CTAB DNA extraction buffer is more suitable for extracting DNA from the plant tissues. Because of the high content of the secondary metabolites, proteins, polysaccharides and polyphenolic compounds into the plant cell CTAB DNA extraction buffer is the first choice in the plant DNA extraction.
The plant DNA extraction process is quite different or we can say difficult than other DNA extraction methods. The routine protocols employed in the DNA extraction for animal cells and bacterial cells are not applicable to the plant DNA extraction.
We will try to answer the question in this article.
In the present article, we will try to understand why extracting DNA is difficult in the plant cell. Also, the core value of the article is the CTAB method for plant DNA extraction.
The content of the article is,
- Introduction to DNA extraction
- Why plant DNA extraction is difficult?
- How to extract DNA from the plant cell?
- CTAB extraction buffer for plant DNA extraction
- CTAB plant DNA extraction method
- Modification of CTAB DNA extraction protocol
- Application of CATB DNA extraction method
- Limitation of CATB DNA extraction method
DNA is the same in all living organisms, made up of Adenine, Thymine, Cytosine and Guanine. The backbone of the DNA is made up the phosphate while the hydrogen bond joins two opposite strands.
Read more on the structure of DNA: Structure of DNA
Some of the organelles such as mitochondria, endoplasmic reticulum, nucleus and nuclear envelope etc are the same in all types of tissue but the structure and the composition are different in different cells. Also, each cell serves a different function in different tissue.
Majorly, two factors are responsible for the difficulties in the plant DNA extraction:
- The difference in the structure of the cell
- The component of the plant cell
The structure of the plant cell is different than the animal cell. The plant cell contains the cell wall while the animal cell does not have the cell wall.
The animal cell contains a soft and smooth cell membrane while the plant cell contains the hard, rough and solid cell wall.
The plant cell wall is made up of the peptidoglycan, pectin, cellulose and chitin like compounds. These types of compound give extra strength to the cell wall.
Due to the rigid nature of the plant cell wall, the shape of the plant cell is fixed. While the cell membrane of the animal cell is smooth and soft, hence it is flexible, adjust as per the requirement.
The components of the plant cells are also different than the animal cells. Importantly, the major difference is the presence of chloroplast in plant cell.
The chloroplast is the organelle involved in the photosynthesis process. With the help of the photosynthesis, it produces secondary metabolites and/or food.
Secondary metabolites cannot be synthesised into the animal cell because of the lack of the chloroplast.
Overall, the structure of the plant cell is more complicated than the animal cell hence DNA cannot be extracted easily from the plant cell.
Another Factor is,
The turgor pressure:
The term “turgor pressure” is commonly used in the plant physiology and in the botany but not common for the genetic student.
In simple words, the water pressure which implemented on the cell wall of the plant cell from the inside of the cell is a turgor pressure.
Turgor pressure helps the plant to stand straight. The animal cell membrane cannot bear the turgor pressure. The cell ruptured.
The polysaccharides present in the cell wall gives the extra strength to the plant cell, to bear the pressure.
“The protocol for plant DNA extraction varies from plant to plant as different species of the plant contains different types of secondary metabolites and different types of cell wall polysaccharides.”
Introduction to DNA extraction
We had already discussed on DNA extraction in our previous article.
Lets quickly look at the process of DNA extraction,
Extracting DNA from the cell by disrupting the cell wall/ cell membrane and nuclear membrane with the help of the chemical, physical or mechanical methods is called DNA extraction.
In the year 1980, Murray & Thompson developed an inexpensive and simple protocol for the plant DNA extraction with the help of the CTAB.
Dellaporta (1983) and Saghai Maroof and coworkers (1984) are the pioneers in developing plant DNA extraction methods.
The major modification was done by the Edwards and coworkers. They used CATB as well as SDS for DNA extraction and extracted DNA from the plants such as corn and soybeans.
Major steps in plant DNA extraction are,
- Lysis of cell wall with the help of the physical methods and chemicals such as grinding and liquid nitrogen.
- Separating DNA from the other organelles by CTAB and SDS like chemicals.
- Precipitation of the DNA by alcohol.
- Purification of extracted DNA by 70% alcohol.
- Dissolving DNA by Tris and EDTA buffer.
Read more on,Role of alcohol in DNA extractionTE buffer in DNA extraction DNA sequencing
What is CTAB and why it is used in plant DNA extraction?
CTAB, cetyltrimethylammonium bromide, also called as Cetrimonium bromide or hexadecyltrimethylammonium bromide. The molecular formula of CTAB is [(C16H33)N(CH3)3]Br. It is a cationic detergent commonly used in DNA extraction. It is soluble in water as well as in alcohol. The CTAB is corrosive and toxic in nature.
The generalised and specialised structure of the CTAB is given into the figure below,
Apart from its use in DNA extraction, the CTAB is the best antiseptic and commonly used against bacteria and fungus.
CTAB is the major ingredient in cosmetics and household use as a detergent.
SDS is also a detergent just like the CTAB but why only CTAB is effective in plant DNA extraction?
As we discussed in the early sections, the plant cell contains so many cell wall polysaccharides, proteins and secondary metabolites.
The cell wall contains mainly hydrophilic end groups, the detergents such as CTAB can dissolve both polar as well as groups.
CTAB can bind with the cell wall polysaccharides and protein and by complexing it, it coprecipitates the macromolecules along with the DNA which is removed by the centrifugation.
“Do you know the CTAB is used in the SDS-PAGE?
The positively charged CTAB can readily bind with the negatively charged glycoproteins during protein electrophoresis. Instead of fuzzy bands clear and, sharp bands can be achieved by using a pinch of CTAB into the SDS PAGE. “
How to extract DNA from the plant cell?
Plant DNA extraction is a tedious process.
First, we have to prepare a sample before proceeding further.
The plant have solid and rigid tissue, we must have to make is homogeneous before DNA extraction.
The combination of physical method and a chemical method such as grinding and Liquid nitrogen treatment helps to prepare plant sample before DNA extraction.
The tissue or the leaf or any part of the plant is taken and grind it with the help of mortar and pestle.
Role of liquid nitrogen in plant DNA extraction:
The liquid nitrogen is predominantly used in the plant DNA extraction.
During the grinding, the heat is generated which activates enzymes such as endonuclease and exonucleases which cleaves our DNA into small pieces.
Adding liquid nitrogen prevents the enzymatic reaction. Also, the liquid nitrogen froze the tissue and helps in making a fine powder of the tissue.
Liquid nitrogen helps in making the homogeneous mixture of the tissue. It instantly froze the tissue to the point where it becomes ash.
Addition of the extraction buffer to the sample prevent our sample from other damage.
Tannin, the villain of plant DNA extraction
The tannin is the long chain of the phenolic ring which are major constituents of a polyphenolic compound. It is naturally occurring in the plant but it is secreted during the tissue damage as well. The polyphenolic compounds are the major inhibitors of PCR reaction.
The polyvinyl pyrrolidone (PVP) binds to the phenolic ring of the tannin and prevents it’s interaction with DNA and protects it.
Hence PVP is the main ingredient along with our CTAB for the plants which contains a high level of tannin or polyphenolic compounds.
Beta mercaptoethanol plays a supporting role in this method. It is used as an antioxidant which prevents the oxidation of the polyphenolic compounds and helps in removing it.
CTAB DNA extraction buffer for plant DNA extraction
Preparation of CTAB buffer for plant DNA extraction is varying from protocol to protocol because different plant species contain different amount of cell wall polysaccharides and secondary metabolites.
Depending upon the type of the starting tissue major ingredients of the CTAB extraction buffer are CTAB, Tris, EDTA, beta-mercaptoethanol, PVP (polyvinylpyrrolidone) and NaCl etc.
Other chemicals such as SDS and combination of phenol: chloroform: isoamyl alcohol can also be used in combination with the CTAB DNA extraction buffer.
We had already discussed the role of Tris, EDTA, SDS and beta-mercaptoethanol in the previous articles.
Read the role of different chemicals in DNA extraction: Different types of DNA extraction methods.
Importance of Tris and EDTA in the DNA extraction: TE buffer in DNA extraction.
For CTAB extraction buffer preparation, we have to prepare the CTAB solution first.
10% CTAB solution:
10% CTAB 100g/ 1000ml
0.7 M NaCl 41g/ 1000ml
d H2O to 1000ml
In general 2% CTAB solution is used in the plant DNA extraction protocol for that the recipe of the 2% CTAB is:
2% CTAB 20g/ 1000ml
0.7 M NaCl 41g/ 1000ml
d H2O to 1000ml
The 2% CTAB solution is readily used in the CTAB DNA extraction buffer.
CTAB DNA extraction buffer:
- 2 % CTAB
- 100 mM Tris pH 8
- 20 mM EDTA
- 1.4 M NaCl
- 1-2 % PVP polyvinylpyrrolidone 40
- 0.2 % Beta mercaptoethanol Add just before use; (20 µl per 10 ml solution)
2X CTAB extraction buffer
2% CTAB (w/v) 20g
100mM Tris(PH 8.0 , 1M) 100ml
20mM EDTA(PH 8.0, 0.5 M) 40ml
1.4 M NaCl 81.8g
d H2O upto 1000ml
|Components||10X stock solution||2X working solution||Volume for 1000mL|
|Mercaptoethanol||2%||0.4% (roughly)||Add just before use|
|D/W||–||Up to 1000ml final volume|
2X CTAB DNA extraction buffer can be readily used for the plant DNA Extraction.
While preparing the CTAB extraction buffer, keep your eyes on some important points.
Prepare only 10% CTAB solution instead of 2% CTAB,
Because dissolving CTAB every time is time-consuming.
Then store it at room temperature or at 37°C. Storing at a low temperature will precipitate the CTAB.
Same way prepare TE buffer and 10% PVP in the stock solution.
Prepare the working solution freshly every time from the stock reagents. If you have a heavy workload, prepare 10X CTAB DNA extraction buffer for the whole day or for two days.
Never use more than 2 days old CTAB extraction buffer.
Use of salt is another important factor in plant DNA extraction. Scientists do not pay attention to the role of salt. The DNA, as well as the polysaccharides both can be soluble in a salt solution or both, can be insoluble. It depends on the concentration of salt in the CTAB extraction buffer.
DNA is insoluble at low salt concentration while polysaccharides can be insoluble at a high salt concentration or vice verse.
So use an appropriate amount of salt in CTAB buffer.
Read further,Gene editing and CRISPR-CAS9 Role of Telomeres in ageing DNA data storage
CTAB plant DNA extraction method
The CTAB DNA extraction method is simple and effective. Beside, the CTAB buffer, other ingredients are RNase, proteinase K, SDS and PCI (optional).
- Take 5gms of fresh plant tissue and cut it in the small pieces.
- Add liquid nitrogen to the tissue and roughly grind the sample into the mortal and pastel.
- After the tissue becomes powder, add 500 μL of CTAB extraction buffer and beta-mercaptoethanol, grind it again followed by the vortexing for 5 minutes.
- After it becomes homogeneous, take the mixture into the 2ml Eppendorf tube.
- Boil the sample for 30minutes at 60°C to 65°C.
- Centrifuge the sample at 25,000rpm for 5 to 8 minutes and transfer the supernatant into another tube.
- Be careful, take the only supernatant, don’t mess the supernatant with the debris.
- Now add 5 μL to 10 μL of RNase solution to the supernatant and incubate at 37°C for 25 minutes.
- The proteinase K step is additional (you can use it if necessary).
- Centrifuge the sample at 10,000rpm for 2 minutes and take supernatant to another tube.
- Now add chilled isopropanol (70%), a pinch of NaCl to the supernatant and precipitate the DNA by inverting the tube several times.
- Centrifuge the precipitate for 10,000rpm for 2 minutes and collect the pellet (remove the supernatant).
- Now wash the DNA pellet with alcohol two times at 9000 rpm for 2 minutes.
- After the clear pellet appears, add 500μL of TE buffer or elution buffer to the pellet and dissolve the DNA in it.
Bonus tips: if the DNA precipitates are not observed after the addition of the chilled alcohol, then place the tube into -20°C for 15 to 20 minutes and precipitate it. The precipitate will surely appear.
Modification in CTAB DNA extraction protocol
Many modifications are possible with the CTAB DNA extraction protocol. The protocol is not always working for all type of plants. Though DNA is obtained, the quality and the quantity of the DNA yield might not be satisfactory.
For different types of plant species such as the plants with a high level of polyphenolic or alkaloids compound, required extra skills and modification into the protocol.
The first modification is the use of SDS. The sodium dodisylsulphate is the powerful anionic detergents.
In the year 1991 Edwards et al., used SDS in combination with the CTAB in the plant DNA extraction. They had used 0.5% of SDS with Tris, NaCl and EDTA in their protocol.
Note: store the beta-mercaptoethanol at 4°C temperature.
Another modification is the use of the proteinase K enzyme in the CTAB DNA extraction method.
The enzyme proteinase K digests the proteins present into the plant cell as well as cell wall protein because the plant contains a high amount of different types of protein as well.
Proteinase K gives the best results in a combination with the RNase. After the RNase treatment, proceed the sample for the proteinase K digestion for overnight or for 2 hrs at 60°C.
Another modification is the best method for routine use. We can use PCI, phenol: chloroform: isoamyl alcohol.
The proportion of the PCI is same as given into the previous article. Prepare phenol freshly and follow the guide given into the article: Phenol, chloroform and isoamyl alcohol DNA extraction method.
The best modification is the use of all three techniques viz. Mechanical lysis, proteinase K method and PCI method in a single protocol along with the CTAB.
PCI can be used after the proteinase K digestion step followed by the centrifugation at 2500rpm for 20 minutes.
Urea is another excellent component which can be a game changer in your plant DNA extraction protocol. We can use urea during the grinding of the tissue in the very first step.
The urea is so powerful that it along can capable of doing DNA extraction, eliminating the need for organic solvents such as phenol or CTAB.
Use of Urea in plant DNA extraction:
- Grind the tissue in mortal and pestle and add Urea buffer (contains Urea, Tris, EDTA and NaCl).
- Add liquid nitrogen and homogenise it.
- Take a sample in the Eppendorf tube and centrifuge it at 12,000rpm for 2 minutes.
- Discard pellet and take supernatant in another tube
- Add chilled ethanol or ammonium acetate
- Wash the sample with the alcohol (minimum 2 wash)
- Dissolve the DNA in 500μL of TE buffer.
The use of urea/ urea plant DNA extraction method is easy, fast and reliable for routine PCR and restriction digestion. However, the quality of DNA obtained by this method might not be sufficient for DNA sequencing and microarray analysis.
Source: Genomic DNA isolation from Maize by Kristen A. Leach, Paula C. McSteen, David M. Braun
Bonus tips: the process of grinding is the king for achieving success in plant DNA extraction. Grind well, hard and very roughly and make a super and even homogeneous solution of the tissue.
Proper grinding will definitely give you the best DNA yield.
The CTAB DNA extraction method is simple yet effective. It is cheap and most reliable in the DNA barcoding and RAPD analysis of different plants.
The combination of CTAB, SDS, proteinase K and PCI is the best for any kind of DNA extraction.
The CTAB extraction method is an organic solvent-based method. The harmful nature of organic solvents can be a major limitation of the present technique.
The phenol is volatile and can burn the skin.
The chloroform can make us faint or unconscious.
CTAB is hazardous and dangerous for human health.
Also, the use of liquid nitrogen can damage human tissue and burns the skin.
Finally, last but not least is the importance of autoclave.
As like the other chemicals, the process of the autoclave is also very crucial and important. Because DNAse can be a major threat for DNA, autoclave all the reagents after preparation.
Autoclave, CTAB solution, CTAB extraction buffer and SDS solution. Further, autoclave all the plasticware and glassware before use.
Remember, never autoclave alcohol and phenol.
Also, use DD/W or nuclease-free water during the preparation of chemicals.
Do some research before DNA extraction,
understand the physiology and anatomy of plant species you selected. Collect information on your sample, it’s cell wall structure and composition. Also, note down the concentration and type of secondary metabolites and polyphenolic compound the plant secrets.
Based on the data, select your own plant DNA extraction protocol.
Our Articles on DNA extraction:
- Different types of DNA extraction methods
- Phenol chloroform DNA extraction method
- Proteinase K DNA extraction method
- Role of alcohol in DNA extraction
- How to prepare lysis buffer for different types of DNA extraction methods?
Plant DNA extraction is tedious and very challenging technique. The variability in the structure of the cell wall and the level of secondary metabolites into plant restricts DNA extraction protocol.
The DNA extraction protocol standardised for one plant species will surely not work for the other plant species.
Still, the use of CTAB is a clever decision always. CTAB extraction buffer is recommended for any type of plant sample, however, modifications are always required.
For more specialised and sensitive reactions such as microarray and DNA sequencing, you can use the ready to use kit which gives you good results but not 100% surely.