The utility of molecular genetic analysis of museum specimens in studying deep-sea fish

Kazic, Amra (2008). The utility of molecular genetic analysis of museum specimens in studying deep-sea fish. PhD thesis The Open University.



The damaging effect of formalin on DNA and the inhibition of PCR are serious problems in molecular studies. The aims of the project were to investigate the possibility of using formalin-fixed, Steedman's preserved museum specimens in molecular investigations, especially organisms with unstudied genomes. A number of DNA extraction protocols and different pre-washing/drying regimes were tested. These gave different levels of success, but a guanidinium-based protocol developed in this study gave the best results. RAPD-PCR methodology was employed to test its applicability on preserved specimens, and it was used as a test of the efficiency of DNA extraction/amplifications and for developing species-specific PCR primers. Attempts to amplify mitochondrial DNA sequences with the six mitochondrial genes were mostly unsuccessful. Sporadic amplifications were obtained with primers of 16S and COIII genes.

This study provided the first molecular data on deep-sea fish (Nezumia aequalis and N. micronychodon) exclusively using formalin-fixed, Steedman's preserved museum specimens. Two genomic sequences of these fishes were determined and submitted to the GenBank database under accession numbers AY826774 - AY826792. Three specific primer sets (RAPD-derived) for Nezumia aequalis and N. micronychodon were designed to amplify PCR product sizes 300 bp - 350 bp. This study has demonstrated that an appropriate strategy and molecular approach could lead to the successful use of museum and other formalin-fixed archival collections even on organisms with unstudied genomes.

Supplementary evidence, related to the method of preservation, the usage of particular DNA extraction protocol and PCR marker system, was obtained from ten differently preserved mackerel (Scomber scombrus) specimens.

This study confirmed that the DNA extracted from preserved specimens possesses unique characteristics that make molecular investigations very difficult. Because of this, it is proposed that DNA extracted from preserved specimens should be referred to as "archival DNA (arDNA)".

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