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DNA extraction from a partially-eaten fruit

forensics DNA extraction preservation

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#1 Onibaku

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Posted 28 August 2013 - 08:24 PM

Hi everybody,

 

I would like to try to extract DNA from a fruit that was partially eaten by somebody (i.e. I want to extract the DNA of possible buccal cavity cells lying on the surface of the eaten part of the fruit for subsequent PCR). Sounds pretty difficult, but I was wondering if any of you would have an idea of how to maximize the chances of getting something? Which extraction procedure would you recommend? (Also, how shall the sample be preserved before analysis to avoid losing DNA?)

 

Thanks a lot in advance! Looking forwarding to reading your comments.

 

Cheers,

Chris


Edited by Onibaku, 28 August 2013 - 08:26 PM.


#2 bob1

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Posted 29 August 2013 - 05:49 PM

This is a difficult one, as the answer will depend on whether you can isolate the buccal cells and then extract the DNA or if you have to extract the DNA from the fruit itself and look for "contaminating" human DNA. If you can get the cells, the extraction is easy, any DNA extraction method should work, just scale it appropriately for the amount of cells you have.

If you need to extract the fruit DNA and look for the human, then you need a specialized plant DNA extraction kit that will remove much of the polysaccharides and other compounds found in plant material. There are many extraction protocols out there, but you will need one that maximizes DNA out put as the contaminating DNA will only be a tiny fraction of the total DNA.

#3 Onibaku

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Posted 30 August 2013 - 07:22 AM

Hi Bob1,

 

Thanks for the reply. I would prefer to isolate the cells, but I am not sure how to best perform this. Using a mouth swab doesn't seem optimal. I heard about using filter sheets with anidinethiocyanate (GuSCN) to absorb DNA. Does this make sense? Any other idea maybe?

 

Cheers,

Chris



#4 bob1

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Posted 30 August 2013 - 06:10 PM

I don't know for sure, but there are likely to be papers in forensics journals about this sort of thing.

 

I would have thought that multiple washes in a a buffer then spinning the cells down would be the easiest.



#5 Onibaku

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Posted 30 August 2013 - 07:23 PM

Hmm... I thought the same and I have been trying to find some forensics mailing-list or forum to post this question in, but so far I am out of luck. Will continue searching though.

 

Actually, I am also a bit afraid of losing genetic material between collection on the field and extraction in the lab (i.e. losing the cells in the solution; not sure if this fear is reasonable, I am maybe becoming a bit paranoid about all this). For the record, this duration will be around 1 month. So far, I have been preserving the first samples in 99.5% ethanol (and I am thinking about switching them to silica now as in the 2-step method by Reddy et al. 2012 - doi:10.1371/journal.pone.0046732). May I ask your opinion about this?



#6 bob1

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Posted 01 September 2013 - 01:53 AM

If you have put the cells into ethanol, the DNA will be fine for as long as you need to keep them, so long as it stays in the ethanol.  I don't know about the method as such without reading the paper, but silica based extraction methods are quite common in labs, so there shouldn't be any problems ther.







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