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

Rapid decline of PCR amplification from genomic extracts of DESS-preserved, slide-mounted nematodes AbstractMany studies use integrative methods to study both morphology and genesequences of nematode species, yet there is little evidence to indicate theoptimum criteria for merging taxonomic and molecular protocols. Preliminaryevidence suggests that standard methods of desiccation and slide mountingnematode specimens in glycerin can sporadically result in degradation ofgenomic DNA. A time series experiment was constructed in order to assesswhether this degradation of genomic DNA could be recorded and quantified.Two groups of nematode specimens were desiccated, mounted on slides, andstored 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 the18S rRNA gene at each time point. The resulting gel photographs were used torecord the outcome of PCR and quantify the strength of amplification ifsuccessful. Results from slide-mounted specimens were compared to PCRproducts derived from unmounted nematodes extracted directly from thepreservative solution at specified time intervals. The desiccation and slidemounting process appears to reduce overall band intensity after 1 day inslide mounts. Unmounted specimens consistently exhibit high success rates ofPCR and a high overall DNA content per band at all time points, whereasslide-mounted nematodes show a decrease in the number of successful PCRs andweakening band intensity as time progresses. Results clearly indicate asteady degradation of genomic DNA in nematodes stored in slide mounts formore than 2 weeks, whereas unmounted specimens extracted from preservativesolution showed no decline in PCR success or quality. We suggest a maximumstorage period of 2 weeks on slides if mounted nematodes are to be used formolecular analyses. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nematology Brill

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

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Publisher
Brill
Copyright
Copyright © Koninklijke Brill NV, Leiden, The Netherlands
ISSN
1388-5545
eISSN
1568-5411
DOI
10.1163/156854109X422922
Publisher site
See Article on Publisher Site

Abstract

AbstractMany studies use integrative methods to study both morphology and genesequences of nematode species, yet there is little evidence to indicate theoptimum criteria for merging taxonomic and molecular protocols. Preliminaryevidence suggests that standard methods of desiccation and slide mountingnematode specimens in glycerin can sporadically result in degradation ofgenomic DNA. A time series experiment was constructed in order to assesswhether this degradation of genomic DNA could be recorded and quantified.Two groups of nematode specimens were desiccated, mounted on slides, andstored 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 the18S rRNA gene at each time point. The resulting gel photographs were used torecord the outcome of PCR and quantify the strength of amplification ifsuccessful. Results from slide-mounted specimens were compared to PCRproducts derived from unmounted nematodes extracted directly from thepreservative solution at specified time intervals. The desiccation and slidemounting process appears to reduce overall band intensity after 1 day inslide mounts. Unmounted specimens consistently exhibit high success rates ofPCR and a high overall DNA content per band at all time points, whereasslide-mounted nematodes show a decrease in the number of successful PCRs andweakening band intensity as time progresses. Results clearly indicate asteady degradation of genomic DNA in nematodes stored in slide mounts formore than 2 weeks, whereas unmounted specimens extracted from preservativesolution showed no decline in PCR success or quality. We suggest a maximumstorage period of 2 weeks on slides if mounted nematodes are to be used formolecular analyses.

Journal

NematologyBrill

Published: Jan 1, 2009

Keywords: INTEGRATED METHODS; MOLECULAR; GLYCERIN; DNA; MORPHOLOGY; DESS

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