutsubo for the use of his spectrometer. The data of DNA extraction efficiency of the Extrap Soil DNA Kit Plus ver.2 was supplied by Nippon Steel & Sumikin Eco-Tech Corporation. This research was partly supported by a Grant-in-Aid for Scientific Research (No. 2550019) from the Japan Society for the Promotion of Science and the Sasakawa Scientific Research Grant from The Japan Science Society.
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