科研之友
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摘要
Genomic DNA isolation is fundamental step to study genetic diversity, phylogeny of species and cloning of desired genes. The preferable protocol needs to be efficient, less time consuming, economic and non-destructive particularly for the endangered species. The current study was aimed at economic isolation of considerable quantity of DNA manually from a particular tissue of the fish without any potential damage to the animal. For this purpose fin tissue was preferably selected over scale due to its universal occurrence in fish. A total of 180 samples of fin tissues belonging to twenty nine species of fresh water fish preserved in different preservatives, were used for DNA isolation. DNA could not be isolated from the fin tissues preserved in formalin, alcohol, air dried and ice preserved fins more than one week by any of two previously described methods including salt extraction and urea treatment. However, introducing some modifications in salt extraction method resulted in large quantity of high quality DNA from those fin tissues that were isolated from the living animal and immediately preserved in absolute ethanol. The modifications include use of low quantity of proteinase K and slight increase in incubation period due to hard nature of fin tissue. The extracted DNA by the modified salt extraction method was very pure, of high quality and resulted in successful PCR amplification by Random Amplified Polymorphic DNA primer and cytochrome oxidase subunit 1 gene specific primers. This modified procedure results in highest yield of isolated DNA reported so far. The method is particularly suited for DNA isolation from endangered and sparse species as it requires minute quantity of fin tissue, without any detrimental effect on fish.
- 出版日期2016-11
