SS2.05 Phylogenetic and Physiologic Successions in Aquatic Bacterial Communities
Date: Monday, June 10, 2002
Time: 3:45:00 PM
Location: Carson C
 
Devol, A, H, University of Washington, Seattle, USA, devol@u.washington.edu
Van MooyB, University of Washington, Seattle, USA, bvm@ocean.washington.edu
Keil, R, G, University of Washington, Seattle, USA, rickkeil@u.washington.edu
 
THYMIDINE INCORPORTATION BY SPECIFIC BACTERIOPLANTKON IN PUGET SOUND, WASHINGTON: A QUANTITATIVE, PHYLOGENTICALLY-BASED METHOD TO SEPARATE DNA
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A novel molecular method to quantitatively capture specific, targeted DNA from a solution of bulk community DNA was developed and this method has been optimized for the analysis of 3H-labled DNA recovered from thymidine incorporation experiments. Fundamentally this technique involves labeling the targeted DNA with biotin, so that it may be separated from the bulk mixture of DNA by affinity binding to streptavidin. DNA polymerase, along with specific primers and biotin-labeled nucleotides, are used to label the targeted gene (16S rRNA) multiple times. The yield of 3H in DNA is determined by normalizing to the recovery of 33P-labelled internal DNA standards added at the beginning of the procedure. These DNA standards are synthesized such that the 33P radioactivity of standards is distributed in the same DNA size ranges as the 3H radioactivity in the sample DNA. This technique was successfully developed and tested the laboratory using synthetic mixtures of 3H-labeled DNA from organisms in pure culture. Field measurements were made in Carr Inlet, Washington and it was determined that 38 +/- 6% of the total 3H-thymidine incorporation was by alpha- and gamma-proteobacteria.