ITS1/ITS4 and ITS1/ITS2 - (Mar/20/2010 )
Hi all!
I am a student, and temporaly I am working on a project of molecular identification of microorganisms. However, I am trying to amplify the ITS1/ITS4 region of the fungi comunity in my samples. But, it is not working. I never get a product (the positive control with a fungi pure culture is OK, so the primers are also OK). However, when I use the primers ITS1/ITS2 - I do amplify this region.
I tried also to change the annealing temperature, but it does not help. I also use BSA, because of the possible inhibitors.
Has anybody an idea why the ITS4 primer is not working?
Thx in advance!
Rancher on Mar 20 2010, 08:58 AM said:
I am a student, and temporaly I am working on a project of molecular identification of microorganisms. However, I am trying to amplify the ITS1/ITS4 region of the fungi comunity in my samples. But, it is not working. I never get a product (the positive control with a fungi pure culture is OK, so the primers are also OK). However, when I use the primers ITS1/ITS2 - I do amplify this region.
I tried also to change the annealing temperature, but it does not help. I also use BSA, because of the possible inhibitors.
Has anybody an idea why the ITS4 primer is not working?
Thx in advance!
Nobody has any idea?
depends on the community you are targeting for (ie. basidiomycetes, ascomycetes, zygomycetes etc). If its basidiomycetes I would use ITS1-F and ITS4-F, these primers are commonly used for community checks, but as far as I have seen have a huge bias towards certain basidiomyceteous genera.
However when using ITS-primers I prefere ITS4/ITS5 and not ITS1. I get better results with these primers. And I personally use V9G/LS266 where possible as these primers are located some 100 bp deeper in the conserved SSU/LSU and as far as I have experienced have less bias towards certain taxa than the ITS primers.
BUT: have you tried to optimise PCR-conditons (i.e. template/primer/enzyme concentration, different BSA concentrations, Mg) or is it possible that you have bad quality DNA (fragmented)....the ITS1/2 primer pair gives a quite short product compared to the ITS1/4......so amplification times etc could be critical as well.