We present a simple strategy for isolating and accurately enumerating target DNA from high-clay-content soils: desorption with buffers, an optional magnetic capture hybridization step, and quantitation via real-time PCR. With the developed technique, g quantities of DNA were extracted from mg samples of pure kaolinite and a field clay soil.Isolating and characterizing DNA sequences for use in molecular methods are integral to evaluating microbial community diversity in soil (6,21,22,24,37). Any isolation protocol should maximize nucleic acid isolation while minimizing copurification of enzymatic inhibitors. Although several methods that focus on extraction of total community DNA from environmental soil and water samples have been published (7,21,26,34), the lack of a standard nucleic acid isolation protocol (32) reflects the difficulty in accomplishing these goals, most likely due to the complex nature of the soil environment.DNA extraction is especially difficult for soils containing clay (3, 5), given the tight binding of DNA strands to clay soil particles (7,10,20). Additionally, extracellular DNA binds to and is copurified with soil humic substances (10), which inhibit the activity of enzymes such as restriction endonucleases and DNA polymerase (6, 13, 23). Although clay-bound DNA can be PCR amplified in the absence of inhibitors (1), it is often the case that inhibitors are present in the soil environment, among them bilirubin, bile salts, urobilinogens, and polysaccharides (40). Of these inhibitors, humic substances have been found to be the most recalcitrant (36).A promising technique for isolating specific target sequences from soil particles and enzymatic inhibitors is the magnetic capture hybridization-PCR technique (MCH-PCR) presented by Jacobsen (19) and used to obtain high detection sensitivities (11, 38).We have found no evidence in the published literature of the use of MCH-PCR on soils that have high clay contents and here present a three-step strategy for isolating specific DNA sequences from the most difficult soil environment-clay that contains humic substances-and enumerating a specific target sequence from the crude extract.Kaolinite clay: DNA binding. To determine whether kaolinite would be a suitable clay type for use in optimizing DNA desorption protocols, the nature of DNA binding to pure kaolinite (KGa-1b) (4) (Clay Minerals Society; Chantilly, VA) was determined from binding isotherms. The DNA used was a 1,040-bp region of the gfp gene (GC content, ϳ38%) PCR amplified from pGFPmut3.1 (3, 9, 14) by using primers specific for this gene (5Ј-ACTGGAAAGCGGGCAGTG and 5Ј-AAA CGCGCGAGACGAAAGGG). Kaolinite was sterilized by gamma irradiation by exposure to Cobalt-60 until 70 kGy was delivered (Radiation Science and Engineering Center, Penn State University, University Park, PA), a dose sufficient to kill all living microbes (25) and to directly and indirectly damage DNA (18,30).By use of the PCR-generated gfp DNA fragment, adsorption data were fit to the Langmuir (33) and Freundlich (29) models ...