Rapid decline of PCR amplification from genomic extracts of DESS-preserved, slide-mounted nematodes

Abstract

Summary -Many studies use integrative methods to study both morphology and gene sequences of nematode species, yet there is little evidence to indicate the optimum criteria for merging taxonomic and molecular protocols. Preliminary evidence suggests that standard methods of desiccation and slide mounting nematode specimens in glycerin can sporadically result in degradation of genomic DNA. A time series experiment was constructed in order to assess whether this degradation of genomic DNA could be recorded and quantified. Two groups of nematode specimens were desiccated, mounted on slides, and stored at either 4 • C or at room temperature for specified time intervals. Genomic DNA was extracted and PCR was conducted to amplify a section of the 18S rRNA gene at each time point. The resulting gel photographs were used to record the outcome of PCR and quantify the strength of amplification if successful. Results from slide-mounted specimens were compared to PCR products derived from unmounted nematodes extracted directly from the preservative solution at specified time intervals. The desiccation and slide mounting process appears to reduce overall band intensity after 1 day in slide mounts. Unmounted specimens consistently exhibit high success rates of PCR and a high overall DNA content per band at all time points, whereas slide-mounted nematodes show a decrease in the number of successful PCRs and weakening band intensity as time progresses. Results clearly indicate a steady degradation of genomic DNA in nematodes stored in slide mounts for more than 2 weeks, whereas unmounted specimens extracted from preservative solution showed no decline in PCR success or quality. We suggest a maximum storage period of 2 weeks on slides if mounted nematodes are to be used for molecular analyses.Keywords -DESS, DNA, glycerin, integrated methods, molecular, morphology.The use of molecular data in nematode studies is now ubiquitous, yet it is still routine for researchers to corroborate DNA sequences with morphology in order to assess the biological relevance of molecular patterns (Griffiths et al., 2006;Stock & Nadler, 2006;Bhadury et al., 2008). DNA barcoding studies promote visual identification of specimens as well as the retention of 'morphological voucher images' to record taxonomic features before nematodes are destroyed for DNA extraction (De Ley et al., 2005;Bhadury et al., 2006a). These barcoding studies have also provided empirical evidence concerning the resolution of different genetic loci used in species identification, with the nuclear 18S ribosomal subunit gene currently recommended as the ideal barcoding locus for nematodes (Bhadury et al., 2006a).Despite the frequent integration of morphological and molecular protocols in nematode studies, there is little * Corresponding author, e-mail: h.bik@nhm.ac.uk published information to suggest how these two disparate approaches might best be optimised to obtain the most robust data. Taxonomic protocols are designed to maximise the clarity of specimens viewed under a microsco...

Three in one: fixing marine nematodes for ecological, molecular, and morphological studies

“…The sample should not be stained with rose bengal; if staining is necessary, then part of the sample should be kept unstained. Given the fast denaturation of DNA in glycerin slides (Bik et al 2009), it is suggested that part of the fauna should be kept in the fixative for identification and subsequent DNA extraction procedures. …”

Molecular Diversity of Fungal Phylotypes Co-Amplified Alongside Nematodes from Coastal and Deep-Sea Marine Environments

“…For deep-sea nematodes, fungal sequences were most often obtained after specimens had been stored in slide mounts for long periods of time. Subsequent methodological investigations revealed that the slide mounting process can effectively degrade nematode DNA during storage [27] , allowing instead for fungal amplicons to dominate PCR reactions. Generally, if nematode DNA is well-preserved when extracted, the signal from any fungal PCR amplicons is silenced by the overwhelming majority fraction of nematode PCR amplicons during protocols for direct sequencing.…”

Assessing DESS solution for the long-term preservation of nematodes from faecal samples

“…In order to corroborate that DNA remained viable in the samples stored for 2 years in DESS (as reported by Bik et al (2009) for unmounted nematodes stored for 6 months), one specimen was randomly selected to be analysed. Genomic DNA was firstly extracted from faecal samples by the NZY Tissue gDNA Isolation kit (NZYTech).…”

“…Refrigerating or freezing samples that are combined or not with preserver solutions is also recommended for preserving samples, but is sometimes impractical under non-laboratory conditions (Kozlova et al, 2020). Other promising options are to use saturated solutions composed of dimethyl sulphoxide, disodium EDTA and saturated NaCl (hereinafter DESS), which have been successfully tested to preserve adult nematodes and their DNA for at least 1 year (Bik et al, 2009;Chałańska et al, 2016;Yoder et al, 2006). Therefore, the development and assessment of feasible preservation protocols should be promoted.…”