Formalin-fixed DNA
22 September 2006: I am now lecturing atDes Moines Area Community College full time and so I have not had much time to update this page. I have received many inquiries about this page and I thought that I would try to continue to update as I can. One issue was that the Dayton protocol link was broken but I have fixed that. I am now aware of a few more protocols and will add them as I have time.-Curtis
Some things that I have come to a conclusion on after getting so much feedback...
- The method using RAPDs is hit or miss at best. This is the technique used by myself and Walsh in 1997. This manuscript has been put on somewhat of a permanent hold because of the issues of the technique. Basically it appears that RAPDs markers can amplify verly large to very small fragments of DNA but it is likely that it only hits highly repetitive DNA and may not be useful in the end. The data used by myself and Walsh was consistent when used to reconstruct a phylogeny of snake species but I don't believe it is enough to stamp this and say this is the answer. However, the positive side of this is that it convinces me that DNA is not destroyed in the fixation process but just 'unavailable'. I believe that in the case of the RAPDs technique that the reason it works at all is because of the fact that you basically boil the DNA thus making it available (my hypothesis is that the extreme heat breaks any cross bindings between parts of the DNA that lower heats can't).
- I believe the key to successfully isolating DNA from such tissues lies in techniques such as that from Cathy Dayton below and others, where emphasis is placed on removing as much of the preservative as possible from the sample through soaking. Somewhere in the 'soaking' process there appears to be a point at which DNA is made more accessible.
- Finally, I am now firmly convinced as I mentioned above that formalin preservation does not break up DNA but in fact may preserve it more completely then we were aware of before. The fact that many have now been able to amplify large fragments from these tissues is testament to that.
Thank you for your interest in getting DNA from Formalin preserved tissues. I typed out all of the protocols that I am aware of. Below are the references to the different protocols. Click on the Author to go to the protocol in their paper.
Shiozawa, D. K., J. Kudo, R. P. Evans, S. R. Woodward, and R. N. Williams. 1992. DNA extraction from preserved Trout Tissues. Great Basin Naturalist, 52(1):29-34.
Shedlock, A. M., M. G. Haygood, T. W. Pietsch and P. Bentzen. 1997. Enhanced DNA extraction and PCR amplification of mitochondrial genes from formalin-fixed museum specimens. BioTechniques, 22:394-400.
Chase, M. R., R. J. Etter, M. A. Rex, and J. M. Quattro. 1998. Extraction and amplification of mitochondrial DNA from formalin-fixed deep-sea mollusks. BioTechniques, 24:243-247.
Eckerman, C. M. and E. J. Walsh. 1997. Breaking the formalin barrier: Development of molecular techniques for genetic analysis of museum specimens. Unpublished.
*NEW* Protocol from Cathy Dayton. US Fish and Wildlife. (PDF version of this protocol)
The protocol that I have used (see above) is very easy and fast. However, it has not proven to be successful 100% of the time. Here I want to mention the chelex protocol that I used and the successes and failures I have had.
The use of Chelex to extract DNA has been criticized by others. The DNA yield is considered to by unpure and problematic. It is also suggested that DNA does not last long because the chelex solution is slightly basic. Other authors have diluted the final supernatant and kept it at -80 degrees C to minimize the effects of the basic solution. Personally I have not experienced inferior DNA problems. DNA amplifications tend to be in sufficient quantities and subsequent sequencing of some of the products are clean.
I have come to realize that the extraction process is the key. All of the protocols I have sent to you use normal amplification techniques but have modified extraction techniques. It is suggested that the reason traditional chloroform-phenol extraction techniques do not work is because the formalin reacts with the proteinase K and the chloroform-phenol solution itself. The techniques using the chloroform-phenol extraction which were successful spent a large portion of the protocol trying to extract the formalin out of the tissue first. The fact that these protocols work is contradictory to the notion that DNA is either sheared or becomes inaccessible due to excesive cross-linkage. In fact the literature now suggests that formalin cannot cause cross-linkage to occur and may actually preserve the DNA but in the process make it difficult to access.
My own speculation as to why the chelex technique works is that the DNA is essentially boiled. Hopefully I will be able to explore this further. At this point I should mention that I boiled the DNA by placing the tubes in a beaker of boiling water. This causes some problems because the tops will pop off of the microcentrifuge tubes and placing a hole in the top can cause water to enter the sample from the beaker. I might suggest using a heat block instead but have not used it myself.
To test this method I used RAPD (Randomly Amplified Polymorphic DNAs) primers. Recently I have also tried targeting part of the 18S gene and part of the Cytochrome b gene. Both fragments are about 500 bp. This procedure was successful most of the time but not 100%. I have generally found that when I have gone back to run out a gel of the whole DNA supernatant that those times that it did not work is when I did not get a visible smear on the whole DNA gel. Subsequent extractions generally worked. I have tried this on only a few specimens so far. It has worked for the few fishes and reptiles that I have tried.
Finally, I want to mention that I have started to write up a review of all these techniques. If you have your own technique for extracting DNA from formalin fixed tissues than I urge you to share them. There are other techniques used in the medical field that I can forward to you if you are interested, however, many of these protocols have to deal with the extra difficulty of the tissues being embedded in parafin.
I will make an effort to forward any new protocols that I become aware of. I am also interested in hearing of your successes and failures in using these techniques.
If you have questions or want to share your own techniques then please email me.
Thank you,
Curtis Eckerman