The effect of the 13 C abundance of soil microbial DNA on identifying labelled fractions after ultracentrifugation.

DNA-based stable isotope probing (DNA-SIP) technology has been widely employed to trace microbes assimilating target substrates. However, the fractions with labelled universal genes are sometimes difficult to distinguish when detected by quantitative real-time PCR. In this experiment, three paddy soils (AQ, CZ, and NB) were amended with 0.1% glucose containing 13 C at six levels, and DNA was then extracted after a 7-day incubation and subjected to isopycnic gradient centrifugation. The results showed that the amount of labelled DNA was notably related to the 13 C-glucose percentage, while the separation spans of 18S rRNA and 16S rRNA genes between labelled and unlabelled treatments became notably clearer when the δ 13 C values of the total DNA were 90.9, 61.6, and 38.9‰ and 256.2, 104.5 and 126.1‰ in the AQ, CZ, and NB soils, respectively. Moreover, fractionated DNA was also labelled by determining the δ 13 C values while adding only 5 atom% 13 C-glucose to the soil. The results suggest that the optimal labelling fractions were not always those fractions with the maximal gene abundance, and detecting the δ 13 C values of the total and fractionated DNA was beneficial in estimating the results of DNA-SIP. KEY POINTS: • Appropriate 13 C-DNA amount was needed for DNA-SIP. • Detecting the 13 C ratio of fractionated DNA directly was an assistant method for identifying the labelled fractions. • Fractions with the maximal 18S or 16S rRNA gene abundance always were not labelled.