Several different types of DNA extraction methods are Phenol-chloroform isoamyl alcohol, Proteinase K, CTAB method, spin column-based methods and magnetic bead-based technique. However, which method to use depends on the sample type and Purity & yield of DNA obtained.
DNA extraction or DNA isolation is a process to isolate or obtain high-quality DNA from biological samples.
Depending upon the type of sample, every DNA extraction method varies, for example, the DNA extraction method for plant DNA is different from that of the blood. Likewise, the bacterial DNA isolation method is different from other types. Meaning, we need varieties of DNA isolation technique for different sample.
This article is focused on DNA extraction; what is it and how to do it. Along with this, we have explained different methods of DNA extraction in detail. We also have explained the advantages and disadvantages of each method. In addition, we have compared methods to clarity the topic more accurately.
We first understand some basics regarding the topics and will proceed further. let us start with the history of DNA extraction.
History of DNA extraction
The first DNA extraction attempt was performed by Friedrich Miescher in 1869. He has isolated the cell material and named it the “nuclei” later on his student named it, a “nucleic acid”. Although he accidentally developed a method for nucleic acid isolation, 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 Nurnberger 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. The present method is most popular among researchers. Also, the yield, purity and consistency of this method are pretty decent.
What is DNA extraction?
The simplest explanation of DNA extraction is, “extracting DNA from cells”.
We can explain it in different ways based on the method, chemical and assay used. 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 we get DNAs from cells.
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- deoxyribonucleic acid 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:
Meaning, the whole process of DNA isolation is of three important steps; cell lysis, precipitation and dissolving DNA. The outline of the whole method is as followed.
Lysis of cell wall/ cell membrane:
- Chemical disruption
- enzymatic disruption
- Mechanical disruption
Lysis of nuclear membrane:
- Chemical lysis
- Enzymatic lysis
Removing cell debris
different chemicals and combinations of different chemicals are utilized for various purposes. The nuclear membrane and cell membrane are 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 them. 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 them. 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 henceforth 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 on 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 the 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 a good quality of result in PCR.
However, the addition of a simple lysis buffer during heating will increase the yield and quality of DNA. The composition of different cell lysis buffer is explained in this article:
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 plant cell wall.
Some of the fungus, algae and bacteria also have hard cell wall for surviving in harsh conditions. For extracting DNA from this type of cell, we have to modify the protocol with a combination of mechanical- chemical- enzymatic methods.
Mechanical lysis is an important step in the isolation of DNA from pectin-rich cells. For doing this; mortar pestle, grinding and liquid nitrogen are used. Our article on Plant DNA extraction here: CTAB DNA extraction buffer for plant DNA extraction.
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 excellently.
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 to get good results. The combination of mechanical- chemical and enzymatic lysis helps to extract good quality 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.
The chemical DNA extraction methods are also known as solution-based methods whilst the solid-phase DNA extraction is a type of physical method.
Chemical or solution-based DNA extraction method:
Chemical or solution-based method uses various organic and inorganic solutions. Note that the main steps remain similar among all methods, viz, cell lysis, precipitation and elution.
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 present method is less recommended. Notwithstanding, the phenol-chloroform method is the best among all.
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.
The proteinase K-based method is an inorganic solution-based technique for DNA isolation.
The 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 DNA extraction. However, the method gives excellent results in combination with proteinase K.
One of the major limitations of the salting-out method is the purity, though enough yield can be obtained, the quality obtained might not be good. We had prepared two different salt solutions 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 referred to as a phenol-chloroform and isoamyl alcohol or 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 the lysis of the cell membrane as well as the nuclear envelope. The organic component of the technique- phenol and chloroform denature the protein portion of cells.
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 the lysis of the cell membrane.
EDTA– EDTA is a chelating agent and can be used to block DNase activity. DNase is an enzyme that lyses the DNA. However, every enzyme required a cofactor to work efficiently. 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 sulfate is an anionic detergent that helps cell membranes and nuclear envelopes to break open.
NaCl– the Na+ ion of NaCl creates the ionic bond with the negative charge of DNA and neutralizes it. It will help DNA comes together and protect from denaturation.
MgCl2– overall, it protects the DNA. MgCl2 blocks the negative charge of the lipoproteins of the cell membrane. After cell lysis, there is no compartment in the cell hence it protects DNA by mixing with other cell organelles.
Phenol– 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. The role of alcohol in precipitation is explained in a separate article, a link is given somewhere in the article.
After lysis and precipitation, we have to dissolve DNA in either TE buffer or water in the final step. It’s an organic solution method for DNA extraction.
The PCI method of DNA extraction is widely accepted. Even the forensic departments trust the PCI method rather than the Kit method. The quantity of DNA obtains by the 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 samples required for PCI DNA extraction is high. It is difficult to isolate DNA from the samples such as hair and nail. Also, the purity of DNA becomes a major issue if not performed well. Read more on Phenol chloroform DNA extraction: Basics, preparation of chemicals and protocol.
Enzymatic method of DNA extraction:
Actually, this method is a combination of a salt method as well as an 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 the sample, all other cell debris is 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.
Silica is a solid substance that 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 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 the sample many times and have to collect the 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 image represents the general process of the Spin column method for DNA extraction.
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 pass through the silica column.
Now the DNA can be washed twice for improving the purity. The aqueous phase contains the impurities that 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 DNA extraction.
The positively charged chelax 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 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 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 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.
Purpose of DNA extraction or DNA isolation:
The main purpose of DNA extraction or DNA isolation is the same, to provide pure DNA. Direct bodily cells or tissue can not be used in DNA testing.
For doing a DNA test for DNA fingerprinting, DNA heritage testing or DNA diagnosis testing we should require a pure DNA. Using different chemicals and solutions, all the other organelles and debris of cells are isolated from the DNA and pure DNA is extracted.
The pure DNA must have a ~1.80 260/280 ratio. Impurities in DNA may result in PCR inhibition.
Thus, the purpose of DNA extraction is to provide pure, unfragmented and highly concentrated DNA for doing a DNA test or PCR.
My ultimate Guide for DNA extraction
I had worked with all three types of DNA extraction methods, yet, PCI DNA extraction method is one of my favorites because only the PCI method gives you the freedom to do something different with your work.
If you are working with a 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 given protocol. You cannot do something new with that.
Instead, in PCI you can play with the protocol (once you have achieved that expertise). 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 DNA extraction method. 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, even 20µL of a sample is sufficient for getting good DNA. 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 a PCI DNA extraction method.
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 to 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 to 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.
- Eslami G, Khalatbari-Limaki S, Ehrampoush MH, Gholamrezaei M, Hajimohammadi B, Oryan A. Comparison of Three Different DNA Extraction Methods for Linguatula Serrata as a Food-Borne Pathogen. Iran J Parasitol. 2017;12(2):236–242.
- Dilhari et al. “Evaluation of the impact of six different DNA extraction methods for the representation of the microbial community associated with human chronic wound infections using a gel-based DNA profiling method. ”AMB Expr (2017) 7:179. DOI 10.1186/s13568-017-0477-z.
- Kelly M. Elkins. “Chapter 4: DNA extraction.” Forensic DNA biology (2013): 39-52.