Comparative analyses of amplicon migration behavior in differing denaturing gradient gel electrophoresis (DGGE) systems

Thornhill, D. J., Kemp, D. W., Sampayo, E. M. and Schmidt, G. W. (2010) Comparative analyses of amplicon migration behavior in differing denaturing gradient gel electrophoresis (DGGE) systems. Coral Reefs, 29 1: 83-91. doi:10.1007/s00338-009-0550-4


Author Thornhill, D. J.
Kemp, D. W.
Sampayo, E. M.
Schmidt, G. W.
Title Comparative analyses of amplicon migration behavior in differing denaturing gradient gel electrophoresis (DGGE) systems
Journal name Coral Reefs   Check publisher's open access policy
ISSN 0722-4028
1432-0975
Publication date 2010-03
Year available 2009
Sub-type Article (original research)
DOI 10.1007/s00338-009-0550-4
Volume 29
Issue 1
Start page 83
End page 91
Total pages 9
Place of publication Heidelberg, Germany
Publisher Springer
Collection year 2011
Language eng
Subject C1
970106 Expanding Knowledge in the Biological Sciences
0601 Biochemistry and Cell Biology
060503 Microbial Genetics
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. © Springer
Keyword Co-migration
DGGE optimization
ITS2 rDNA
Microbial diversity
Symbiodinium
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ
Additional Notes Published online: 9 October 2009

Document type: Journal Article
Sub-type: Article (original research)
Collections: 2010 Higher Education Research Data Collection
School of Biological Sciences Publications
Centre for Marine Studies Publications
 
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Citation counts: TR Web of Science Citation Count  Cited 10 times in Thomson Reuters Web of Science Article | Citations
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Created: Sun, 21 Feb 2010, 00:09:37 EST