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Extracting DNA from whole organism homogenates and the risk of false positives in PCR based diet studies: A case study using spiny lobster larvae

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O'Rorke, R and Jeffs, A.G and Fitzgibbon, QP and Chow, S and Lavery, B (2013) Extracting DNA from whole organism homogenates and the risk of false positives in PCR based diet studies: A case study using spiny lobster larvae. Journal of Experimental Marine Biology and Ecology, 441. pp. 1-6. ISSN 0022-0981

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Abstract

Better understanding the diet of small marine predators such as the planktonic larvae of spiny lobsters is important
for our awareness of interactions within marine assemblages and for species commercialisation. In
DNA-based diet studies of small organisms there is a risk that any DNA contaminating the outside of an
organism will be detected and falsely assumed to originate from the gut. Experiments with terrestrial predators
have overcome the problem of exogenous contamination by treating the exterior of the predator with
bleach (sodium hypochlorite). However, the use of bleach is a risky strategy when treating either a rare predator
or aquatic predators, which are generally more permeable than terrestrial animals. Many plankton studies
have not reported how they dealt with exogenous contamination, or do not use a control during PCR to
detect false positives due to exogenous contamination. One approach is to wash the predator with MilliQ filtered
water or ethanol and to use the final wash as a PCR template to detect residual DNA. In the present
study we report that washing has variable success at removing exogenous contaminants and that using the
final wash as a control for exogenous contamination consistently fails. Based on our results we recommend
using DNA extracted from a swab of the exterior of the predator as a control for exogenous contamination. We
also report on the benefit of using a novel syringe technique to obtain gut content that minimises contact with
the predator surface, and therefore the risk of exogenous contamination.

Item Type: Article
Keywords: Contamination, Diet, Larvae, PCR, Phyllosoma, Plankton
Journal or Publication Title: Journal of Experimental Marine Biology and Ecology
Page Range: pp. 1-6
ISSN: 0022-0981
Identification Number - DOI: 10.1016/j.jembe.2013.01.003
Additional Information:

Copyright 2013 Elsevier

Date Deposited: 25 Nov 2013 02:51
Last Modified: 18 Nov 2014 04:56
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