ITS gene amplification and PCR tests were done on 2 fungal samples of unknown identity. The ITS gene amplification results show a difference between these 2 samples (bands did not align) while the PCR results show that the samples are similar (bands aligned).
Im not sure what this means because of what I understood is that they both create several copies of the gene and there is a difference in laboratory conditions.
Hi Caren,
while your PCR will show unique bands at apparently same size for both strains because the primers will target a specific region of a unique gene, ITS are used for taxonomy and phylogeny because they can evolve in sequence, size and number. that's why you get some differences between both techniques. see the wikipedia topic for more informations (https://en.wikipedia.org/wiki/Internal_transcribed_spacer#cite_note-5).
fred
they can't be from the same species since ITS is different!
Nowadays differentiation is done by NGS, which is largely more precise compared to ITS or PCR gel bands observation.
fred
Hi Caren,
What sequence are you amplifying by PCR? If by ITS you mean you are analysing internal transcribed spacers in ribosomal genes, these sequences have less constraints and are more variable in sequence than mature rRNAs or mRNAs, I expect more variability in the first than in the latter..
The sizes of the products is also much less informative than the exact DNA bases from each of your PCR products (Note: you used PCR to amplify your ITS sequence and another gene(s) you didn't specify. PCR is a technique).
Sequencing your samples will give you much more specific information you can use to identify your species. Good luck!
- Badges
- Science method