Intragenomic Profiling Using Multicopy Genes : The rDNA
Internal Transcribed Spacer Sequences of the Freshwater
Sponge Ephydatia fluviatilis
Liisi Karlep , To˜nu Reintamm, Merike Kelve*
Department of Gene Technology , Tallinn University of Technology, Tallinn, Estonia
Abstract
Multicopy genes, like ribosomal RNA genes (rDNA), are widely used to describe and distinguish individuals. Despite
concerted evolution that homogenizes a large number of rDNA gene copies, the presence of different gene variants within
a genome has been reported. Characterization of an organism by defining every single variant of tens to thousands of rDNA
repeat units present in a eukaryotic genome would be quite unreasonable. Here we provide an alternative approach for the
characterization of a set of internal transcribed spacer sequences found within every rDNA repeat unit by implementing
direct sequencing methodology. The prominent allelic variants and their relative amounts characterizing an individual can
be described by a single sequencing electropherogram of the mixed amplicon containing the variants present within the
genome. We propose a method for rational analysis of heterogeneity of multicopy genes by compiling a profile based on
quantification of different sequence variants of the internal transcribed spacers of the freshwater sponge Ephydatia fluviatilis
as an example. In addition to using conventional substitution analysis, we have developed a mathematical method, the
proportion model method, to quantify the relative amounts of allelic variants of different length using data from direct
sequencing of the heterogeneous amplicon. This method is based on determining the expected signal intensity values
(corresponding to peak heights from the sequencing electropherogram) by sequencing clones from the same or highly
similar amplicon and comparing hypothesized combinations against the values obtained by direct sequencing of the
heterogeneous amplicon. This method allowed to differentiate between all specimens analysed.
Citation : Karlep L, Reintamm T, Kelve M (2013) Intragenomic Profiling Using Multicopy Genes: The rDNA Internal Transcribed Spacer Sequences of the Freshwater
Sponge Ephydatia fluviatilis. PLoS ONE 8(6): e66601. doi:10.1371/ journal .pone.0066601
Editor : Mary Bryk, Texas A&M University, United States of America
Received January 24, 2013; Accepted May 7, 2013; Published June 18, 2013
Copyright : ß 2013 Karlep et al. This is an open - access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium , provided the original author and source are credited.
Funding: This work was supported by the Estonian Ministry of Education and Research (Grant No. 0140108) and the Estonian Science Foundation (Grant
No. 9185) (www. etis .ee). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist .
* E-mail: [email protected]
Introduction
(based on the peak heights of different nucleotides in the same
position on the electropherogram) have been focused on analysing
Ribosomal RNA genes (rDNA) have been widely used in
alleles that differ by nucleotide substitutions [5,6]. Whereas, if the
taxonomy, biogeographic and phylogenetic analyses. A eukaryotic
gene variants differ due to insertion or deletion events , and
genome has tens to thousands of rDNA copies, containing genes
substitutions may be absent, a different strategy is needed to
for 18S, 5.8S, and 28S rRNAs. Between these genes, on either side
adequately analyse the heterogeneity of the gene pool. We hereby
of the 5.8S rRNA gene, the internal transcribed sequences (ITS),
describe a method that also allows for the quantification of the
ITS1 and ITS2, are located [1]. In order to preserve the
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