Comparative analyses of amplicon migration behavior in differing denaturing gradient gel electrophoresis (DGGE) systems |
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Authors: | D J Thornhill D W Kemp E M Sampayo G W Schmidt |
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Institution: | (1) Department of Biology, Bowdoin College, 6500 College Station, Brunswick, ME 04011, USA;(2) Odum School of Ecology, University of Georgia, Athens Georgia, 30602, USA;(3) Centre for Marine Studies, University of Queensland, St. Lucia, QLD, 4072, Australia;(4) Department of Biology, Pennsylvania State University, University Park, PA 16802, USA;(5) Department of Plant Biology, University of Georgia, Athens GA, 30602, USA |
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Abstract: | Denaturing gradient gel electrophoresis (DGGE) is commonly utilized to identify and quantify microbial diversity, but the
conditions required for different electrophoretic systems to yield equivalent results and optimal resolution have not been
assessed. Herein, the influence of different DGGE system configuration parameters on microbial diversity estimates was tested
using Symbiodinium, a group of marine eukaryotic microbes that are important constituents of coral reef ecosystems. To accomplish this, bacterial
clone libraries were constructed and sequenced from cultured isolates of Symbiodinium for the ribosomal DNA internal transcribed spacer 2 (ITS2) region. From these, 15 clones were subjected to PCR with a GC
clamped primer set for DGGE analyses. Migration behaviors of the resulting amplicons were analyzed using a range of conditions,
including variation in the composition of the denaturing gradient, electrophoresis time, and applied voltage. All tests were
conducted in parallel on two commercial DGGE systems, a C.B.S. Scientific DGGE-2001, and the Bio-Rad DCode system. In this
context, identical nucleotide fragments exhibited differing migration behaviors depending on the model of apparatus utilized,
with fragments denaturing at a lower gradient concentration and applied voltage on the Bio-Rad DCode system than on the C.B.S.
Scientific DGGE-2001 system. Although equivalent PCR–DGGE profiles could be achieved with both brands of DGGE system, the
composition of the denaturing gradient and application of electrophoresis time × voltage must be appropriately optimized to
achieve congruent results across platforms. |
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