Degradation of DNA Samples

[megalodon]

Hi,

we have a new project in our lab where we take sediment samples from river systems, extract DNA and then sequence the samples by 454 or Illumina. We are taking several samples per day from different locations. The sampling and DNA extractions are done by a single person right now. Because of that, sometimes, samples sit in the freezer for a few days before the DNA is extracted. The problem that we are experiencing is that samples for which DNA is not extracted right away degrade very rapidly and in some cases, the DNA fragments that we are getting are only a few hundred bp long. It is simply not possible for us to do the DNA extraction for all samples right after sampling, so I'm wondering if anybody has any suggestions for us on what we could do to prevent degradation from happening even if the samples are stored for a while prior to DNA extraction. Any help would be greatly appreciated!


Here is some more information about the sampling:
At the site where samples are taken, samples are placed on ice and taken to the lab where they are stored at -20C in a freezer until DNA extraction. Samples are typically stored in the freezer for 12h to 2 days prior to DNA extraction. After DNA extraction, DNA extracts are kept at 4C in the fridge. 260/280 ratios are usually between 1.5-1.9. 260/230 ratios tend to be very low (usually well below 1).


Thanks in advance,

Daniela

[pmiguel]

Hi Daniela,
I guess what would be more useful is to know the conditions under which the DNA does not degrade.

One possibility is that it is the slow freezing to -20 oC that is causing the degradation. If so, then you could store the samples at 4 oC, instead of -20 oC until they are processed. Alternatively, you could "quick freeze" the samples somehow either in the field or in the lab prior to storage. This could be done by placing the samples in powdered dry ice or liquid nitrogen in the field or in lab.

The idea here is that DNA, by itself, is fairly stable. So if you are seeing degradation, it is probably caused by enzymes in the organisms you are sampling. The organisms, if viable, will have intact DNA. But if you slow freeze them (put them in the freezer), many will lyse and during lysis, DNAses will have time to degrade their DNA.

So either don't freeze them at all, or freeze them fast enough that there is no time for degradation to occur. Obviously the same issue might occur during thawing as well. So again you would want to minimize the time during which DNAses can degrade the DNA.

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Phillip

[megalodon]

Hi Phillip,

thank you so much for once again answering one of my questions, I really appreciate it!

The samples that are not degraded (by not degraded, I'm talking about 8-10kb fragments, we usually don't get larger fragments from those samples) are treated the same way as the ones that are degraded. The only thing we have noticed is that the ones that do badly degrade were taken in industrial areas, so maybe there is something in the water that causes degradation? However, we also have extracts from those areas that are not degraded, so the degradation is rather inconsistent.

I will suggest to the person who is doing the sampling to either store the samples in the fridge until he does the extractions or quick freeze them. Maybe this is really the problem. The other thing that came to my mind is if it is possible that we are co-extracting DNases and/or some substances that are in the sediment/water that may cause DNA degradation? If this is a possibility, would you recommend storing the extracted DNA in the freezer instead of the fridge?

Once again, thanks for taking your time to help with this!

Daniela

[pmiguel]

What DNA prep method are you using?
We mostly store DNA in a freezer. Used to be common lore that freezing genomic DNA "sheared" it, but in my experience, this is bogus.

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Phillip

[megalodon]

We are using the FastDNA Spin Kit for Soil from MP Biomedicals for our extractions.

I'm usually storing my DNA samples in the freezer as well and was a bit surprised when I was told that these samples are stored in the fridge...

[pmiguel]

Yes, I would store the DNA after extraction frozen. Also, make sure to do a clean-up (eg minelute or Ampure) after the fragmentation step of library construction. With completely pure DNA, this may not be necessary. But any DNA that is degrading at 4 oC probably is not all that pure. Contaminating nucleases will cause big problems for the a-tailing steps that most protocols now use prior to ligation of adapters.

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Phillip

[megalodon]

Thanks, Phillip!

Daniela