This lesson centers around isolating DNA from plants. For some educators, this lesson may help to fulfill certain mandated proficiencies, however the basic act of isolating DNA is not our intended main objective. There are multiple objectives, with DNA isolation being just one. Overall, we are hoping to provide a lesson that accomplishes the following:
- Show examples of different occupations that are related to biology
- Provide examples of different types of microorganisms and their potential as patogens
- Demonstrate how applied science is not isolated to disciplines, but relies on many disciplines that are highly interwoven
- Provide information on critical elements of biology in an alternative learning framework based on practical application as opposed to a text book format
The main theme of this lesson is isolating DNA from plants, however there are many tangents that can be pursued based on the structure of the guide. The importance of epidemiology, plant pathology, microbiology, genetics, and agronomy are all highlighted within this lesson. Our focus is DNA, and hence molecular biology, but some students may be excited about other avenues, and in time the curricula will be expanded so they can explore that line of inquiry. But for now, we are looking at DNA.
Molecular biology has made its way into all levels of education. It is commonly used in many college curricula, in AP and general biology classes in high schools, and at times in middle schools and earlier. Any kid that has watched Jurassic Park (or the sequels) has some idea of the role of DNA in living things. Thus, isolating DNA in a laboratory course is a common occurrence. When choosing a source of DNA, a benign group of organisms that can be tapped that won’t cause controversy, as it is neither an animal nor microbe, are plants. From experience there are parents and students wary of microorganisms, and of course there is significant resistance to using animals in the lab, but not many people will have much compassion for corn leaves or similar leafy vegetables.
When isolating plant DNA, safety is a concern when using common extraction methods. Alternatively, OPS Diagnostics’ Synergy™ chemistry is a great tool to use when teaching plant molecular biology labs. It is simple, fast, and safe, but retains the fundamental steps associated with DNA isolation protocols. For plant molecular biology, it follows the same chemistry, though in a unique format, as the traditional CTAB and phenol/chloroform method. Figure 1 shows a comparison of the phenol/chloroform method and Synergy™ protocol. The chemistry also can be used to highlight important concepts, such as action of surfactants, micelle formation, phase extraction, function of chaotropes, solubility, and lysing methods. The explanation and application of these concepts are often glossed over in lab exercises.
Phenol/Chloroform Method
Synergy™ CTAB Method
Figure 1. A comparison of cryogenic grinding with phenol/chloroform extraction and the Synergy™ DNA isolation process.
If you teach plant molecular biology labs, the Synergy™ isolation process is worth reviewing. Below are notes, a basic lesson plan, and a link to a PowerPoint presentation that are useful for either teaching about DNA isolation in general, or more specifically about isolating DNA from plants.
Background for the Instructor
Though plants can be an excellent specimen for the isolation of DNA, biochemically they are a notoriously diverse group. This can make the isolation of DNA difficult. However, OPS Diagnostics’ Synergy™ Plant DNA Isolation kits simplify the isolation, making the teaching exercise efficient and safe. Aside from the basics, the Synergy™ kits can be used to extend the lesson to beyond the act of isolating DNA and be used to explain the chemistry of the process and some important characteristics about plant biology.
Prior to delving into the lab, one suggestion we put forth is on how to teach this material. An approach we often use is to treat laboratory activities in context, i.e., demonstrating a practical use of the laboratory exercise. For students who are questioning the relevance of the exercise, contextual learning will help address such issues. Though the teaching environment is academic, much of what is taught in classrooms has real world application. By focusing on practical uses of lab exercises, the activities not only demonstrate real world uses, but also present possible areas students may like to explore for careers. Some real-world applications are:
- Isolation of plant DNA for genotyping
- Harvesting all nucleic acids to check for bacterial pathogens (this can be done with a mock pathogen)
- Isolation of total nucleic acids, including RNA, to look for RNA viruses (e.g., TMV)
- Protection of intellectual property by looking for engineered genes in plants.
In the classroom, contextual objectives can be made to justify the isolation of plant DNA, which in many cases, will be followed by another common process, such as PCR. Thus, laboratories that combine isolation and PCR make a good story.
We have probably all asked ourselves why are we learning “this stuff” when taking an assigned class. Often the relevance of the course topic is not apparent. These lessons use contextual learning to stress the practical, real-life application of science. In this case, harvesting nucleic acids from a plant to check for a pathogen.
Storyline: You are a plant pathologist working for the USDA. A new strain of bacterial wilt has been introduced into the fields of spinach from seeds imported from abroad. Your job is to identify infected plants in order to assess the dissemination of the pathogen.
This storyline is very realistic, and aside from the DNA isolation activity, can be used to address many basic lab principles, such as aseptic technique, sample storage, cross- contamination, and the list goes on.
Aside from the mechanics of benchwork, lab exercises can be used to highlight important chemical and biological principles. For instance, the isolation process can be dissected into the following concepts:
- Sampling leaves demonstrate browning, which is the action of polyphenol oxidase as it polymerizes monomers, such as catechol, into chains (polyphenols) that can be toxic to invading microorganisms. However, as well as a plant defense, the polyphenols can also interfere with subsequent uses of the isolated DNA. Thus, the simple browning of a leaf opens up an analysis of plant defense, effect of polyphenols on pathogens, and enzyme inhibition.
- The chemistry used in this lab is based on the Synergy™ DNA Isolation kit, a CTAB buffer approach, which is very common when isolating nucleic acids from plants. Leaf tissue and CTAB buffer are commonly used together. What is in the buffer and why? The breakdown of a standard CTAB buffer is as follows:
a. CTAB – cationic detergent that solubilizes proteins, causes polysaccharides to precipitate, and associates with DNA to keep it in solution.
b. Polyvinylpyrrolidone (PVP) – effectively prevents the synthesis of polyphenols by polyphenol oxidase by binding up substrate (phenolics). Polyphenols can interfere with downstream applications, such as inhibiting Taq polymerase.
c. Ethylene diaminetetracetic acid (EDTA) – chelate bivalent cations, most importantly magnesium, integral to the function of DNases.
d. Sodium Chloride (NaCl) – in very high concentrations (1.4 M) works with CTAB to solubilize DNA while causing polysaccharides to precipitate.
e. Tris Buffer – Stabilizes the pH and ensures the phosphates of nucleic acids are ionized, thus allowing for binding to silica membranes or MNPs in subsequent steps.
- Sample disruption, i.e., grinding/ shearing/ beating – Plant samples can be disrupted, or homogenized, many ways. For a thorough description of sample disruption, see Guide to the Disruption of Biological Samples – 2012. This lab uses bead beating as it is simple and effective. However, it has limitations in that DNA is sheared during processing. Aside from breaking open cells, what else can be learned from this process?
a. Isolating a plasmid and running a gel is a standard exercise for many students. Isolating DNA from a plant is the next level up. However, comparing DNA from this lab to a plasmid or molecular weight marker on an agarose gel provides a visual opportunity. Genomic DNA, once isolated, never looks like a nice clean band. The DNA is typically fragmented and is a continuum of sizes, which on a gel looks like a smear. If the DNA is isolated cryogenically, as with the older mortar and pestle cryogenic CTAB method, the smear is tighter and very close to the well. Bead beating breaks the DNA down into fragments most visible in 2 to 7 Kb size range, so the final product on an agarose gel will show a smear.
b. Bead beating demonstrates the fragility of DNA and the importance of fragment size in subsequent steps. For PCR and Illumina sequencing, sheared DNA is fine, but most long read sequencing requires much large fragments. So how does one isolate large, or high molecular weight, DNA. This introduces cryogenic grinding and its value. Though this laboratory moves away from liquid nitrogen grinding, that method has value, which can be highlighted. So the discussion points here are clonal sequencing, long read sequencing, HMW DNA, cryogenic grinding, etc.
c. CTAB, cetyltrimethylammonium bromide, is a very interesting surfactant. Like most surfactants, it forms micelles, in this case at concentrations above 0.9 mM, or the CMC (critical micelle concentration). Synergy™ purification requires bead beating, which catalyzes a reaction between the white grinding matrix and CTAB. The ammonium head of the CTAB binds to the matrix and results in the hydrophobic tale extending into the buffer. Over the surface of the matrix, a hydrophobic lawn is created. This is the organic layer that mimics the phenol/chloroform. Hydrophobic moieties of proteins and other biomolecules stick to this surface.
Some things to consider:
i. If the bead beating is insufficient, i.e., if foam is still present in the tube following bead beating, then a significant level of CTAB is still in the buffer. Foam indicates the concentration is above the CMC, which causes micelle formation. Hydrophobic molecules will dissolve into the micelles and stay in solution. Most noticeable will be chlorophyll, which will show a green supernatant. Additional grinding, followed by centrifugation, will show the chlorophyll in the pellet. At this point the CTAB concentration is now below the CMC.
ii. If time permits, a simple little experiment that demonstrates micelle formation can be performed. In this experiment, a dye that dissolves in micelles is mixed with CTAB above the CMC. The dye disappears as it dissolves in the micelles. When the CTAB is diluted with water, so the concentration is lower than 0.9 mM (i.e., the CMC), the dye added to the solution stays visible. For an experiment demonstrating micelle action, see this link.
iii. An interesting assignment that can be used to explore CTAB is to ask students to research how CTAB was originally used to isolate DNA. The original protocols exploited a unique property of CTAB that allows polysaccharides to precipitate in the presence of CTAB at high concentrations of NaCl (these buffers normally have around 1.4 M NaCl). If the NaCl concentration drops below 600 mM, the DNA precipitates. Thus, CTAB was originally used to clear the sample of polysaccharides and then precipitate the DNA by lowering the salt concentration. The original method dates back around 60 years.
- Phase Separation – centrifugation of the homogenized sample. Normally when a homogenization tube is centrifuged, you at most see a pellet, however the Synergy™ tube shows a dramatic result. Though the chemistry is proprietary, the Synergy™ gel reacts with CTAB during bead beating and yields essentially a solid phase extraction gel, i.e., a reverse phase chromatography resin. As noted above, the insoluble gel is coated with the C16 tails of the CTAB and mimics an organic phase, very similar to the phenol and chloroform extraction. Hydrophobic molecular and protein motifs dissolve in this C16 layer. Upon centrifugation, the “junk” from the leaf pellets. Normally this wouldn’t be that dramatic, except the chlorophyll dissolves in the C16 layer, resulting in a green pellet and clear supernatant (Figure 2).
Figure 2. Bead beating leaf tissue with Synergy™ gel on the left and zirconium disruption beads on the right. CTAB binds to the Synergy™ gel and traps contaminants while micelles are formed on the right side thus solubilizing chlorophyll. This solid phase extraction is widely used in the lab and industrial processes. It would be worth asking the student to seek out other examples of organic extraction that are commonly used.
- Collecting DNA – Here there are choices; alcohol precipitation, binding DNA to a spin column, or using silica coated magnetic nanoparticles (MNP) to capture DNA. Employing the classic method of capturing DNA in the presence of salt and alcohol, spin columns can be used. Silica-coated MNPs with handheld magnets also work, and in contrast is a homogeneous solution method compared to solid phase binding. There is a whole lesson on kinetics with these two methods. The washing steps help to demonstrate the concept of partition co-efficient, while the final elution is a solvent change. In short, this is classic normal phase chromatography. A simpler option is simply centrifuge the lysate after the addition of alcohol to emphasize how salt and alcohol reduced DNA solubility causing the precipitation of the nucleic acids.
Summary
There is meaningful chemistry behind every step when nucleic acids are isolated from plants. The beauty of Synergy™ is it allows students to analyze each step without the risks of burns or carcinogens. Aside from the mechanics that can be taught in a lesson using Synergy™, there are also the details associated with each step of the process. For any student moving on to bigger and better science, understanding the process will be useful. Even if kits are part of their future, at least they may be able to troubleshoot problems when they arise.
So, while Synergy™ may be packaged as a kit, it’s much more than that. It’s a bridge between hands-on learning and conceptual understanding, something every future scientist can benefit from.
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