Coral Reefs

, Volume 29, Issue 1, pp 83–91

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

  • D. J. Thornhill
  • D. W. Kemp
  • E. M. Sampayo
  • G. W. Schmidt
Report

DOI: 10.1007/s00338-009-0550-4

Cite this article as:
Thornhill, D.J., Kemp, D.W., Sampayo, E.M. et al. Coral Reefs (2010) 29: 83. doi:10.1007/s00338-009-0550-4

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.

Keywords

Co-migration DGGE optimization ITS2 rDNA Microbial diversity Symbiodinium 

Supplementary material

338_2009_550_MOESM1_ESM.tif (2.3 mb)
DNA sequence alignments of ITS2 sequences recovered from cloned rDNAs from cultured isolates of Symbiodinium subclades and employed for subsequent DGGE analyses. Data presented include clones from Symbiodinium ITS2 types A3 (a), B1 (b), C1 (c), D1a (d), and E2 (e). The nucleotide sequences listed include the only internal regions amplified by the ITS2for/ITS2clamp primers (LaJeunesse and Trench 2000). The most frequently recovered and dominant ITS2 rDNA copy (A3.1003 [a], B1.1068 [b], C1.11280 [c], D1a.1020 [d], and E2.2070 [e]; Thornhill et al. 2007) are used as the standards against which rare polymorphic sequences of other cloned amplicons are compared. A nucleotide deviation (A, C, G, or T) in place of a dot (.) denotes base pair substitutions. Dashes indicate the ITS2 sequence of clone C1.1126 where the variant of the gene region has undergone a 9 base pair deletion. Supplementary material 1 (TIFF 2357 kb)
338_2009_550_MOESM2_ESM.tif (869 kb)
Supplemental Figure 2: PCR-DGGE profiles of GC-clamped amplicons of variant ITS2 rDNA sequences cloned from five Symbiodinium cultures (one clone per culture) on two independent gels (a, b). Samples were electrophoresed under identical conditions including 45-80% denaturing gradient gels for 10 h at 150V on a C.B.S. Scientific DGGE 2001 model system. Note the similarity in relative amplicon position between the two independent gels. Gel profiles presented as reverse images and images are presented at the same scale. Supplementary material 2 (TIFF 868 kb)
338_2009_550_MOESM3_ESM.tif (2.3 mb)
PCR-DGGE profiles of variant ITS2 rDNA sequences from five clonal Symbiodinium cultures (three clones per culture) under intermediate combinations of the gradient and electrophoretic conditions presented in Figure 1, on a CBS Scientific DGGE 2001 model system apparatus. (a) Profile on a 45-80% denaturing gradient, electrophoresed for 5h at 130V. Note that only clones which denatured at a low melting temperature (high gel position; i.e., clones from ITS2 types A3 and C1) appeared as distinct, albeit poorly separated, bands in the gel profile, suggesting insufficient electrophoresis time for most clones to sufficiently migrate and denature. (b) Profile on a 25-55% denaturing gradient, electrophoresed for 10h at 150V. Note that all clones appeared as smeared profiles regardless of electropheretic time, suggesting that denaturing conditions had not been reached even after 1500Vh of eletrophoresis. Gel profiles presented as reverse images. Supplementary material 3 (TIFF 2396 kb)

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • D. J. Thornhill
    • 1
  • D. W. Kemp
    • 2
  • E. M. Sampayo
    • 3
    • 4
  • G. W. Schmidt
    • 5
  1. 1.Department of BiologyBowdoin CollegeBrunswickUSA
  2. 2.Odum School of EcologyUniversity of GeorgiaGeorgiaUSA
  3. 3.Centre for Marine StudiesUniversity of QueenslandSt. LuciaAustralia
  4. 4.Department of BiologyPennsylvania State UniversityUniversity ParkUSA
  5. 5.Department of Plant BiologyUniversity of GeorgiaGAUSA