RATIONALELiquid chromatography/isotope ratio mass spectrometry (LC/IRMS) is currently the most accurate and precise technique for the measurement of compound-specific stable carbon isotope ratios (C-13/C-12) in biological metabolites, at their natural abundance. However, until now this technique could not be applied for the analysis of nucleic acids, the building blocks of the carriers of genetic information in living cells and viruses, DNA and RNA.
METHODSMixed-mode chromatography (MMC) was applied to obtain the complete separation of nine nucleotides (eight originating from DNA/RNA and one nucleotide (inosine monophosphate) that may serve as an internal standard) in a single run using LC/IRMS. We also developed and validated a method for DNA and RNA extraction and an enzymatic hydrolysis protocol for natural samples, which is compatible with LC/IRMS analysis as it minimizes the carbon blank. The method was used to measure the concentration and stable carbon isotope ratio of DNA and RNA nucleotides in marine sediment and in the common marine macro alga (Ulva sp.) at natural abundance levels as well as for C-13-enriched samples.
RESULTSThe detection limit of the LC/IRMS method varied between 1.0nmol for most nucleotides and 2.0nmol for late-eluting compounds. The intraday and interday reproducibility of nucleotide concentration measurements was better than, respectively, 4.1% and 8.9% and for C-13 measurements better than, respectively, 0.3 and 0.5 parts per thousand. The obtained nucleic acid concentrations and nucleic acid synthesis rates were in good agreement with values reported in the literature.
CONCLUSIONSThis new method gives reproducible results for the concentration and C-13 values of nine nucleotides. This solvent-free chromatographic method may also be used for other purposes, such as for instance to determine nucleotide concentrations using spectrophotometric detection. This sensitive method offers a new avenue for the study of DNA and RNA biosynthesis that can be applied in various fields of research. Copyright (c) 2014 John Wiley & Sons, Ltd. |
