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Revealing genetic diversity of eukaryotic microorganisms in aquatic environments by denaturing gradient gel electrophoresis

By E.J. van Hannen, M.P. van Agterveld, H.J. Gons and H.J. Laanbroek

Abstract

A new Eucarya-specific 18S rDNA primer set was constructed\ud and tested using denaturing gradient gel electrophoresis\ud to analyze the genetic diversity of eukaryotic microorganisms\ud in aquatic environments. All eukaryal lines\ud of descent exhibited four or fewer nucleotide mismatches in\ud the forward primer sequence, except for the Microspora line\ud of descent. The reverse primer annealed to a more conserved\ud region with fewer than two nucleotide mismatches. Genomic\ud DNA from test organisms with different numbers of\ud nucleotide mismatches were amplified to test primer specificity.\ud Relatively low annealing temperatures allowed the\ud amplification of sequences with up to four nucleotide mismatches\ud while still maintaining specificity for the eukaryal\ud domain. Denaturing gradient gel electrophoresis was used\ud to separate similarly sized PCR products of environmental\ud samples, and the obtained banding patterns were converted\ud to a binary format for statistical comparisons. Cluster analysis\ud of these patterns showed similar results to a cluster\ud analysis based on environmental variables. This approach\ud provides an analytical tool to study the population structure\ud and molecular ecology of eukaryotic microbial communities\ud inhabiting aquatic environments

Topics: Biologie, 18S rRNA, aquatic environments, community structure, DGGE, Eucarya, genetic diversity, nucleotide mismatches, Southern blot hybridization
Year: 1998
OAI identifier: oai:dspace.library.uu.nl:1874/25594
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