Different types of DNA extraction methods are available for different cell types. For example, the DNA extraction method for plant DNA is different from that of the blood.
In this article, we are going to discuss different types of DNA extraction methods which are mostly used in genomic labs. We will focus on the principle and the mechanism of each type of DNA extraction method.
We will compare each DNA extraction method based on their performance and yield. Further, we will also discuss the advantages and limitation of different DNA extraction methods.
Outline of this article:
- History of DNA extraction
- Definition of DNA extraction
- How to obtain DNA
- Different methods of DNA extraction
- Phenol-chloroform method for DNA extraction
- Enzymatic DNA extraction method
- Silica- (spin)Column based DNA extraction method
- DNA extraction using anionic resins
- Magnetic bead method of DNA extraction
- CsCl density gradient DNA extraction method
History of DNA extraction
The first DNA extraction attempt had performed by Friedrich Miescher in 1869. He had isolated the cell material and named it as the “nuclei” later on his student named it as a “nucleic acid”. Although he accidentally developed a method for isolation of nucleic acid, he was not sure that what he isolated was DNA or not.
Later on, in 1958 Meselson and Stahl developed a full-function protocol for DNA extraction. The density gradient centrifugation protocol was the first protocol described by isolating DNA from E.coli bacteria.
The protocol of the proteinase K enzyme method of DNA extraction was developed by Lahiri and Nurenberger in 1991. They also modified the protocol by using the Nonidet P40 and SDS. However, the use of proteinase K in DNA extraction was reported earlier by Miller et al., in 1988.
The phenol chloroform isoamyl alcohol method which is most popular in recent days was developed by Joseph Sambrook and David W. Russell.
What is DNA extraction
“Extracting DNA from the cell” this is the simplest explanation for DNA extraction.
Several definitions of DNA extraction are enlisted here,
“Isolating DNA by disrupting cell wall/cell membrane and a nuclear membrane is called a DNA extraction”.
“Isolation of DNA by breaking the cell membrane and nuclear membrane with the help of chemicals, enzyme or physical disruptions is defined as a DNA extraction”.
Or simply, “isolating nucleic acid from rest of the cell organelle is called nucleic acid extraction or DNA extraction”.
How to obtain DNA
Well, I don’t have the exact label to define this section, but still, I want to discuss this portion with you because it is important to understand how DNA is obtained from the cell.
First, we will understand the anatomy of the cell, roughly. The cell is made up of cytoplasm and cell membrane/ cell wall. The cytoplasm contains several organelles such as mitochondria, ribosomes, nucleus, endoplasmic reticulum etc.
The animal cell does not have the cell wall, plant cell and (most) bacterial cells contain the cell wall. I am not going to discuss each and every component of the cell wall or cell membrane because that is not the point of discussion.
Ok, coming to the point,
So if we want to isolate DNA we have to break cell wall/ cell membrane and nuclear envelope as well. Also, we have to remove other cell organelle debris. In the final step, we have to precipitate and purify the DNA.
Our articles on DNA precipitation:
This is the outline, we have to follow these steps for obtaining a good quality of DNA.
Lysis of cell wall/ cell membrane:
- Chemical disruption
- enzymatic disruption
- Mechanical disruption
Lysis of nuclear membrane:
- Chemical lysis
- Enzymatic lysis
Removing cell debris
The nuclear membrane and cell membrane is made up of protein and lipids almost. Hence the same types of chemicals can work for both.
Chemicals such as SDS, CTAB, Tris and other detergents can lyse the cell wall/ cell membrane by solubilizing it. Each chemical has a different function to play which we will discuss separately in each method.
Enzymes such as proteinase K, peptidase, protease disrupt proteins by digesting it. The enzyme works better than any other chemicals because it directly targets bonds between the amino acids and digests the protein.
Once the cell wall or cell membrane lysed, there are no compartments inside the cell hence all the cell organelles are mixed into the solution. By doing high-speed centrifugation DNA remains in the solution and the other cell debris settled into the bottom of the tube.
Moreover, the DNA extraction method varies depending upon the type of cells. Take look at some example here,
The cell having a soft cell wall: some of the bacteria have a very smooth and soft cell wall. For example, M.tuberculosis has a smooth cell wall. By only heating the bacterial solution we can lyse cell wall.
The supernatant can directly be used for the PCR. Even, by putting the bacterial culture directly into the PCR tube for 15 minutes at initial denaturation we can directly get the good quality of result in PCR.
However, the addition of a simple lysis buffer during heating will increase the yield and quality of DNA.
Our series of articles on PCR,
- A Complete Guide of the Polymerase Chain Reaction
- PCR reaction: Ten secrets that nobody tells you
- What is TB (Mycobacterium Tuberculosis) PCR?
The cell having a harder cell wall: Plant cells have pectin and other polysaccharides present in their cell wall. This pectin protects the cell from mechanical damage. Therefore pectin provides additional strength to the cell wall of the plant.
Some of the fungus, algae and bacteria also have hard cell wall for surviving in harsh conditions. For extracting DNA from this type of cells, we have to modify protocol with a combination of mechanical- chemical- enzymatic method.
Mechanical lysis is an important step in the isolation of DNA from pectin-rich cells. For doing this mortal pestle, grinding and liquid nitrogen is used.
Read more on plant DNA extraction method.
The cell having a cell membrane: The cell wall is not present in animal cells. A combination of the enzymatic and chemical method is most suitable for the DNA extraction from animal cells, However, each method individually performs the best.
The phenol-chloroform method is one of the best choices for animal cells.
How to do mechanical lysis
Take plant tissue and grind it with a mortar and pestle. Grind it well, and add liquid nitrogen carefully and again grind it even harder until the tissue becomes powder. Finally, add lysis buffer or DNA extraction buffer. Grind it till it becomes homogeneous. The tissue is ready for DNA extraction.
Additionally, we can add an enzyme to this homogenized solution and incubate it for an hour. The combination of mechanical- chemical and enzymatic lysis helps to extract good quality of DNA from the plant cell. This combination most probably gives the best result.
Different types of DNA extraction methods
DNA extraction methods are broadly categorized into two categories:
- Chemical based DNA extraction method.
- Solid phase DNA extraction method.
Chemical or solution based DNA extraction method:
Different types of organic and inorganic solutions are used in the chemical or solution based DNA extraction method.
The steps of the DNA extraction remain the same in all the types of DNA extraction methods.
SDS, CTAB, phenol, chloroform, isoamyl alcohol, Triton X100, guanidium thiocyanate, tris and EDTA are several common chemicals used in the solution based DNA extraction method.
The solution based ( also called chemical) DNA extraction method is subdivided into organic solvent-based DNA extraction and inorganic solvent-based DNA extraction.
The organic solvent-based DNA extraction method is based on the use of organic substances such as phenol and chloroform. Due to the harmful nature of the phenol and chloroform, the method is restricted.
Nonetheless, phenol-chloroform DNA extraction method is one of the best methods among all.
Among the inorganic DNA extraction, two are most popular: use of proteinase K and use of salt.
The proteinase K DNA extraction method facilitates high DNA yield but the method is time-consuming. Also, if not maintained well in a cold chain, the proteinase K cannot be utilized for a longer period of time.
The lower stability of the enzyme is another major issue in this method.
Salting out DNA extraction method is safer than the PCI method. The use of salts such as sodium chloride, potassium acetate and ammonium acetate helps in the DNA extraction.
However, the method is more aggressive in combination with proteinase K.
Use of the different salt in DNA extraction can increase the yield but the purity is not good enough. We had prepared two different salt solution in this article: phenol-chloroform DNA extraction method.
Phenol-chloroform method of DNA extraction:
This method is one of the best methods of DNA extraction. The yield and quality of DNA obtained by the PCI method is very good if we perform it well. The method is also called as a phenol-chloroform and isoamyl alcohol, PCI method of DNA extraction.
The major chemicals of PCI DNA extraction methods are lysis buffer, Phenol and chloroform.
The lysis buffer contains Tris, EDTA, MgCl2, NaCl, SDS, and other salts. Here the components of lysis buffer help in lysis of cell membrane as well as the nuclear envelope. The protein portion of the cell denatured with the help of chloroform and phenol which are organic in nature.
Role of chemicals:
Tris: DNA is pH sensitive, Tris buffer maintains the pH of the solution. Also, it interacts with the lipopolysaccharides of the cell membrane and makes them permeable, this will help in lysis of the cell membrane.
EDTA: EDTA is a chelating agent and can be used to block DNase activity. DNase is an enzyme which lyses the DNA. However, every enzyme required cofactor to work properly.
The chelator EDTA blocks the activity of DNase by blocking the cofactor binding site. It will work best in combination with Tris.
SDS: Sodium dodecyl sulphate is an anionic detergent which helps cell membrane and nuclear envelope to break open.
NaCl: the Na+ ion of NaCl creates the ionic bond with the negative charge of DNA and neutralize it. It will help DNA comes together and protect from denaturation.
MgCl2: overall, it protects the DNA. MgCl2 block the negative charge of the lipoproteins of the cell membrane. After the lysis of cell, there is no compartment in the cell hence it protects DNA by mixing with other cell organelles.
Phenol: it precipitates the protein impurities.
The SDS removes the negative charges from the amino acid and disrupts the confirmation of a protein. Therefore, the protein loses its structure and stabilized by using the SDS.
The combination of phenol, chloroform and isoamyl alcohol helps in the removal of protein. After centrifugation, the phenol settles in the bottom of the tube and DNA in the aqueous phase while the denatured protein remains between both layers as a whitish cloud.
So care must be taken to collect nucleic acid from this method. The collected nucleic acid is precipitated with the help of chilled alcohol (isoamyl alcohol). We can add salt as well to increase the yield of the DNA.
Finally, the DNA is dissolved in TE buffer. Because of the use of the organic solvents, this method is also named as an organic solvent-based DNA extraction method.
The PCI method of DNA extraction is widely accepted. Even the forensic departments trust PCI method rather than a Kit method. The quantity of DNA obtains by PCI method is very high. We can obtain 800 to 900 ng of DNA with great quality.
If we prepare all chemicals very well and perform it sincerely we can always get a good result by this method.
However, the amount of sample required for PCI DNA extraction is high. It is difficult to isolated DNA from the samples such as hair and nail. Also, the purity of DNA becomes a major issue if not performed well.
Some of the interesting articles,
An enzymatic method of DNA extraction:
Actually, this method is a combination of a salt method as well as enzymatic method. Here the extraction buffer is used before going further on enzymatic digestion.
The extraction buffer composition may vary from lab to lab, however, the major components are Tris, EDTA, NaCl, sodium lauryl and SDS. Here phenol, chloroform or isoamyl alcohol is not used. Instead, the enzyme proteinase K is utilized for digesting the sample.
The sample is incubated with proteinase K for 2 hours this will digest all the protein present inside the sample.
Immediately after the proteinase K digestion, the sample is precipitated by chilled alcohol. By centrifuging sample, all other cell debris are removed. Finally, the DNA pellet is dissolved in TE buffer.
This method of DNA extraction is rapid and easy. We can use ready to used DNA extraction buffer. Even the yield is very high. However, the quality of DNA is a major concern for this method.
We had covered a whole article on this method. Read it here: Proteinase K DNA extraction method
Solid phase DNA extraction method:
Nowadays all the DNA extraction kits available are based on the unique chemistry of the solid/ liquid phase DNA extraction.
The silica is the solid substance which binds with DNA during purification along with it, different solutions are used to purify the DNA.
The solid phase silica is one of them.
The main advantage of silica gel based DNA extraction is that it is rapid and gives “PCR ready DNA” for the downstream applications. No extraction or precipitation steps are required therefore this method is superior among all.
Silica column based DNA extraction method
The silica column based DNA extraction method is very unique and different from other DNA extraction methods.
In the PCI or proteinase K method we have to centrifuge sample many times and have to collect aqueous phase or pellets depending upon the step of extraction. Sometimes we have to collect aqueous phase or sometimes we have to collect pellets.
The silica-based DNA extraction method works on the unique chemistry of interaction between silica and DNA. A positively charged silica particles bind with the negatively charged DNA and hold it during centrifugation.
The method was first described by McCormick in 1989. However, the idea was developed in 1979, when silica was used in DNA purification by Vogelstein.
The silica-based solid phase DNA extraction method is now commercially available and it is most routinely used in diagnostic laboratories. Because of its good quality DNA yield and minimal simple operating system, it is widely accepted.
The lysis buffer breaks the cell membrane and the nuclear envelope. The proteinase K digests all the protein.
In the very first step, the sample is incubated with a cell lysis buffer or called a DNA extraction buffer.
Along with it, a small amount of proteinase k is added to the sample. All the other impurities are removed by centrifugation. Here the DNA remains bounded with silica and other impurities passes through the silica column.
Now the DNA can be washed twice for improving the purity in it. The aqueous phase contains the impurities are discarded by discarding the collection tube.
Finally, the DNA is dissolved into the TE buffer. The method is fast reliable, accurate and consumes less time as compared to other methods.
Other DNA extraction methods are DNA extraction using the anionic resins, magnetic bead DNA extraction method and CsCl density gradient DNA extraction method.
DNA extraction using the anionic resins:
The Chelex the product of Bio-Rad laboratories is the best example of the anionic resin used in the DNA extraction.
The positively charged chelex binds to the negatively charged phosphate of DNA and helps in the extraction of DNA. However, sometimes several negatively charges contaminants also interfere with DNA extraction by binding with the anion.
The chelex is made up of the styrene-divinylbenzene copolymers. The method for using chelex as an anionic resin for DNA extraction was first described by Walsh et al., in the year 1991, the sample they had used was the blood.
Another resin used by Seligson et al., was the diethylaminoethyl.
In this method, the column of the tube is filled with positive resins. The cell lysate passes through the matrix and DNA binds to the positively charged resins.
By applying the low concentration salt buffer, proteins and other impurities or debris are washed off and only DNA remains into the matrix.
In the final step filling the matrix with the high concentration salt buffer the DNA is eluted from the resins and precipitated using the alcohol.
The method is also called as DNA extraction through anion exchange chromatography.
DNA extraction by magnetic beads:
Personally, I had never worked with magnetic bead DNA extraction but I know little about the magnetic bead method of DNA extraction. Positively charged magnetic beads attract the negatively charged DNA. The DNA is separated under the magnetic field. DNA extraction buffer is needed in this technique also.
CsCl density gradient method of DNA extraction:
In this method, the DNA is separated based on the density of it with the centrifugation.
In the high-speed centrifugation, at the isopycnic point where the density of the DNA and the gradient (CsCl) become same, the DNA band will appear.
However, the method is tedious and hard to perform as it required hi-speed centrifugation (10,000 to 12,000rpm) for more than 10 hours.
Still, the plasmid DNA can be isolated more easily than the supercoiled DNA.
Another major limitation of the density gradient centrifugation is the use of the carcinogenic EtBe in the DNA extraction.
The EtBr intercalates between the strands of DNA and separates supercoiled DNA from the non-supercoiled.
My ultimate Guide for DNA extraction
I had worked with all three types of DNA extraction methods, yet, PCI DNA extraction method is one my favourite because only the PCI method gives you the freedom to do something different with your work.
If you are working with ready to use-kit, the buffers, spin column tubes and other utilities are provided by the kit manufacturer, you have to just follow the steps as per the protocol is given. You cannot do something new with that.
Instead, in PCI you can play with the protocol (once you have achieved that expertize). You can do changes to buffer composition, you can use different chemicals, different physical methods etc to enhance your result and that is the beauty of working with PCI.
Nonetheless, care must be taken while performing PCI. The phenol is a volatile organic chemical which can burn your clothes and skin.
Also, chloroform is dangerous to health. So you have to be careful while dealing with PCI.
Our PCI protocol is very precise, we got a result from using the only 20µL of a sample. I will give you my DNA extraction protocol of PCI later on in another article.
Phenol can be oxidized under the sunlight hence always store phenol in a brown coloured umber bottle.
Wear a lab coat, gloves, goggles and mouth cape while performing PCI method of DNA extraction.
In the case of the enzymatic method of DNA extraction, Store proteinase K at 4ºC temperature after reviving it otherwise the activity will reduce over a period of time.
Mixing sample with buffer or chemical is very crucial, don’t take it granted. As you mix the sample well, the purity and quantity of DNA will automatically increase.
DNA extraction step
Lysis of cell wall/ cell membrane and Lysis of nuclear membrane
Tris, MgCl2, EDTA, NaCl, SDS, CTAB, Triton X100
Digestion of protein
CTAB, SDS, phenol, chloroform, Nonidet P40, different chaotropic, urea, guanidium isothiocyanate, guanidium thiocyanate, N-Lauroyl sarcosine
Precipitation of DNA
Isopropanol, ethanol, methanol, NaCl, sodium acetate
Washing of DNA
TE buffer, distil water
Conclusively, we can say that the choice fo DNA extraction method entirely depends on us. So many modifications fo DNA extraction methods are available in the literature. We have to select our own method from available different types of DNA extraction methods.
I have enlisted different chemicals used in each step, you can choose the appropriate chemical and design your own protocol.
Detailed PCI DNA extraction method, DNA extraction method for plant tissue and for other tissues I will explain you in the next article.
Read another article: CTAB DNA extraction buffer for plan DNA extraction
Till then, stay connected with this article.
Comment in the comment box below and contribute to increasing the content of this article.
Read more on agarose gel electrophoresis,
Conclusively, what types of DNA extraction we want to use depend on our requirement. However, the best method in the research lab is the PCI method and for diagnosis, the spin column ready to use DNA extraction kit is the unmatched choice.