Abstract
BackgroundCultures of heterotrophic protists often require co-culturing with bacteria to act as a source of nutrition. Such cultures will contain varying levels of intrinsic bacterial contamination that can interfere with molecular research and cause problems with the collection of sufficient material for sequencing. Measuring the levels of bacterial contamination for the purposes of molecular biology research is non-trivial, and can be complicated by the presence of a diverse bacterial flora, or by differences in the relative nucleic acid yield per bacterial or eukaryotic cell.Principal FindingsHere we describe a duplex PCR-based assay that can be used to measure the levels of contamination from marine bacteria in a culture of loricate choanoflagellates. By comparison to a standard culture of known target sequence content, the assay can be used to quantify the relative proportions of bacterial and choanoflagellate material in DNA or RNA samples extracted from a culture. We apply the assay to compare methods of purifying choanoflagellate cultures prior to DNA extraction, to determine their effectiveness in reducing bacterial contamination. Together with measurements of the total nucleic acid concentration, the assay can then be used as the basis for determining the absolute amounts of choanoflagellate DNA or RNA present in a sample.ConclusionsThe assay protocol we describe here is a simple and relatively inexpensive method of measuring contamination levels in nucleic acid samples. This provides a new way to establish quantification and purification protocols for molecular biology and genomics in novel heterotrophic protist species. Guidelines are provided to develop a similar protocol for use with any protistan culture. This assay method is recommended where qPCR equipment is unavailable, where qPCR is not viable because of the nature of the bacterial contamination or starting material, or where prior sequence information is insufficient to develop qPCR protocols.
Highlights
A major obstacle to molecular research on free-living, heterotrophic protists is their mode of nutrition
Guidelines are provided to develop a similar protocol for use with any protistan culture
The principle of the assay is that, at saturation, the relative amount of each of the two amplified products generated in the duplex polymerase chain reaction (PCR) will depend on the relative concentrations of the two rRNA target sequences in the template, and this provides a measure of the relative amounts of eukaryotic and prokaryotic material in the original nucleic acid sample
Summary
A major obstacle to molecular research on free-living, heterotrophic protists is their mode of nutrition. A possible solution to this problem is to create a monoxenic culture containing the protistan species plus one unique strain of prey bacteria, and to separate DNA obtained on the basis of GC content [3]. Developing such methods is a lengthy and difficult process. Cultures of heterotrophic protists often require co-culturing with bacteria to act as a source of nutrition Such cultures will contain varying levels of intrinsic bacterial contamination that can interfere with molecular research and cause problems with the collection of sufficient material for sequencing. Measuring the levels of bacterial contamination for the purposes of molecular biology research is non-trivial, and can be complicated by the presence of a diverse bacterial flora, or by differences in the relative nucleic acid yield per bacterial or eukaryotic cell
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