I am working in Cyperaceae and suspect that in some cases its well known high silica content (or possibly lignin) may be causing problems with getting high quality DNA during extraction using typical easy extraction kits. Has anyone had similar problems? How did you determine that it was in fact silica or lignin that caused the problem? Did you develop a protocol to overcome the problem?
Do You use a commercial DNA extraction kit, because for plant it is better than home made extraction protocols? I recommend to You Bioline ISOLATE II Plant DNA Kit, BIO-52069 and You will not have a problems.
Check the following links all of them followed Doyle & Doyle (1987). You may get some useful information
http://www.academia.edu/5561402/Mapania_multiflora_a_distinctive_new_species_of_Cyperaceae_Mapanioideae_from_Borneo
http://archive.bsbi.org.uk/2437Fay.pdf
http://www.uvm.edu/~plantbio/dragon2008.pdf
Recovery of plant DNA using a reciprocating saw and silica-based columns. Issue Molecular Ecology Notes. Volume 7, Issue 1, pages 5–9, January 2007
Silica in general can't cause big problems in DNA Isolation process. Maximum effect from silica - absorbtion of DNA by very big surface and electric charge. If we change electic charge in most cases it will be helpful. Lignin can case some more complex problems becuse lignin is very compex biopoymer. But some techniques for DNA purification from polysaccarides also can be used for samples with grat amount of lignin.
In general electric charges of silica and DNA are both negative, so direct biding is impossible. But if you have any chaotropes in buffers it can cause good binding. To sove this problems you may use protocols for DNA elution from silica columns. Principles of these methods are the same
Most spin-column kits for DNA isolation are based on the ability of silica to bind and release DNA at different conditions. By using a set of solutions those conditions can be controlled. Below the text the references are attached that explain how DNA isolation is achieved (Boom R, papers). My experience is that for difficult material it is better to make solutions for isolation on your own (using modified Boom R, protocols) rather than spend months looking for a perfect kit. I used it for bacterial DNA isolation, parasite DNA isolation from feces and blood, DNA isolation from insects.
1. First of all if you have a lot of polysaccharides (clinical bacterial isolates, plants) those can just block the column, that might be also the case with lignin. If you observe non soluble jelly-like objects in the lyzed material Boom R, methods might be a solution for you.
2. Important factor affecting DNA isolation efficiency is efficient cell disruption. I found mechanical disruption better than enzymatic one, the defect of such approach is you can not get intact high molecular weight DNA (50 Kb or more).
The advantage of the Boom R, method is you can modify the volumes of buffers. Washing can be performed several times, big chunks of aggregated material like polysaccharides can be removed from the solution with the pipet tip.
Mechanically homogenized tissue can be mixed with Boom's lysis buffer, glass beads can be added and additional homogenization by shaking can be done.
When it comes to washing, the home made Boom's approach has another advantage, one of the steps is washing with acetone, which can remove plant pigments.
3. In Booms method you can use diatomaceous earth (silica), cheap and efficient. You can adjust the amount of silica to the expected amount of DNA.
That might seem primitive but was shown to be more efficient than some kits (J Clin Microbiol. 2005 Sep;43(9):4616-22.)
In order to achieve higher purity DNA, I precipitate the DNA obtained by Boom method with sodium acetate and ethanol.
Protocols:
Boom R, et al. Improved silica-guanidiniumthiocyanate DNA isolation procedure based on selective binding of bovine alpha-casein to silica particles. J Clin Microbiol. 1999 Mar;37(3):615-9.
Boom R, et al. Rapid and simple method for purification of nucleic acids. J Clin
Microbiol. 1990 Mar;28(3):495-503.
I modified protocol for parasite DNA isolation from feces J Microbiol Methods. 2009, it was more efficient than commercial spin-column kits
I would agree if you say that getting the DNA from the source is the starting point of your research, then definitely you wouldn't like to waste time and money in trying to standardize methods of DNA extraction. In which case , I agree with Dr. Sirakov and would say that you use the kit he has recommended or similar good performing kit (of course, unless you are trying to develop kits by your self for commercial exploitation).
You'll find some information on removing inhibitory materials, including polyphenols like lignin or lignans, in Gegenheimer (1990) Methods in Enzymology 182,174. The techniques described are for obtaining enzyme preparations free from contaminating inhibitors, but many plant compounds and secondary metabolites inhibit enzymes used for DNA manipulation -- restriction enzymes & ligases.
Jai to clarify, I have been using a kit, but some taxa do not come out well. I suspect that leaf silica content causes the problem. Peter can you attach the publication you mentioned in a message to me?
- Badges
- Science method