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Methods for restriction enzyme analysis of mitochondrial DNA from adult and puerulus rock lobster, Jasus edwardsii (Crustacea:Decapoda:Palinuridae).


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Brasher, Darren John 1992 , 'Methods for restriction enzyme analysis of mitochondrial DNA from adult and puerulus rock lobster, Jasus edwardsii (Crustacea:Decapoda:Palinuridae).', Unspecified thesis, University of Tasmania.

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A relatively inexpensive and rapid technique is described for the isolation and
restriction enzyme analysis of mitochondrial DNA (mtDNA) from adult rock lobster
belonging to the genus Jasus. Using this technique, easily detectable and
reproducible fragment patterns were obtained from mtDNA extracted from antenna!
glands. Mitochondrial DNA from four other tissues — hepatopancreas, oocytes, gill,
heart — was recovered in low yields or contaminated with nuclear DNA. From an
initial screening process involving 29 restriction enzymes, only five generated
interpretable fragment patterns from antennal gland mtDNA following
electrophoresis and autoradiography. Re-screening unsuccessful enzymes with four
enzyme enhancers (magnesium chloride, Triton X-100, bovine serum albumin
[BSA], spennidine) provided 11 additional useful enzymes. From these 16
enzymes, the six most informative were chosen and their activity and reliability
further optimized with systematically varied combinations and concentrations of
BSA and spermidine.
Preliminary experiments showed that the standard technique employed to extract
and analyze the mtDNA of adult rock lobster could not be applied to the puerulus
stage. An alternative technique, in situ gel hybridization, was used for the detection
of mtDNA restriction fragments from a puerulus. Due to a low degree of sequence
homology, puerulus mtDNA restriction fragments could not be detected using
probe synthesized from cloned honeybee mtDNA. Probe synthesized from gel
purified adult mtDNA enabled partial genetic characterization of a puerulus larva.
This puerulus was identified as having three restriction fragments in common with
an adult specimen. Diffusion of small fragments from the gel meant that a small
portion of the pueulus mtDNA (— 10%) could not be characterized. However, by
selecting restriction enzymes that produce large fragments from variable regions of
the genome, informative genetic markers could be routinely analyzed.

Item Type: Thesis - Unspecified
Authors/Creators:Brasher, Darren John
Copyright Holders: The Author
Additional Information:

Dept. of Zoology M.Sc. Qual. Thesis, 1992

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