Extracting DNA from plant leaves or seeds is not a novel concept; it is critical to developing future plant-based technologies. There are many downstream molecular biology techniques, such as PCR and NGS, that rely on plant DNA, and nucleic acid extractions are central to the workflows of many academic researchers and commercial breeding programs.
In modern agrobiotechnology and agrigenomics, generating accurate and actionable data for agricultural research relies on high-quality plant DNA, which makes the techniques, as well as the time and cost required to execute them, essential for many plant researchers.
Today, we at Molecular Biology Products will tell you everything you need to know about plant leaves DNA extraction. We are here to provide innovative solutions to the life science industry while establishing a personal connection with each of our valued customers. We are known for reasonably priced, high-quality PCR Tubes with Flat Caps, Cell Culture Plates, and 10ul Filter Tips.
Steps For Plant DNA Extraction
Plant DNA extraction and purification can be divided into six steps in general:
- Disruption/homogenization of the tissue
- Cell lysis in DNA extraction buffer
- Separation of DNA from other cellular components
- DNA precipitation
- DNA washing
- DNA collection/resuspension for downstream processing
While it may appear biochemically simple, it comprises unique technical challenges that even experienced scientists may find difficult to overcome. For this purpose, several plant DNA extraction methods have been developed and used for various applications.
What To Consider Before Choosing A Plant DNA Extraction Method?
With various DNA extraction methods and protocols to choose from, it can be challenging to know which is suitable for your practical application and plant species. Here are some critical considerations for determining a plant DNA extraction method.
Protocols In Terms Of Usability And Length
CTAB (Cetyl trimethyl ammonium bromide) has been the traditional method for plant DNA extraction, but it requires a significant amount of equipment, reagents, and labor-intensive steps. Therefore, performing with a small number of samples can be time-consuming, while processing a large number of samples can be difficult. Even experienced researchers can take several hours to complete this method.
Column and magnetic bead-based protocols are much easier to use and are available commercially as kits with most reagents pre-prepared. Furthermore, the only tools required are a centrifuge or magnetic stand and a bead beater. These two methods can also be used to implement scalable, automated workflows, which would be impossible with the many hands-on steps required for CTAB protocol execution.
Efficiency Of Lysis
Plant cells must be lysed in order to extract DNA and separate it from all other cellular components. The efficiency of cell lysis is proportional to the amount of DNA extracted. Since lignified and thick plant cell walls can be resistant to disruption by CTAB, there can be significant variation in lysis efficiency across samples when using the CTAB protocol. However, bead beating methods, which use physical force and agitation to disrupt plant cell walls, have assisted in overcoming these obstacles by providing a more homogeneous and consistent tissue disruption technique. Bead beating has also enabled difficult-to-lyse plant species more efficiently.
Removal Of Downstream Inhibitors
A diverse range of polysaccharides, polyphenols, lipids, and other secondary metabolites can contaminate purified DNA and inhibit enzyme function, making downstream experiments difficult. However, CTAB protocols, with additives such as polyvinylpyrrolidone, can remove many of these compounds.
In general, it can be challenging to remove all contaminants. Many CTAB protocol changes have been published, particularly for plant species high in these problematic compounds. Immobilization of DNA on a membrane or magnetic bead allows for more efficient washing of extracted DNA and removes many contaminants and downstream inhibitors.
Quality Of DNA
Poor DNA quality can limit its utility in downstream molecular analysis. For instance, low-quality DNA can result in unreliable data or failed sequencing runs. The presence of high molecular weight fragments in high-quality DNA is typically characterized by agarose gel electrophoresis or the use of a Bioanalyzer. Furthermore, it will be free of RNA, salts, phenol, and other secondary metabolites, which can be determined using a spectrophotometer.
As previously stated, protocols based on columns or magnetic beads enable more efficient washing than protocols based on phase separation, DNA precipitation, and pelleting, such as the CTAB protocol.
Conclusive Thoughts
In a nutshell, the plant tissue is lysed by efficient mechanical disruption via bead beating, followed by a series of centrifugation steps using spin-columns to purify the DNA and remove downstream inhibitors. The eluted DNA is ideal for various downstream molecular biology and sequencing applications.
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