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Old 11-23-2009, 05:43 PM   #21
Nitrogen-DNE-sulfer
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One can design assays for DNA damage using enzymes which target it and exploit the products the enzymes create. We've used antibodies to 8-oxoG to measure photo damage on arrays and it lights up like a X-mas tree without proper scanning buffers. So we dont have to live with this. It can be measured but requires alot of time and effort tuning a quantitative assay. Most assays are specific for an a priori lesion of interest so this is a long slog and quanting total DNA next to amplifiable DNA is probably too imprecise but should be able to pick up 90% effects like you mention (1 lesion eliminates a whole molecule).

Thymidine dimers from light through windows is one angle. I'd also point out low bind tubes and DNA adherence. While you are at, dont ship your DNA through certain zip codes or airports as the E-beam dosage varies. We're going through the steps which assume perfect DNA and seeing how poor these efficiencies are and then pushing them to highest we can achieve. If the simple hypothesis fails, we'll no doubt be digging into the damage more as the numbers in regards to loss are higher than anyone can explain.

great thread.. very pertinent to the field.
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Old 12-01-2009, 05:42 AM   #22
pmiguel
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Quote:
Originally Posted by Nitrogen-DNE-sulfer View Post
One can design assays for DNA damage using enzymes which target it and exploit the products the enzymes create. We've used antibodies to 8-oxoG to measure photo damage on arrays and it lights up like a X-mas tree without proper scanning buffers. So we dont have to live with this. It can be measured but requires alot of time and effort tuning a quantitative assay. Most assays are specific for an a priori lesion of interest so this is a long slog and quanting total DNA next to amplifiable DNA is probably too imprecise but should be able to pick up 90% effects like you mention (1 lesion eliminates a whole molecule).

Thymidine dimers from light through windows is one angle. I'd also point out low bind tubes and DNA adherence. While you are at, dont ship your DNA through certain zip codes or airports as the E-beam dosage varies. We're going through the steps which assume perfect DNA and seeing how poor these efficiencies are and then pushing them to highest we can achieve. If the simple hypothesis fails, we'll no doubt be digging into the damage more as the numbers in regards to loss are higher than anyone can explain.

great thread.. very pertinent to the field.
Naively, I would think that a method that hydrolyzes a DNA sample into mononucleotides followed by some flavor of mass spec would give the most global overview of DNA damage. Caveats there, of course. But damage introduced by the protocol itself could be discovered and corrected for, probably.

As to pyrimidine dimers, I seem to remember that there was an enzyme (from celery?) that reversed the dimerization process. Once we get to the point where we are repairing our DNA samples prior to assays, that would be a good enzyme to deploy. All the bacterial remedies for the pyrimidine dimer problem sounded far less appealing, if I remember this correctly.

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Old 12-04-2009, 08:24 AM   #23
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A side comment:
We found that the protocol for finishing the single stranded library did not neutralize the solution sufficiently and we were not recovering enough molecules here. Once we found that the pH was off and we had to add a lot more Na Acetate, our recovery sky-rocketed.
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Old 12-25-2009, 07:06 PM   #24
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good challenge! i have been worrying about the same problem, too.
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Old 01-26-2010, 02:27 PM   #25
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Super-interesting thread. One obvious thing to reduce DNA damage in library prep is to use a visible light stain and a white light box for gel based size selection versus EtBr and UV. I have had good success with nile blue sulfate, an inexpensive visible light stain. I have not directly compared an identical library processed with EtBr/UV versus Nile Blue/white light so don't know the quantitative effect of UV damage though.
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Old 01-27-2010, 04:01 AM   #26
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Originally Posted by greigite View Post
Super-interesting thread. One obvious thing to reduce DNA damage in library prep is to use a visible light stain and a white light box for gel based size selection versus EtBr and UV. I have had good success with nile blue sulfate, an inexpensive visible light stain. I have not directly compared an identical library processed with EtBr/UV versus Nile Blue/white light so don't know the quantitative effect of UV damage though.
Yes, we don't use EtBr or UV light boxes in the lab at all. We just use SYBR Safe and one of those "Dark Reader" boxes. We bought the latter from Clare Chemical.

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Old 02-14-2010, 08:33 AM   #27
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Some of the DNA is damaged during shearing. My experience has been under certain conditions, only half of the ends can be repaired on one molecule. I don't know the reason yet, but I think the DNA was turned into single stranded. The damages,I think are pretty much mechanical, not chemical. Hydro shear is pretty low speed but will generate just a little better quality DNA. Some of the damages just can be repaired. I tried to repair the single-end adaptor ligated product, but it didn't go beyond that no matter how much enzyme I put in.
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Old 02-15-2010, 05:20 AM   #28
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Some of the DNA is damaged during shearing. My experience has been under certain conditions, only half of the ends can be repaired on one molecule. I don't know the reason yet, but I think the DNA was turned into single stranded. The damages,I think are pretty much mechanical, not chemical. Hydro shear is pretty low speed but will generate just a little better quality DNA. Some of the damages just can be repaired. I tried to repair the single-end adaptor ligated product, but it didn't go beyond that no matter how much enzyme I put in.
Could you give us a little more detail on what the conditions were where only "half of the ends can be repaired on one molecule"?

Any 5' or 3' overhand (locally single stranded) can be blunted by T4 polymerase's 5'->3' polymerase or 3'->5 exo-nuclease activity (respectively). The polymerase activity does rely on a 3'-OH, but T4-PNK should remove any 3' phosphate that would otherwise block the polymerase.

So, if your end repair regimen includes both T4 polymerase and T4 polynucleotide kinase (and most do) the remaining culprit would be non-phosphate, non-hydroxyl DNA fragment ends.

I have been able to find little in the literature about this possibility, the classic Richard and Boyer paper [1] mentions it and I summarized their results here:

http://seqanswers.com/forums/showthread.php?t=2759

As far as a comparison to the hydroshear, it seems like the results published by Oefner, et al. in their 1996 publication describing the prototype of the hydroshear [2] where 20-40% of fragments produced could be ligated with no end repair suggests it is qualitatively different from sonication.

1. Richards OC, Boyer PD (1965) Chemical Mechanism of Sonic Acid Alkaline and Enzymic Degradation of DNA. Journal of Molecular Biology 11: 327-240.
2. Oefner PJ, HunickeSmith SP, Chiang L, Dietrich F, Mulligan J, et al. (1996) Efficient random subcloning of DNA sheared in a recirculating point-sink flow system. Nucleic Acids Research 24: 3879-3886.

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