Biotechnology and Bioinformatics (1+1)

Isolation of genomic DNA from fish tissues

1. Isolation of genomic DNA from fish tissues

In principle the isolation of nucleic acids from any prokaryotic or eukaryotic cells (DNA,RNA and plasmids) two major steps are followed viz, cell disruption and separation of nucleic acids from the associated proteins. In step one, the cells are broken and the nucleic acids are dissociated from other cellular components. This can be done by osmotic shock, freezing-thawing and ultrasonic sound treatment. Quite often surface-active agents such as Sodium Dodecyl sulfate (SDS) and lysozyme are used to disrupt cells. In the second step the cellular debris are removed by centrifugation. The associated proteins are removed from the nucleic acids either by high salt or phenol extraction.

The phenol extraction gives fairly undegraded DNA. In this method, chromatin proteins are dissociated by treatment with SDS. The proteins are denatured by phenol-chloroform treatment. The denatured proteins become insoluble and can be separated by centrifugation.

Isoamyl alcohol reduces foaming during the extraction and facilitates the separation of the aqueous and organic phases. The aqueous phase will contain DNA which can be precipitated with ethanol. Since DNA is a high molecular weight polymer, care should be taken not to break or shear the DNA. Pipetting through narrow bore pipettes should be avoided. Tissues chosen for DNA extraction can be frozen in 100% ethanol and kept at -20 ° C. The tissues can be stored even in room temperature but the DNA may get degraded.