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Old 06-27-2013, 04:05 PM   #1
floem7
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Default DNA isolation & organelle DNA issue

Hi All
I need to prepare genomic DNA for maize resequencing.
What kit would you reccomend?

I've read (http://www.biomedcentral.com/1472-6750/11/54) that reduction of plastidal and mitochondrial DNA during isolation is beneficial.

What do you think? Is it worth to use procedure from the article or commercial
Kit would be ok?

Would magnetic-bead technique (invitrogen kit) be better than columns?

I plan to isolate DNa from leaves (most obvious choice I think), but maybe roots (softer tissue) would be better (or introduce bacterial DNA from soil/growth medium)?

Maize genome is big ca. 3Gb and highly repetitive. Would organellar DNA compromise sequencing results?
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Old 06-27-2013, 04:15 PM   #2
chadn737
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I can't speak for Maize, but from Arabidopsis leaves we have not had to do any reduction of organelle DNA. Granted the genome is so much smaller that perhaps this negates the issue, but I find it hard to imagine that Maize would have significantly more chloroplasts or mitochondria than Arabidopsis.

I think I would stick to leaves unless you isolate from roots grown on a sterile medium. Leaves of course will have some amount of bacteria, but it will certainly be easier than trying to wash off soil. On the other hand roots may have few if any plastids, reducing the amount of chloroplast DNA....I have no clue regarding mitochondrial DNA.
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Old 06-30-2013, 01:43 PM   #3
floem7
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Thanks.
I've read that more copies of organellar DNA than genomic DNA will cause that the former will have much higher coverage in NGS. So maybe it is a matter of reduction of organellar DNA amount - after all it is interesting.

So maybe two step procedure - isolation of nuclei and DNA from them would be good choice?
In that way number of organellar DNA copies would be lowered and would have similar coverage as genomic DNA in NGS.

I just don't know what is the standard - so most people don't care for organellar DNA and use commercial kits for DNA isolation?

As you wrote, I'll use leaves.

Last edited by floem7; 06-30-2013 at 03:36 PM. Reason: revision
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Old 06-30-2013, 08:31 PM   #4
SNPsaurus
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Fresh leaves have a higher percentage of nuclear DNA. Some plants/samples can have extraordinarily high chloroplast DNA levels, so depending on your project it might be worth running a few tests.
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Old 07-01-2013, 01:53 AM   #5
floem7
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So you suggest to extract DNA, choose plastidal, mitochondrial and genomic gene and to run qRT-PCR to see how much copies of each I'll get?

In the article I've been citing in my first post they have 278 copies of chloroplast and 16 copies of mitochondrial DNA per one nuclear DNA. In material from leaves.
Isolation of nuclei reduced organellar DNA by half.
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Old 07-01-2013, 11:34 AM   #6
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Yes, something like that would give you an idea if you will be wasting too much sequencing on the stuff you don't want. The amounts you list don't sound so bad... 278 copies of the chloroplast genome will be just 38 megabases, so a small fraction of the nuclear genome. Probably not cost or labor-efficient to try to get rid of them.
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Old 08-16-2013, 09:50 PM   #7
rnaeye
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Hi all,
This is old thread but I thought it might be useful for someone. New England Biolabs has a microbiome enrichment kit which also seperates organaller DNA from host DNA.
https://www.neb.com/products/e2612-n...enrichment-kit
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