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how to amplify a fungus which is in the host tissue


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#1 azadeh12345

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Posted 07 November 2012 - 12:12 AM

hi
how can I amplify a fungus ITS region which is in the host tissue? my fungus is a biotroph and cannot be cultured, and I have to amplyfy it from the host tissue.
I have extracted the DNA of my infected leaves in which I have both the DNA of the fungus and plant, and I have used primer ITS1 and ITS4, I have got two bands , I extracted the bands from gel, and sequenced them. one was the ITS of plant and the other was the ITS of my fungus.
but is there any other way to amplify only the fungal band?

#2 bob1

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Posted 07 November 2012 - 12:18 AM

If you know the sequence of the fungus, you can design fungus specific primers for the region of interest. If you don't know the sequence, you may need to look at some primer walking where you sequence from a primer, then use that to design another primer further along the DNA which you then sequence from...etc.

#3 El Crazy Xabi

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Posted 07 November 2012 - 04:15 PM

Fungal ITS specific primers
Check this article
http://www.biomedcen.../1471-2180/5/28

#4 azadeh12345

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Posted 20 November 2012 - 01:44 AM

Thank you , I ordered specific primers for ITS region from the article you suggested




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