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Amplfication of fungal DNA


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#1 chat25

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Posted 08 September 2009 - 11:53 PM

Hi all,

i have isolated the DNA from a fungus ( a species of fusarium) i wanted to amplify that DNA, so i have synthesised a primer. but the genome of that species of fusarium is not sequenced in NCBI genebank. so i have used a 28s rRNA sequence to design the primer..

but atlast DNA didn't amplify.. i really want to know where it went wrong.

To amplify the DNA is the primer should match exactly with the perticular species or genus is enough?

please help me to solve this out?

thanks....

#2 ivanbio

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Posted 09 September 2009 - 08:01 AM

Hi chat25,

What you are trying to do is always tricky since you are blindly trying to amplify something whose DNA sequence you do not know. It should be possible to identify a region that is 100% identical in fungi, which I assume you did for the 28S sequence. If that is indeed your assay, then it is always possible that the PCR did not work because the PCR itself is not working (bad primers, not ideal PCR conditions, etc). If the later is the problem, you should try to optimize the assay and/or design new primers. Finally your DNA is of critical importance. Unless you are 100% sure you have very high quality DNA, then I suggest you isolate more, ideally using a commercial kit.

Good luck

Ivan
Carlsbad, CA

#3 chat25

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Posted 10 September 2009 - 12:23 AM

Hi chat25,

What you are trying to do is always tricky since you are blindly trying to amplify something whose DNA sequence you do not know. It should be possible to identify a region that is 100% identical in fungi, which I assume you did for the 28S sequence. If that is indeed your assay, then it is always possible that the PCR did not work because the PCR itself is not working (bad primers, not ideal PCR conditions, etc). If the later is the problem, you should try to optimize the assay and/or design new primers. Finally your DNA is of critical importance. Unless you are 100% sure you have very high quality DNA, then I suggest you isolate more, ideally using a commercial kit.

Good luck






Thanks ivan..

Actually i'm a fresher to this feild and i don't have much experiences regarding this. i have done a similarity searching through BLAST and maximum identity was 100%. and also i have isolaed the DNA by using a commercial kit as well as C-Tab - Nacl method. but the kit didn't work properly. so i proceed with the 2nd way.
Is this primer is matching with the Fusarium genus, is it work with the any species of that genus or it should be specific???

thanks again...

#4 gebirgsziege

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Posted 10 September 2009 - 01:36 AM

I assume you got your DNA from pure culture?

You should not start at the end of the process. Use some fungal unversal primers to get the seq of your fungus first. Try the White et al primers. You can find them here, but be careful one or two of them used to be in the wrong direction. Another good source for tested fungal primers is the aftol homepage (although sometimes tricky to find the right ones). If these do not work (esp. ITS1/4, ITS5/4 for the ITS region or NS1/8, NS3/6 for the 18S region, NL1/4 for the 28S) then you have a problem with your DNA. Fusarium sp. is quite at tricky genus to work with.....often standard kits do not work for DNA extraction.

If you have a sequence of your region of interest THEN you can go for specific primers. You are wasting your time to try the other way round.
A man cannot be too careful in the choice of his enemies. (Oscar Wilde)

#5 Adrian K

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Posted 13 September 2009 - 09:33 PM

Hi chat25,
I used to work with some ascomycetes such as aspergillus and ceratocystis sp. ribosomal gene amplification with ITS primers.
I use ITS1F and ITS4A combination. it is good enough for me.

ITS1F is fungal specific, and ITS4A is ascomycetes specific. In the article below also mentioned ITS4A + ITS5 works for fusarium, but ITS1F ITS4A amplify a weak band (but I think you can still adjust the PCR conditions to incease the intensity).

Article:
I. Larena et al. Journal of Biotechnology 75 (1999) 187194
Design of a primer for ribosomal DNA internal transcribed spacer with enhanced specificity for ascomycetes
http://linkinghub.el...168165699001546

Tell me if you don't have access the article and I'll PM it you.
:) all the best.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#6 gebirgsziege

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Posted 13 September 2009 - 10:44 PM

I do not like the ITS1F and ITS4A very much....they are giving inconsistent results for lot of fungi. Better to use the "original" ITS1(5)-ITS4 or V9G-LS266 for the ITS1-5.8S-ITS2 region. much more consistent.
But as far as I am aware (correct me if I am wrong) you are using D1/D2 of the 28S rDNA rather than the ITS region in Fusarium, as it contains more taxonomic information.....So better to use a set of NL primers found in the link in my previous post....
A man cannot be too careful in the choice of his enemies. (Oscar Wilde)

#7 chat25

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Posted 15 September 2009 - 12:12 AM

hi

thanks alot for all the advices....

Edited by chat25, 15 September 2009 - 12:58 AM.


#8 chat25

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Posted 15 September 2009 - 01:08 AM

Hi chat25,
I used to work with some ascomycetes such as aspergillus and ceratocystis sp. ribosomal gene amplification with ITS primers.
I use ITS1F and ITS4A combination. it is good enough for me.

ITS1F is fungal specific, and ITS4A is ascomycetes specific. In the article below also mentioned ITS4A + ITS5 works for fusarium, but ITS1F ITS4A amplify a weak band (but I think you can still adjust the PCR conditions to incease the intensity).

Article:
I. Larena et al. Journal of Biotechnology 75 (1999) 187194
Design of a primer for ribosomal DNA internal transcribed spacer with enhanced specificity for ascomycetes
http://linkinghub.el...168165699001546

Tell me if you don't have access the article and I'll PM it you.
:P all the best.



Hi Adrian,

I was unable to get that article... it's a big help if you can send it to me...

Thanks alot for your advices...

#9 Adrian K

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Posted 15 September 2009 - 06:57 PM

Hi chat25,
check your PM for the paper.
Regarding the questions you asked me in your PM I will reply it shortly. :D
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434




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