University Of Tasmania

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

posted on 2023-05-27, 07:46 authored by Brasher, Darren John
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 ‚ÄövÑvÆ hepatopancreas, oocytes, gill, heart ‚ÄövÑvÆ 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 (‚ÄövÑvÆ 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.


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

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