If you’ve figured out how to extract high-quality DNA from an elephant, chances are that without too much trouble, you’d be able to do the same from a moose, a mouse or even a meerkat. However, if you’ve figured out how to extract DNA from an Arabidopsis plant, well that might be about all you’ve figured out. That’s because plants have developed something akin to chemical warfare in order to survive a variety of climactic extremes, pathogens, and predators, without the luxury of being mobile. As a result, plants harbor an enormous variety of organic compounds, some with antifungal and antimicrobial properties and some which make them taste bad to herbivores. Other structures are complex networks of polymers that store water and nutrients for both feast and famine.

 

It’s all good and well for the plants, but many of these substances muck up DNA extractions....

 

Some, such as polyphenols, bind almost irreversibly to DNA, interfering with downstream enzymatic applications. Others, like polysaccharides, also bind to DNA and in addition, can form a gelatinous mess during the extraction making the DNA concoction akin to alien slime. Complicating matters, plants in the same family, genera or even species can contain radically different varieties and amounts of these substances making it problematic to generalize techniques that work with one plant to work with another.  It’s enough to give plant molecular biologists nightmares.

 

No wonder we get a lot of technical phone calls from weary plant scientists, skeptical that we have anything off the shelf that can be of use to them. And while our DNeasy PowerPlant Pro and RNeasy PowerPlant Kits may not be the end all and be all for every plant out there, they are a step in the right direction towards botanical bliss. Our DNeasy PowerPlant Pro and RNeasy PowerPlant Kits can help you produce high-quality DNA and RNA from a wide variety of specimens while avoiding some of the cumbersome methods that have traditionally plagued plant extractions such as liquid nitrogen, CTAB, phenol, and chloroform treatment.

 

 

Get the DNA out

 For any DNA extraction, the first step is to break open the cells so that the DNA is accessible. Plants, of course, are no exception. Unlike animal tissue, however, plant cell walls are tough and hearty against osmotic pressure.  So to get at their DNA, you’ve got to get rough. Traditional methods use liquid nitrogen and a mortar and pestle to grind up the frozen tissue or the blades of a blender to slash the smithereens out of it. These work but they can be either time consuming or risk sample cross-contamination.  In our plant kits, we use a method of mechanical lysis called bead beating. With this technique, a small amount of sample tissue is placed inside a tube with beads and some lysis buffer and is shaken at high velocity either on a vortex with a vortex adapter or on a specialized high powered bead beating instrument. The beauty of this method is each sample is homogenized inside its own sterile tube.  For optimal homogenization of plant tissue, we’ve found that a few steel and ceramic beads between 2-3 mm in diameter are very effective at breaking down the cells.

 

Polyphenols and Polysaccharides

Once the plant cells are broken apart then you’ve got to deal with the issue that your DNA is free to mix with all those complex plant molecules mentioned earlier. Polyphenols like flavonoids, anthocyanins, lignans, and tannins may be great for lowering your cancer risk, but they are nasty for DNA extractions. And because of all the positive press that polyphenols have recently received, scientists are really focusing on studying those plants with the highest levels of these compounds. In the past six months, we’ve received calls regarding plant such as soybeans, chocolate, coffee, strawberries, orange peels, sunflower seeds, and corn, all with very high levels of polyphenols.

 

 

When plant material is macerated in order to release the DNA, polyphenols become exposed to oxygen and react with enzymes, most importantly polyphenol oxidases (PPO). [These are the same enzymes that turn apples and potatoes brown.] It is these polyphenol oxidation products that can covalently bind to nucleic acids, making them virtually impossible to remove. So, it’s better to try to prevent the two from associating at the get-go.  Common methods involve using detergents (CTAB & SDS), antioxidants (bME, Ascorbic acid, & DTT) or certain polymers (polyvinylpyrrolidone (PVP) & polyvinylpolypyrrolidone (PVPP). Detergents help by solubilizing lipids and enzymes that complex with DNA making them easier to remove. Antioxidants work by denaturing and suppressing the activity of the PPO enzymes slowing down their breakdown of polyphenols. PVPP and PVP work by binding up the polyphenols and preventing them from reacting with the DNA.

 

In both our DNeasy PowerPlant Pro and RNeasy PowerPlant Kits, we’ve included a specially formulated Phenolic Separation Solution (PSS) that can be added to the bead tube before homogenization. It is very effective at keeping phenolics at bay. We have observed that the affect is variable, however.  For some samples, it greatly improves the nucleic acid yield and in other cases, it has no effect.  It’s part of the variability of plants.  So it’s best to try a test run with and without the PSS to see how your sample type will respond.

 

 

 Polysaccharides, used for food storage in plants, are the other great offenders in plant DNA extractions.

Acidic polysaccharides can be removed from DNA during the prep under high salt conditions. The DNA can be out-competed with a cationic detergent such as cetyltrimethyl ammonium bromide (CTAB).  The CTAB: polysaccharide complex can then be preferentially precipitated out. A few disadvantages of the technique are that it is time-consuming, expensive, and it is difficult to keep CTAB in solution while it’s hanging out in the lab. Plant polysaccharides can be enormous and complex.  DNA can get all bound up in them, often adding a visible viscosity to the DNA slurry.  People who study the effects of polysaccharides on downstream enzymatic reactions have found it useful to categorize them as either neutral or acidic. Acidic polysaccharides inhibit the enzymes involved in PCR and restriction digests, while neutral polysaccharides don’t. Some common examples of acidic polysaccharides are pectin, xylan, and carrageenan. Some neutral polysaccharides are dextran, gum locust bean, starch, and inulin.

 

Our DNeasy PowerPlant Pro and RNeasy PowerPlant Kits avoid the use of CTAB with the use of our Inhibitor Removal Technology® (IRT).  They use a combination of chemistry in the lysis buffer and in the subsequent step after bead beating that is very effective at removing polysaccharides. For samples that are very high in polysaccharides, however, it might be necessary to use less starting material, since large amounts of polysaccharides might overwhelm the chemistry.  When polysaccharides are combined with alcohol they can precipitate into a gelatinous blob making it difficult to work with, for example when loading it onto the spin column.

 

All Parts Are Not Created Equal

One last thing to keep in mind with plant DNA extractions is that levels of polyphenols and polysaccharides will vary in different parts of a plant and even in the same plant at different times in its development. For some plants, the levels of polyphenols may be very high in the leaves but low in the roots.  For others, the stem might contain a lot of stored sugars but have little in the leaves. So if one part doesn’t yield good results you may need to try another. It’s all par for the course in the plant world. Usually, younger plants have the least amount of offending substances so these are often easier to work with. But, of course, it’s not always possible to be choosy. If you want to study RNA expressed in a certain part of the plant or some embedded fungal DNA, you may not have that luxury. In that case, you will need to depend on the power of chemistry to give you the best results in your nucleic acid isolation.

 

References Used:

 

BioTechniques 13: 52-56; (1992). A quick and inexpensive method for removing polysaccharides from plant genomic DNA. Fang G, Hammar S, and Grumet R.

 

Plant Mol Biol Rep 17:1-8.; (1999) An improved and rapid protocol for the isolation of polysaccharide- and polyphenol-free sugarcane DNA. Aljanabi SM, Forget L, and Dookun A

 

J Plant Mol Biol Biotechnol 2011 2: (2) 1-7; Biochemical Role of Ascorbic acid during the Extraction of Nucleid Acids in Polyphenol Rich Medicinal Plant Tissues; Tushar BORSE1,2*, Prashant JOSHI 2 and Sushama CHAPHALKAR1,2

 

Biotechniques. 1991 Feb;10(2):162, 164, 166. A simple technique for removing plant polysaccharide contaminants from DNA. Do N, Adams RP.

 

Phenol-Explorer: an online comprehensive database on polyphenol contents in foods. Database, doi: 10.1093/database/bap024.Neveu V, Perez-Jiménez J, Vos F, Crespy V, du Chaffaut L, Mennen L, Knox C, E isner R, Cruz J, Wishart D , Scalbert A. (2010)

 

HortScience,Dec 1992; 27: 1316 - 1318. A Rapid Procedure for the Isolation of RNA from High-phenolic-containing Tissues of Pecan; Amnon Levi, Glenn A. Galau, and Hazel Y. Wetzstein;

 

PROTOCOLS Isolation of milligram quantities of nuclear DNA from tomato (Lycopersicon esculentum), A plant containing high levels of polyphenolic compounds. Daniel G. Peterson, Kevin S. Boehm and Stephen M. Stack

 

Plant Mol. Biol. Rep. 23: 297a-297i. (2005) Angeles JGC, Laurena AC, Maetecson-Mendoza E. Extraction of genomic DNA from the lipid-, polysaccharide-, and polyphenol-rich coconut (Cocos nucifera L.).

 

Kim CS, Lee CH, Shin JS, Chung YS, and Hyung NI (1997) A simple and rapid method for isolation of high quality genomic DNA from fruit trees and conifers using PVP.  Nucleic Acids Res 25: 1085-1086