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Optimizing methods for PCR-based analysis of predation.
- Source :
-
Molecular ecology resources [Mol Ecol Resour] 2011 Sep; Vol. 11 (5), pp. 795-801. Date of Electronic Publication: 2011 Apr 20. - Publication Year :
- 2011
-
Abstract
- Molecular methods have become an important tool for studying feeding interactions under natural conditions. Despite their growing importance, many methodological aspects have not yet been evaluated but need to be considered to fully exploit the potential of this approach. Using feeding experiments with high alpine carabid beetles and lycosid spiders, we investigated how PCR annealing temperature affects prey DNA detection success and how post-PCR visualization methods differ in their sensitivity. Moreover, the replicability of prey DNA detection among individual PCR assays was tested using beetles and spiders that had digested their prey for extended times postfeeding. By screening all predators for three differently sized prey DNA fragments (range 116-612 bp), we found that only in the longest PCR product, a marked decrease in prey detection success occurred. Lowering maximum annealing temperatures by 4 °C resulted in significantly increased prey DNA detection rates in both predator taxa. Among the three post-PCR visualization methods, an eightfold difference in sensitivity was observed. Repeated screening of predators increased the total number of samples scoring positive, although the proportion of samples testing positive did not vary significantly between different PCRs. The present findings demonstrate that assay sensitivity, in combination with other methodological factors, plays a crucial role to obtain robust trophic interaction data. Future work employing molecular prey detection should thus consider and minimize the methodologically induced variation that would also allow for better cross-study comparisons.<br /> (© 2011 Blackwell Publishing Ltd.)
- Subjects :
- Animals
Base Sequence
Coleoptera physiology
DNA metabolism
DNA Primers genetics
Digestion genetics
Feeding Behavior physiology
Gryllidae genetics
Molecular Sequence Data
Sensitivity and Specificity
Sequence Analysis, DNA
Spiders physiology
Coleoptera genetics
DNA isolation & purification
Food Chain
Polymerase Chain Reaction methods
Spiders genetics
Temperature
Subjects
Details
- Language :
- English
- ISSN :
- 1755-0998
- Volume :
- 11
- Issue :
- 5
- Database :
- MEDLINE
- Journal :
- Molecular ecology resources
- Publication Type :
- Academic Journal
- Accession number :
- 21507208
- Full Text :
- https://doi.org/10.1111/j.1755-0998.2011.03018.x