Internal transcribed spacer region

The Internal transcribed spacer region represents a nucleotide sequence of the 5.8S rRNA gene and the adjacent ITS (Internal Transcribed Spacer) in the chromosome.

The Internal transcribed spacer region and the surrounding ITS are used in eukaryotes to study phylogenetics. For example, the sequence analysis of this region is also used for the identification of the genus and species of fungi.

By comparing the ITS sequence determined and the 5.8S rRNA gene sequence of a fungal strain to be identified, sequence similarities are determined which serve as a basis for assignment to a taxonomic group.

The nucleotide sequence of the ITS region is about 500-600 base pairs. In addition to highly conserved regions where the sequence is identical for all fungi, there are also variable regions. These variable regions can be characteristic as signatures for a species, genus or group of fungi. Thus, the analysis of these partial sequences allows a taxonomic assignment of fungal strains.

After extraction of the genomic DNA from a pure culture of the fungus under investigation, the sequence of the 5.8S rRNA gene and the adjacent ITS genome region is amplified with the primer pair ITS-1/ ITS-4 by PCR. After purification of the PCR product, it is sequenced with the specific PCR primers in the further course of the investigation. The genomic DNA is isolated from a freshly grown pure culture of the fungal sample to be examined. Commercial kits are suitable for the extraction and purification of genomic DNA from fungi.

Evaluation of the sequencing: For further evaluation, the edited DNA sequences are analysed using a reference database (e.g. the NCBI database).

1. White TJ et al (1990) Analysis of phylogenetic relationships by amplificationand direct sequencing of ribosomal RNA genes. PCR protocols: A Guide to Methods and Applications, pp. 315-322, New York, Academic Press.
2. National Center for Biotechnology Information. Basic Local Alignment Search Tool (blast) http://www.ncbi.nlm.nih.gov