Internal DNA standards for isopycnic centrifugation of environmental DNA in quantitative stable isotope probing.

Element assimilation rates or the DNA replication rate of microbial taxa can be measured in environmental samples through quantitative DNA-stable isotope probing (DNA-qSIP). Here, we introduce a set of DNA standards that may be used to quantify the density of DNA extracted from environmental samples after isopycnic centrifugation. The standards are approximately 9 Kbp PCR products with either isotopically enriched (98 atom% 15N, 13C) nucleotides or natural abundance nucleotides and have densities that differ by 0.051 g/mL. The internal standards were tested in a DNA-qSIP analysis of bacterial populations in soil exposed to 63 atom% H218O, performed in two different laboratories with different equipment and protocols. While fractionation results, including number of fractions taken and differences in density between adjacent fractions, between the two laboratories were different, the internal standards allowed the two data sets to be compared, and both research groups found similar Excess Atom Fraction (EAF) of oxygen-18 in the DNA of bacterial taxa. These internal DNA standards allow direct comparison of DNA-qSIP results from different experiments regardless of operator, tracer enrichment levels, protocol or equipment used and can support the creation of a large global database that contains qSIP results from many different laboratories.