I know what you are thinking, everything is made of cells, so how different can DNA extractions be in plants? The answer is… sort-of different. The overall concept is the same. Cell membranes are lysed, DNA is separated from other cell materials, washed a few times, and then resuspended in water or Tris-EDTA buffer (aka TE). The difference, though, is plants are distinct not only in cell structure but also in cellular components. These differences can lead to some frustrating results at the end of a DNA extraction if they aren’t accounted for. Let’s walk through some of those differences.
Mr. Pestle, Tear Down This Wall!
Plants can stand up because of water pressure on the inside of their cells pushing against adjacent cells, known as turgor pressure. In bacterial or mammalian cells, this pressure would rupture the cell membrane, but plant cells are surrounded by the cell wall that can withstand this water pressure. The robustness of the cell wall comes from being composed of polysaccharides (which we will talk about again soon). While this is great for the plant, it isn’t so great for any researchers trying to get inside the cells themselves. The cell wall has to be removed before the cell membrane can be accessed and lysed open.
The easiest way to get past the cell wall is physical removal. The most common method is grinding with a mortar and pestle. Tissue is first frozen in liquid nitrogen and then ground into a powder. Care must be taken to do this quickly, so the tissue isn’t allowed to warm too much. If the tissue becomes warm, then the endogenous nucleases can become active. Alternatively, the tissue can be ground with ball bearings in a shaker. The tissue is suspended in buffer with a ball bearing and placed into a shaker specifically designed for the task. While the yield is generally much lower as a result of the smaller tissue sample than the manual method, the ball bearing method lends itself better for high throughput applications. Samples can be processed in 96 well plates, allowing hundreds of samples to be processed in a day.
Forgive Your Contaminants, but Never Forget Their Names
Depending on the species, plant cells can also contain lots of contaminants that have the potential to inhibit downstream reactions. Polysaccharides, polyphenols, and tannins are all common in plant samples and are usually difficult to remove if allowed to persist all the way through the extraction process. For the most part, the cell wall and other larger structures are removed via centrifugation during the extraction process. However, structures like polyphenols can still be present. As all of these different chemicals can inhibit downstream applications, you should use a method that can remove them.
The most common techniques use a combination of cetyltrimethyl ammonium bromide (CTAB), beta-mercaptoethanol (BME), and polyvinylpyrrolidone (PVP). CTAB can solubilize the cell wall components and other membranes, BME can help break down protein bonds, and PVP assists with phenolic compounds. Depending on the protocol you use, and the specific species you are working with, the amounts of each may vary. As each plant species will vary widely in the actual amounts of the different compounds, you should find a protocol specifically designed for your target species. When performing high-throughput extractions on corn, my study species, for instance, I have found that it is possible to obtain PCR-quality DNA without a CTAB buffer at all.
Pick a Method. If It Fails, Then Try Another
Luckily, for most species a simple PubMed search will yield dozens of protocols. I have found it best to try a few different methods until you come up with what works in your hands for your application.
For instance, in corn, I use different methods depending on application. If I need high quality DNA for a southern hybridization or sequencing, then I might use a kit. For routine screening and PCR, then I generally use a protocol developed in the lab next door.1
To give you an example of what this protocol looks like, the basic steps are as follows:
We collect leaf samples with a hole punch into a tube;
Add urea extraction buffer and a small stainless steel bead to the tube;
Pulverize the tissue in a bead shaker and then spin it in a centrifuge;
Move the supernatant to a new tube, leaving the cell debris behind;
Precipitate the DNA in ammonium acetate and then wash it with ethanol;
Air dry the DNA pellet and then suspended it in TE or water.
The final procedure will depend on the species and downstream application, but the bottom line is that plant DNA extraction is generally a simple process. Moreover, when looking for protocols, you might be lucky and find that one of the available plant-specific DNA extraction kits is suitable for you. Some of these even contain ball bearing tissue collection tubes for grinding, and solutions for dealing with compounds such as phenolics. Whatever route you decide to take, if you make sure you are aware of any special compounds your species may contain, you should be able to obtain high quality DNA in no time.
In austerity times, nothing is in excess. Apart from saving reagents, which can be refilled with extra financial injections, there is a commodity that cannot be easily resupplied – tissue samples! If, like me, you have experienced the fear of not having enough sample for performing a qPCR, western blot, and conventional PCR from the […]
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