Purification and PCR amplification procedures for DNA extracted from environmental samples (soil, compost, and river sediment) were improved by introducing three modifications: precipitation of DNA with 5% polyethylene glycol 8000 (PEG) and 0.6 M NaCl; filtration with a Sepharose 4B-polyvinylpolypyrrolidone (PVPP) spin column; and addition of skim milk (0.3% w/v) to the PCR reaction solution. Humic substances' concentration after precipitation with 5% PEG was 2.57-, 5.3-, and 78.9-fold lower than precipitation with 7.5% PEG, 10% PEG, and isopropanol, respectively. After PEG precipitation, Sepharose, PVPP and the combined (Sepharose-PVPP) column removed 92.3%, 89.5%, and 98%, respectively, of the remaining humic materials. Each of the above-mentioned modifications improved PCR amplification of the 16S rRNA gene. DNA extracted by the proposed protocol is cleaner than DNA extracted by a commercial kit. Nevertheless, the improvement of DNA purification did not improve the detection limit of atrazine degradation gene atzA.

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The following study evaluated the diversity and biogeography of 83 new atrazine-degrading bacteria and the composition of their atrazine degradation genes. These strains were isolated from 13 agricultural soils and grouped according to rep-PCR genomic fingerprinting into 11 major clusters, which showed biogeographic patterns. Three clusters (54 strains) belonged to the genus Arthrobacter, seven clusters (28 strains) were similar to the genus Nocardioides and only one strain was a gram-negative from the genus Ancylobacter. PCR assays for the detection of the genes atzA, B, C, D, E, F and trzN conducted with each of the 83 strains revealed that 82 strains (all gram positive) possessed trzN, 74 of them possessed the combination of trzN, atzB and atzC, while only the gram-negative strain had atzA. A similar PCR assay for the two analogous genes, atzA and trzN, responsible for the first step of atrazine degradation, was performed with DNA extracted directly from the enrichment cultures and microcosms spiked with atrazine. In these assays, the gene trzN was detected in each culture, while atzA was detected in only six out of 13 soils. These results raise an interesting hypothesis on the evolutionary ecology of the two atrazine chlorohydrolase genes (i.e. atzA and trzN) and about the biogeography of atrazine-degrading bacteria.

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