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paper on DNA purity and PCR efficiency


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15 replies to this topic

#1 Maddie

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Posted 02 June 2010 - 10:55 AM

Would someone know publications that correlate DNA purity (I guess determined Absorbance at different wavelenghts, or rations of A, or any other technique) and PCR efficiency?
Can't find any.

Thanks

Maddie
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

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#2 pito

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Posted 03 June 2010 - 11:18 AM

check the following links.

But it depends on what you define with purity. Most speak of reaction purity not really the DNA itself, however the DNA itself doenst need to be that pure if they use PCR to detect environmental samples that arent "cleaned" at all.

paper 1
paper2
paper3
google book

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#3 Maddie

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Posted 04 June 2010 - 09:36 AM

Thanks a lot Pito. I will read these carefully :)

But it depends on what you define with purity. Most speak of reaction purity not really the DNA itself, however the DNA itself doenst need to be that pure if they use PCR to detect environmental samples that arent "cleaned" at all.



Yeah I didn't specify what I meant by purity on purpose :D . I have extracts, that could be compared to environmental extracts, that do not amplify well when I extract high amounts of material (by high I mean 1 g). So I'm looking at various things to try to understand what is messing up the PCR efficiency and whether I can do something about it. I analyze the absorbances, A 260, A 280 (and ratio) A230 and A320. Looks like A 320 is an interesting indicator of "purity". Dunno...I'm playing around here. I was hoping that someone had publised something correlating PCR efficiency with absorbance at different wave lenghts. I have a nanodrop but no other instrument available really.
Does it make sense?

Oh and before someone tells me to simply extract less material :( , let me explain that I do need to increase DNA yield, and how can you do that without increasing sample amount?
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

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#4 pito

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Posted 04 June 2010 - 12:49 PM

you should try a hot start pcr then.

Or you should find a paper on environmental samples or samples like you.. when they describe how they did it, it might be helpfull for you.

OH, well some of the papers might be intersting, paper 2 for instance might be valuable.

Good luck with it anyway.

PS. what about increasing cycles?

Edited by pito, 04 June 2010 - 12:49 PM.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#5 Maddie

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Posted 04 June 2010 - 12:59 PM

We already push as hard as possible at the amplification step (increase cycles, etc..).
I work on getting DNA fingerprints from old bones that contain lots of...who knows? residual proteins, residual minerals, humic acids, cross-linked collagen. Who knows? I still need more templates to start the amplification but when I extract more material, I co-extract more crap and at the end, don't get better amps.
If I knew what this "crap" is, maybe I could get rid of it, but I don't have a clue :P
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

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#6 hobglobin

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Posted 04 June 2010 - 01:10 PM

We already push as hard as possible at the amplification step (increase cycles, etc..).
I work on getting DNA fingerprints from old bones that contain lots of...who knows? residual proteins, residual minerals, humic acids, cross-linked collagen. Who knows? I still need more templates to start the amplification but when I extract more material, I co-extract more crap and at the end, don't get better amps.
If I knew what this "crap" is, maybe I could get rid of it, but I don't have a clue :P

320 nm is for turbidity if i remember right, we used it as correction after photometer measurements....
do you use mtDNA?
and there should be several protocols for this as even from Neanderthal bones DNA is isolated...

One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.


#7 pito

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Posted 05 June 2010 - 09:10 AM

We already push as hard as possible at the amplification step (increase cycles, etc..).
I work on getting DNA fingerprints from old bones that contain lots of...who knows? residual proteins, residual minerals, humic acids, cross-linked collagen. Who knows? I still need more templates to start the amplification but when I extract more material, I co-extract more crap and at the end, don't get better amps.
If I knew what this "crap" is, maybe I could get rid of it, but I don't have a clue :(

320 nm is for turbidity if i remember right, we used it as correction after photometer measurements....
do you use mtDNA?
and there should be several protocols for this as even from Neanderthal bones DNA is isolated...


Indeed, like you said: there are protocols out there.

When I saw Maddie's post I wondered why she couldnt get it right since it directly made me think about forensic science.

I think you are looking on the wrong places Maddie. You should try to get in contact with a lab specialised with forenscis science and/or history labs that are specialised in ancient history/DNA techniques.

I however understand that you dont find a lot of papers at first since those are most likely all published in very specialised magazines that arent well known and might not even have online files yet.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#8 Maddie

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Posted 07 June 2010 - 01:00 PM

I am both in the forensic and aDNA community already. If you look at the Neandertal papers, you'll see they start with extremely small amounts of bone powder (10 to 40mg). One can easily do mtDNA with small amounts of material, even if it's 40,000 years-old (that's relatively easy stuff). When it comes to genomic DNA, things get more complicated and if you don't have a next generation sequencing instrument as these guys do, it can be very challenging to get enough gDNA to get a good DNA fingerprint.
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

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#9 pito

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Posted 08 June 2010 - 02:00 AM

I am both in the forensic and aDNA community already. If you look at the Neandertal papers, you'll see they start with extremely small amounts of bone powder (10 to 40mg). One can easily do mtDNA with small amounts of material, even if it's 40,000 years-old (that's relatively easy stuff). When it comes to genomic DNA, things get more complicated and if you don't have a next generation sequencing instrument as these guys do, it can be very challenging to get enough gDNA to get a good DNA fingerprint.


Oh, I see.

Then you can offcourse ignore my post :P

I hope you do find what you need and maybe you can send some samples to a lab that has that thechnology? It might be cheaper to send it 1 time and let them do it then putting all your time in it?

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#10 Maddie

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Posted 08 June 2010 - 09:55 AM

Oh, I see.

Then you can offcourse ignore my post :P
.


Pito, my dear, I would Never do that ;)

I hope you do find what you need and maybe you can send some samples to a lab that has that thechnology? It might be cheaper to send it 1 time and let them do it then putting all your time in it?

ohh believe me, we are trying...if it was that simple.. ;) .
Maybe I should go to the "what if" topic...what if I could have tons of money and use it AS I WANT?
Now that would be B) ...ahhhh sigh.

Edited by Maddie, 08 June 2010 - 10:16 AM.

Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#11 hobglobin

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Posted 08 June 2010 - 01:02 PM

Oh, I see.

Then you can offcourse ignore my post :P
.


Pito, my dear, I would Never do that :P

I hope you do find what you need and maybe you can send some samples to a lab that has that thechnology? It might be cheaper to send it 1 time and let them do it then putting all your time in it?

ohh believe me, we are trying...if it was that simple.. :P .
Maybe I should go to the "what if" topic...what if I could have tons of money and use it AS I WANT?
Now that would be B) ...ahhhh sigh.

Sounds as if you want to be 'better' or 'superior' to those people with lots of money and experience...what's wrong with working on mtDNA? I guess you can also find sequences that are polymorphic enough for fingerprints, though if I remember right microsats are missing..

One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.


#12 Maddie

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Posted 09 June 2010 - 12:40 PM

oh I love mito. Problem is the control region, althought very polymorphic, isn't person specific. You and me may have the same sequence althought not related. For identification purposes, most labs do need to use STR since, in theory, it's as unique as a fingerprint. Anyway. No I don't want to do better, I am trying to find a way to help Forensic labs that work on old bones get better genomic DNA, especially labs (like mine) that don't have access to NGS.
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#13 hobglobin

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Posted 09 June 2010 - 12:58 PM

oh I love mito. Problem is the control region, althought very polymorphic, isn't person specific. You and me may have the same sequence althought not related. For identification purposes, most labs do need to use STR since, in theory, it's as unique as a fingerprint. Anyway. No I don't want to do better, I am trying to find a way to help Forensic labs that work on old bones get better genomic DNA, especially labs (like mine) that don't have access to NGS.

And what about SNPs? I'm not familiar with human mtDNA, but in insects there are 2-3 polymorphic regions on mtDNA, this is control region, and noncoding intergenic regions. If more , I'm not sure...have to look it up

One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.


#14 Maddie

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Posted 09 June 2010 - 01:21 PM

We sometimes use mt SNP when we get a very common profile. My problem here is not what target to chose, we have forensic kits already optimized for loci that are analyzed in all the labs around the world. It's important to make sure everyone works on the same markers in order to create databases (especially crime labs).
So, I'm not trying to get a new method of identification, my only business at the moment is to boost DNA yield without increasing other crap (pardon my French) that ends up inhibiting. Oui?
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#15 hobglobin

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Posted 10 June 2010 - 11:50 AM

We sometimes use mt SNP when we get a very common profile. My problem here is not what target to chose, we have forensic kits already optimized for loci that are analyzed in all the labs around the world. It's important to make sure everyone works on the same markers in order to create databases (especially crime labs).
So, I'm not trying to get a new method of identification, my only business at the moment is to boost DNA yield without increasing other crap (pardon my French) that ends up inhibiting. Oui?

Have you tried magnetic beads then?

One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.





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