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Analysis of Bacterial Assemblage Genetic Diversity in Environmental Samples Using the DCode System

, MO.) was added to the samples followed by a 30 minute incubation at 37 C. Next, 10 ml of lysis buffer (0.1 M NaCl and 0.5 M Tris-HCl) was added and samples were subjected to three cycles of rapid freezing and thawing, in a -70 C ethanol bath and a 70 C water bath. DNA was extracted once with Tris-saturated phenol, once with phenol: chloroform: isoamylalcohol (25:24:1), then once with chloroform: isoamylalcohol (24:1) (Sigma Chemical, St. Louis, MO). Each extraction was followed by a 10 minute centrifugation at 6,000 x g. DNA was precipitated overnight in ice-cold isopropanol. The next day, samples were centrifuged at 10,000 x g and pellets were resuspended in 500 l of Tris-EDTA buffer(TE), pH 8.0. Anhydrous ammonium acetate was added and samples were centrifuged for 30 minutes. Supernatant DNA was precipitated with 1.5 volumes of isopropanol for 2 hours. Samples were centrifuged at 10,000 x g for 10 minutes, and pellets were resuspended in 300 l of TE buffer, pH 8.0. DNA extracts were purified using Sephadex G-200 (Sigma Chemical, St. Louis, MO) spun columns.3

To obtain bacterial 16S rDNA, hot start PCR reactions were performed using a Bacteria specific and a universal primer. The sequence for the forward primer, termed 68F, was 5-TNANACATGCAAGTCGAKCG-3 and it aligned with nucleotides 6585 of E. coli.4 The sequence for the reverse primer, termed 1392R, was 5-ACGGGCGGTGTGTRC-3 and it aligned with nucleotides 14081423 of E. coli4; where N=adenine or thymidine, K=guanine or thymidine, R=adenine or guanine. Reaction mixtures were prepared in thin walled microcentrifuge tubes (Fisher Scientific, Pittsburgh, PA) and contained 1x Stoffel Fragment buffer (Perkin Elmer, Branchburg
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