Museum DNA - Extracting and PCRing ancient DNA (Jun/22/2005 )

I am having problems extracting/PCRing my museum DNA samples. I am using rodent skins.

For the extraction, I have been using a Qiagen Kit with a special (homemade) lysis buffer which contains 10mM Tris-Cl (pH 8.0), 10mM EDTA, 100 mM NaCl, 40mM DTT, 2% SDS, and 250 micro-grams/milli-liter Prot. K. I have been doing my initial digestion with pro-k (and the rest of the reagents) for 24 hours.

For the PCR, I am using microsatellite (nuclear) primers. I have actually successfully amplified my museum samples using mitochondrial primers....so, it does appear that I am successfully extracting mitochondrial DNA. When I PCR using my microsat. primers, I do not get any bands on an agarose gel nor do I get any bands on a polyacrylimide capillary gel. For my PCR, I use Taq beads that contain the following: 2.5 units of PuReTaq DNA polymerase, 10 mM Tris-HCl, (pH 9.0 at room temperature), 50 mM KCl, 1.5 mM MgCl2, 200 µM of each dNTP, stabilizers, and BSA. All of my PCR reactions are run with a positive control, which successfully amplifies.

I am not sure if my problem is with my extraction or with my PCR. I have already tried concentrating (pelleting) down the extracted DNA in a vacuum centrifuge and rehydrating it with 10 micro-liters of water, but the PCR still did not work.

I have successfully extracted/amplified DNA using my microsatellite primers from fresh muscle tissue.

If anyone has any ideas, I would be very, VERY greatful.

Thanks!
Megan

Well, this question is really outside my area but...

1. Always have a positive control for your PCR. Maybe its simply not working.
2. Check your DNA on a gel to ensure your are getting a reasonable amt. of it.
3. If you are not getting alot of DNA, try adding SDS to your lysis buffer. I REALLY am not sure if this will help, but most protocols (all the ones that I have used) for isolating genomic DNA from tissues include SDS to help disrupt the cells (esp. the membranes). Since SDS helps disrupt membranes it may be helpful for isolating mitochondrial DNA.

Hello! Have you been able to get your museum sample DNA to amplify? I am soon to begin work that will involve museum specimens and microsatellites, so I am EAGER to hear what you have learned. Please let me know, and thank you! :-)

CF

Hi, Chad F,
I think the first thing that you can ensure is whether your homemade lysis buffer works well. Did the buffer really improve the isolation of DNA although it's supposed to be? I remember that some studies showed that Qiagen is a satisfactory kid in animal's DNA extraction. And it is proven that this kit will not affect the subsequent reations including PCR and ligation. So rather, I suspect that it's not the problem of extraction. Since the gene in mitochondrial genome can be amplified and the positive control of the gene in nuclear genome can be amplified as well. So I think the nuclear genome in your DNA extract may be degraded. As you know, the mitochondrial genome is about 10 -20 kbp while the nuclear one is more than 10 Mbp depending on organisms. It's relatively easier to shear nuclear DNA, especialy for your specimen in museum. So the main problems is how and how long the specimen was preserved and in what condition are they being stored. You have to figure out what kind of preservative was used (e.g. formalin or ethanol) (it's troublesome to extract DNA from formalin preserved tissue). Even using the same preservative, the amount of preservative or time of fixation will also affect the DNA inside the tissue (better fixed the tissue within 24 hrs). If the preservation is good enough, DNA from tissue over fifty years can still be extracted. I hope these information will help. : )