Quantitative substrate utilization in microbial ecology using stable isotope probing
AssigneeArizona Board of Regents On Behalf of Northern Arizona University
InventorsBruce Hungate, Egbert Schwartz, Rebecca Mau, Jane Marks
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Abstract
Methods modifying conventional SIP so that isotopic incorporation into the genomes of individual microbial taxa can be quantified are described. Further, methods to quantify the baseline densities of the DNA of individual microbial taxa without exposure to isotope tracers and then to quantify the change in DNA density of each taxon caused by isotope incorporation are described. The distribution of DNA of each taxon along a density gradient reflects the influence of isotope incorporation only, without reflecting the guanine-plus-cytosine content.
Claims10
A method to quantify uptake of a substrate by a taxon in a microorganism sample, comprising:
- (a) extracting nucleic acids from a first microorganism sample that has been incubated with at least one isotopically labeled substrate and from a second microorganism sample that has not been incubated with an isotopically labeled substrate, thereby obtaining a labeled sample and an unlabeled sample;
- (b) separating said nucleic acids extracted from said first microorganism sample by density into a plurality of labeled tractions;
- (c) separating said nucleic acids extracted from said second microorganism sample by density into a plurality of unlabeled fractions;
- measuring a density of said nucleic acids from each of the fractions obtained in steps (b) and (c);
- (d) detecting a target sequence of a taxon of a microorganism in the fractions obtained in steps (b) and (c), whereby the presence of the taxon of a microorganism is determined;
- (e) measuring the relative abundance of the taxon of the microorganism in each of the fractions obtained in steps (b) and (c) through sequencing the target sequence;
- (f) quantifying through quantitative PCR an average density of said taxon of said microorganism in said fractions obtained in steps (b) and (c);
- and
- (g) measuring the difference between the average density of said taxon of said microorganism in the plurality of labeled fractions and the average density of said taxon of said microorganism in the corresponding plurality of unlabeled fractions;
- wherein said difference in average density is indicative of the quantity of said substrate taken up by said taxon of said microorganism.
Dependencies are inferred from the claim text.