DNA Stable-Isotope Probing (DNA-SIP)

The overall goal of this procedure is to retrieve DNA from active microorganisms that have consumed a growth substrate of interest without the prerequisite of laboratory cultivation. This is accomplished by first incubating an environmental sample with a stable isotope labeled substrate of interest under conditions that are similar to those found in the native environment. The second step of the procedure is to extract total DNA from this isotope labeled sample.

The third step of the procedure is to subject this DNA to density gradient ultracentrifugation in cesium chloride. The final step of the procedure is to fractionate the density gradient to obtain heavy and light DNA for subsequent molecular characterization. Ultimately, results can be obtained that reveal the identity of active yet uncultured microorganisms involved in the metabolism of a particular substrate through a variety of possible molecular techniques, including fingerprinting, microarray, hybridization, clone, library analysis, and metagenomics.