PCR of degraded DNA

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extari
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PCR of degraded DNA

Post by extari » Wed Nov 18, 2009 5:49 pm

Hello,
I am a phD student who is focused on the genotyping of microsatellites in tuna. My problem is that the dna I'm using is degraded.
I have changed my PCR conditions such as the amount of template, decrease Tm..., but is hasn't been succesfull. Could anyone help me?

Thanks.
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JackBean
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Post by JackBean » Thu Nov 19, 2009 2:00 am

how much is it degraded? Does it work with intact DNA?
http://www.biolib.cz/en/main/

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MrMistery
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Post by MrMistery » Thu Nov 19, 2009 3:31 am

some companies sell these kits especially for "repairing" DNA before PCRing it. I don't know what exactly is in those kits, but they work pretty well at least in some conditions.

Here is the one I have seen before: http://www.neb.com/nebecomm/products/productM0309.asp though I am sure other companies have other products that work in a similar way.

Fun fact: all of these things carry a label saying: not for use if template is heavily damaged. WTF? Isn't that why i am buying your product?
"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter
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JackBean
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Post by JackBean » Thu Nov 19, 2009 4:32 am

It works only for small damage like " including those that block PCR (e.g. apurinic/apyrimidinic sites, thymine dimers, nicks and gaps) and those that are mutagenic (e.g. deaminated cytosine and 8-oxo-guanine)"
In my opinion, he was talking about a little more severe damage, like broken down DNA And that won't this mix fix ;) And that also explains your fun fact ;)
http://www.biolib.cz/en/main/

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extari
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Post by extari » Thu Nov 19, 2009 11:01 am

Hello again,

Some of my DNA is degraded and the highest fragment is about 250bp, the higher fragment I have to obtain in my results (in the PCR) is 240bp length, but the pcr works with intact DNA.
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JackBean
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Post by JackBean » Thu Nov 19, 2009 11:37 am

Yeah, so I understood it well :)
Well, if the largest is about 250 bp, than many will be shorter plus you must count with primers, so it's no wonder, it doesn't work. You would need to do some ligation, but I would expect blunt ends, so it would be unspecific and thus you will get just lot of mess ;)
How, that is your DNA degraded?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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