I'm struggling to find resources or an appropriate method of extraction that can break the beta sheets and still have viable DNA for PCR reactions. Any help or references would be greatly appreciated. The current method of various Qiagen kits do not successfully break the beta sheets, but rather just the alpha helix, which is only about 30% of my samples. SDS, pressure, heat, none are giving up viable DNA. I have been searching for over a year and found one resource on beta sheet keratin and it's not very helpful.
Thanks!
Hi Nichole,
i think instead of SDS why cant you try with UREA which can break the hydrogen bonds asThe Beta sheets in the protiens are maintained by same (hydrogen bonds) . Whereas the SDS can break only hydrophobic interactions.
Is this of any use? http://rspb.royalsocietypublishing.org/content/277/1685/1161.full
Selective biodegradation of keratin matrix in feather... By Theagarten Lingham-Soliar et al.
James, that helped me with the structure, but they allowed it to decompose and checked the structure then. I don't know if I could extract viable DNA after an 18 month decomp stage (it may be a fun test though). Plus, the sheds don't like to decompose except when they mold. Good for me in one way, horrible in another.
Venkata, the proteinase K kit we used with SDS worked in about 12 hours to extract the most viable samples we have, but the weights are low. I'm not too familiar with using UREA. I've had to teach myself this as I go, I'm a neurology nerd, not a molecular savant. Anything I should look out for? It's been a while since I've been in the lab (because I got so frustrated I needed a break), but I believe we were using 1% SDS, I'd have to check my books, but I'm pretty sure that's what we were using.
Nicole, 8 Molar urea is very common for doing exactly what Venkata said- breaking up hydrogen bonding because it hydrogen bonds very well itself, and it is a small molecule that can insinuate itself into proteins. Urea is also extremely inexpensive. See for example: http://pubs.acs.org/doi/pdf/10.1021/bi00167a017 "Equilibrium folding studies of cellular retinoic acid binding protein, a predominantly. beta.-sheet protein" which, at least according to the abstract, describes unfolding and refolding of the beta-sheet structure with the help of 8 M urea. The paper also discusses the use of low pH to switch from beta-sheet to alpha helix (at least for their protein of interest). A Google Scholar search on "8 M urea" "beta sheet" turned up many, many references...
I'm not sure I understand the problem with "the weights being low" after proteinase K treatment- do you mean the DNA is being extracted at less than the full genomic size after partial digestion of the beta-keratin? Why would that matter? You'll be sequencing or amplifying it in pieces, anyway, presumably, unless you are looking at a very small pathogen genome instead of the host... Just because DNA has been sheared doesn't mean you don't have the complete sequence (or nearly the complete sequence). Am I missing something (completely)? Best, Jim
I'm trying to get genomic sizes. So far, I'm getting pieces too small to do much of anything with. There are two shotgun sequences for the species I'm working with, but they are only for specific genes, not the genome. The nearest species that has anything close to a genome is in a different family. I'm still new in this analyzing aspect from non-historical data expectations or a baseline to gauge my results against. I haven't done genomic work, so I'm still trying to figure this part out. The UREA may break the beta sheet, but it denatures what I could extract from it, right? (I'm not a mol bio person, so please bear with me). Thanks!
Yes to your question about urea, it will denature nucleic acids as well as beta sheets, but I don't see why denaturing the DNA is such a big problem. You can always regenerate the double strands by either templated DNA synthesis with primer extension reactins or by re-annealing once the DNA is separated from protein... But I don't do this exact kind of work, just related work, so maybe I'm still missing something. For DNA of the size you are talking about, you do need to be gentle to avoid mechanical shearing while you are breaking up the protein. If you are not being careful about shearing, the small DNA may all be caused by that rather than other problems.
But back to keratin, have you used a reducing agent like beta mercaptoethanol or dtt during your attempts to dissolve or otherwise break up the beta keratin? This is important because you need to break disulfide bonds as well as hydrogen bonds.
Have you looked into forensic techniques for recovering DNA as a possible source of ideas?
I hope we are aking some progress. If not, I can direct you to people who do this kind of thing for a living with avian samples.
Best wishes.
- Badges
- Science method