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How to Isolate Genomic DNA from Plants
Introduction
Extraction and isolation of DNA from animals are comparatively 
easy as different tissues from different types of species will have rather similar characteristics. But in the case of plants, the extraction of genomic DNA from divergent species is extremely difficult because of the production of various metabolites and biomolecules by them. Also, the biochemistry between different species is considerably different.
When it comes to the isolation of DNA from plants two types of plant biomolecules, polyphenols and polysaccharides pose a major problem. These biomolecules interfere with the various steps during the isolation of DNA.
Steps Involved in The Isolation of Plant DNA
In order to isolate DNA from the plants and to choose any method for isolation following things should be kept in mind. They are −
Method used should give the highest concentration of DNA.
The type of tissue selected should give the highest concentration of DNA.
The method which is opted for extraction should be suitable for PCR amplification.
The CTAB method of DNA Isolation
To overcome the difficulty of isolating genomic DNA from the plant tissues CTAB method is used which is also the most commonly used method. CTAB stands for cetyltrimethylammonium bromide.
This method was developed in 1980 by Doyel and Doyel, and from then it is used to extract DNA from plants.
The CTAB Procedure
In the first step the plant tissue is frozen with the help of liquid nitrogen. After the tissues are frozen, they are ground to a fine powder with the help of a mortar and pestle.
In the next step, to the finely grounded powder the CTAB buffer is added. This CTAB buffer helps in the lysis of the plant cell and facilitates the release of genomic DNA. It also stops biomolecules like polysaccharides and phenols from interfering with the isolation procedure.
Once the cell has been lysed, an enzyme called RNase is added to digest the RNA. DNA is also separated from the cellular debris with the help of chloroform and phenol mixture.
The whole mixture is centrifuged to form two distinct layers. The first layer is the organic phase that contains the non-polar molecules and the second layer is the aqueous phase that contains DNA and polar molecules.
The aqueous phase is centrifuged again and again till it becomes clear, and the DNA is collected.
DNA present in the clear aqueous phase is precipitated with isopropanol. This solution is centrifuged again till a small pellet of DNA is formed at the bottom.
This pellet contains additional salts which are removed by washing with 70% ethanol. Once the salts are removed the residual ethanol is allowed to evaporate and the pellet is re-suspended in a suitable buffer.
An alternate method for DNA Precipitation
Phenol and chloroform are generally used for extraction of high molecular weight DNA. But both these compounds are potentially hazardous and pose some serious medical conditions like burns and cancer.
To avoid these health hazards an alternative method is used which skips the usage of phenol and chloroform and can be used to isolate DNA fragments of smaller sizes. This method is known as the solid phase isolation method.
Storage of Extracted DNA
Once the DNA is formed in the form of a pellet it can be suspended in sterile water for a short duration and should be immediately used.
But if DNA has to be stored for a longer duration, then TE (Tris-EDTA) buffer should be used.
If the PCR process follows the DNA extraction, in that EDTA hinders the process, in that case, extra amount of magnesium ions should be added.
Limitations of Plant Genomic DNA Isolation
Extraction of DNA by CTAB method is very time taking, tedious and cumbersome and the product obtained is also very less. Extraction of DNA from one sample takes about 2 hours which increases as the number of samples increases.
Liquid nitrogen is extremely dangerous as its slightest spillage on the skin causes cold burns. Phenol also causes burns and chloroform is potentially carcinogenic and it should be stored carefully and disposed of safely.
Grinding and pasting with mortar and pestle can cause variations in the quality of the DNA obtained. Therefore, lender can be used to make fine paste which can increase the quantity of the DNA.
After the centrifugation step the aqueous phase and the organic phase should be separated carefully and is a tedious process. A slight disruption may lead to the mixing of DNA with the debris again.
Phenol and other salts which are used throughout the isolation procedure may remain with the DNA even after repeated washes with ethanol.
Automated Alternatives
Nowadays automated processes like bead beating and column-based isolation and purification methods are available which can be completed in 15 minutes, are more consistent, DNA isolated is also of high purity, can be streamlined as per the laboratory requirements, and keep the technicians safe from hazardous chemicals.
Conclusion
In most of the laboratories, research facilities, and colleges DNA from plants is extracted by the traditional CTAB method. But due to the lengthy procedure and low throughput, it has been replaced by faster ways of extraction which can save time and give high yield and can be used for plants like Cannabis and cacao whose DNA is difficult to extract enabling novel and rapid advancements in modern plant genomics.
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