Dear all, Thank you very much for sharing your experience concerning my question of how to extract DNA from >100yr toe pads of birds. They were all extremely helpful. Below are all the answers I got. Julia Schroeder I'm the sequencing technician in Irby Lovette's lab and was forwarded your ancient DNA request by one of our postdocs. I've done a ton of ancient DNA work on toe-pads from 50-150 yr old bird toe-pads. I use the qiagen kit for extraction, with a few protocol modifications. On the first day, I chop up the toe-pad as much as possible with a razor blade, and add it to 40ul proteinase and 160 buffer ATL. This digests overnight in a 55 degree incubator on a shaker. The next morning, I vortex the samples and if they still haven't digested fully, I add another 20ul proteniase and let them digest another 4 or 5 hours. On the second day, I add the AL buffer and ethanol as the protocol states, and if there are still chunky bits in the tube, I centrifuge and add just the supernatant to the column. If there is no chunky stuff, add the whole thing as normal. The only other protocol change is the elution amount. I elute with 50ul of the elution buffer, and repeat with another 50ul into a second tube. I don't bother checking DNA yields with gels or spectrophotometry because I don't like "wasting" any DNA from scarce samples, and our ancient DNA samples do not move back and forth between our low-contamination room and the normal lab. If you have any questions, feel free to contact me and I'll do my best to give advice. -Amanda Talaba I used the QIAamp tissue kit kit, supplemented with DTT, for extraction from feathers and also a few toe pads. My samples consisted mostly of feathers from 30-40 years old museum skins, but I also extracted from a few toe pads, and some of my material was up to 100 years old. Not surprisingly, older material yielded DNA of worse quality. I attached two papers on genotyping and sequencing errors that were observed in experiments with the old DNA, maybe you find these useful. Best regards, Kristina Kristina Sefc Department of Zoology University of Graz Universitätsplatz 2 8010 Graz To extract from museum skin or feathers, I digest overnight at 55C with Proteinase K, DDT, and SDS. The next day I do a short phenol/chloroform extraction then put through a modified version of the Qiagen DNA minikit. Cheers Hayley Lawrence I extract DNA from 80-year old museum skins and have successfully used a modification of the DNeasy Tissue kit that was first suggested to me by Tasha Belfiore of UC- Berkeley.I first perform several washes of the samples in 100% EtOH for ~24 hours, which gets rid of any PCR inhibitors, such as the gasoline and butane that were used to prepare the skins I've worked with. I can provide the entire protocol if you are interested. Have you seen the following paper by Mundy et al. Skin from feet of museum specimens as a non-destructive source of DNA for avian genotyping. The Auk. 1997. 114(1): 126-129? That may also be of help. Lynne Mullen Hoekstra Lab Ecology, Behavior, and Evolution Division of Biological Sciences University of California, San Diego 9500 Gilman Drive, MC 0116 La Jolla, CA 92093-0116 Tel (858) 822-0170 Fax (858) 534-7108 email lynne@ucsd.edu I've worked quite a bit with toe pads from bird museum skins. I did my work in Rob Fleischer's lab at the Smithsonian Institution. Maybe someone from his lab has already contacted you. But if not, I'll try to track down an electronic copy of the entire protocol so I can email it to you. But we didn't use a kit-- we made our own extraction solution and did phenol/chloroform extraction followed by Centricon purification. I had really good luck with the procedure, but the key is to design really small, overlapping fragments for PCR (<200 bp or even smaller). I noticed that I had less success with samples that came from museums in tropical locations-- likely the DNA was degraded more from the heat and humidity prior to the invention of air conditioning. I'll be in touch in a few days with the protocol. Dawn Dawn Reding Graduate Student Ecology, Evolution, and Organismal Biology 253 Bessey Hall Iowa State University I extracted DNA from a toe pad of a bird museum sample from, the 1950s. I just give you the details of my protocol: I used the Qiagen DNeasy Tissue Kit and performed all steps in a separated lab only for ancient DNA samples. I took 0.0076g of the toe pad and cut it a bit (as possible :-)) + 180µl ATL-Buffer + 20µl proteinase K 30'' vortex 55°C water bath for overnight during the next day I added 3 times 20µl proteinase K until everything was digested the 3rd day: + 5µl RNA (to increase the crop of DNA) + 240µl AL-buffer vortex incubate for 10' 70°C +240µl EtOH 96%, vortex, put in on the column and follow the washing procedure in the protocol. I eluted the DNA in 2x100µl AE-buffer. My target gene was only amplificable using nested PCR and using primers for very small (~100bp) PCR fragments, so I hope, you are not planning to amplify long markers... But now I am interested in what you are doing and with which birds? Possibly we could exchange knowledge and/or samples? And I would appreciate if you could send me other answers to your EvolDir request, of course :-) . Good luck and best regards Simone Hi, You might try Macherey-Nagel Nucleospin Tissue Kit. I've used it for many difficult applications such as faecal DNA extraction or isolation from 100 years old butterfly legs. I had quite good results in isolation from soft tissues that remained on museum skulls - my record is amplification of mtDNA from over 130 years old wildcat sample. The only thing is that elution of DNA should be performed in 60-80ul instead of 200ul as indicated in protocol. You might also repeat washing steps twice to get rid of more contaminants. I had also very bad experience with Bio'101 Ancient DNA Kit - it used coloidal silica and special filters to collect the silica particles. Unfortunatelly the filters usually broke and I had to use another filter which made this kit the most expensive ancient DNA kit on earth. I needed on average 3-4 filters for 2 isolations so kit for 100 extractions was capable of isolating only 60 samples which gave about 6Euro per sample. Good luck and be careful about carry-over contaminants ;) Maciek Konopin'ski I tried tons of way to isolate DNA in 100 year old bird toe pad pieces. Tried Qiagen, Chelex, Glass milk, to no avail. Best way was long digestion w/ lots of pro-k/shaking/heat, phenol chloroform extractions, and millipore filters (centricon) purification/desalting after. Rob Fleishcher (sp?) used similar protocol in some of his papers. Good luck! Audrey Washburn, Grad Student, University of Florida. Hello Julia, There was an article in the Auk from 1997 describing a protocol for bird museum skins: The Auk 114(1):126-129 1997 If you haven't seen this one, perhaps it will help. cheers oliver Oliver Haddrath Dept. of Natural History Royal Ontario Museum 100 Queen's Park Cres. I use both of these kits for quill and toe pad extractions (yellow warblers). They work fine. Qiagen is a bit more fancy. Depending on how big your samples are, you may have to elute the DNA in 30-50 uL at the end of the procedure. You may also a re-eution step. Let me know if you need more details. Cheers, Marylene Boulet I have worked on samples approaching this age (and some older), and have had very good success with a relatively simple and straightforward protocol (Peregrine falcons; Brown et al., 2007, Mol. Ecol. 16: 327-343). Here is an excerpt from my thesis: "Purification of historical DNA from museum skins was accomplished using DNeasy? tissue extraction procedure (Qiagen, Mississauga) with the following modifications to the manufacturer's suggested protocol: to maximize the amount of DNA recovered, the elution buffer was diluted 1:3 in ddH2O and heated to 37°C prior to elution. DNA was eluted with 150 ?L of the diluted buffer and then concentrated to 50 ?L using an evacuated centrifuge to return salt concentrations to the original buffer. Purified historical DNA was not diluted prior to amplification." The idea behind this is that with a greater elution volume, you will get more DNA, albeit at a much lower concentration. However, concentrating down the eluted DNA is problematic, because concentrations of salts will also increase, and can hinder PCR down the line. So, I diluted my elution buffer, eluted, and concentrated the volume back down to where the salt concentrations were optimal. The warming of the diluted elution buffer was passed to me by someone (Candace Scott, Queen's University) who has done thousands of extractions and swears by it. I cannot tell you how much this improves things, because frankly I have not tried it without the warming. Presumably the warmed elutant helps strip the DNA from the column. Although this procedure had remarkable success for me, I should point out that I was using very small microsatellites (~ 200 bp or less), as well as a small fragment of the control region (~ 400 bp). Amplification of larger fragments may require a more sophisticated protocol. It goes without saying that contamination is of the utmost concern, and sampling and extraction should follow the relevant precautions. Good luck. Joseph. We've had some limited success in this arena; see attached preprint. If you want to know more, you can contact Jamie (who did the lab work) at jarudnic@brookfieldzoo.org . Best, Andrew J. Andrew DeWoody 1159 Forestry Building Purdue University West Lafayette, IN 47907 765-496-6109 765-496-2422 (fax) http://www.fnr.purdue.edu/faculty/dewoody/index.shtml We have had good success with the Qiagen kit and material like this. Beyond that, however, there are many additional considerations, like having a separate laboratory dedicated to extraction from old specimens - the lab should have its own set of equipment (none of which has had any contact with fresh DNA samples or PCR products) and should be completely isolated from the lab in which fresh samples and PCR products are worked with. We also do PCR setup in the "clean" lab and then move these reactions to the post-PCR lab for further processing. It is also likely that you will have to work in small pieces - i.e., amplify short segments of DNA. For things that are 100 years old, you might have to get down to 100-250 base pair fragments in order to get products. Also, there are some issues with data quality (see Sefc et al. in press - let me know if you need a reprint). This is a quick answer, but in general, I would suggest getting into old museum specimens only after carefully considering the methods you are going to use to prevent contamination from "modern" DNA. Best wishes, Mike You might be interested in these - I can send reprints if you need - the cuckoo study used lots of museum specimens... Chilton, G. & M.D. Sorenson. In press. Genetic identification of eggs purportedly from the extinct Labrador duck. The Auk (2007). Sefc, K.M., R.B. Payne & M.D. Sorenson. In press. Single base errors in PCR products from avian museum specimens. Conservation Genetics (2007). Sorenson, M.D. & R.B. Payne. 2005. A molecular genetic analysis of cuckoo phylogeny. Pp. 68-94 in R.B. Payne. Bird Families of the World: Cuckoos. Oxford University Press. Sefc, K.M., R.B. Payne & M.D. Sorenson. 2003. Microsatellite amplification from museum feather samples: the effects of fragment size and template concentration on genotyping errors. The Auk 120: 982-989. Payne, R.B. & M.D. Sorenson. 2003. Museum collections as sources of genetic data. In G. Rheinwald, ed. Bird Collections in Europe: the Challenge of Mutual Cooperation. Bonner Zoologische Beiträge 51 (2002): 97-104. We have just recently used mammalian museum skins for a large scale phylogeography of stoats. The methods are in the enclosed paper (QIAGEN kit), and based on my experience DNA quality from museum skins depends largely on the museum of origin. Anecdotally, I would say that museum deposits from southern (warm) areas are less likely to have usable DNA, but 100+ years old samples are a lottery. I have 44 samples in my stoats database that were sampled prior to WWII, and out of those I was able to sequence only two for the whole region of interest (~1.8kb, EF089001-1930 National Museums of Scotland, Edinburgh; EF088992 - 1938 Natural History Museum, London). Those are for PCR products at least 650bp long. I have shortened the PCR product to half (300-400bp) now, and the results are better. It is still longer than usual length used in ancient DNA studies, but museum skins are contaminated by definition whereas DNA inside of a bone or tooth is protected somewhat better. As you might have noticed, I'm overly too sceptical when it comes to sequence authenticity in these instances. Good luck, Natalia Martinkova I have used passerine toe pads from museum samples as old as 150 years. I chopped the tissue to a fine sand and then used a Gentra PureGene extraction kit. It was necessary to add several lots of ProK to get the tissue to break down sufficiently but yields were surprisingly high. I had previouly sequenced a 1200 bp fragment in fresh tissues of the same species but found that the museum sample DNA was degraded, making it necesary to design new primers to amplify smaller segments (~200 bp). If I can be of any further assistance, please let me know. Lee Ann Lee Ann Rollins PhD Candidate University of New South Wales Biological, Earth & Environmental Science SYDNEY NSW 2052 Back in the old days I used Chelex, see Ellegren (1991) DNA typing of museum birds. Nature 354:113. Good luck with your work. Best wishes, Hans It should work fine, I have worked on much older toe pads no problem. Although you may need to keep your amplicons fairly short. The easiest method I know is the Qiagen DNEasy kit. Just do what it says for animal tissues, although you might want to double the volumes of all the 'digest related' steps...(ie up to the first wash). This method is fairly cheap and works well. A few points 1) At the elution you can do several things to increase DNA recovery. a) leave the elution buffer on the filter for 5 mins at room temp before spinning. b) you can take the elute and put it back on the filter and repeat to try and get even more DNA. c) OR you can simply add another volume of elution buffer to the filter and do a second elution to get more DNA off the filter. I prefer this option...for example I use 50ul of elution buffer twice. 2) The more you cut up the toe pads before you digest them, the better things work (obvious but worth stating). I do have an alternative method that I use a lot that you could also consider that involves making up a buffer then purifying with a Qiagen Qiaquick PCR clean up kit. It can work better on problematic specimens. If you are interested, let me know. Tom Gilbert Dear Julia, I recently saw an article (Rohland and Hofreiter (2007) Comparison and Optimization of ancient DNA extraction. Biotechniques Vol. 42, March, Issue no. 3, p343-352) discussing the best DNA extraction method for ancient tissues, maybe that would be of help? Laurence Laurence Clarke PhD candidate Institute of Conservation Biology University of Wollongong Northfields Ave., Wollongong NSW Australia 2522 E-mail: ljc03@uow.edu.au j.schroeder@rug.nl