Proteinase K is one of the best DNA extraction methods to date. The method is accurate, reliable and fast. The proteinase K DNA extraction method is also called as an enzymatic method of DNA extraction because of the important role of proteinase K enzyme in the DNA extraction.
In this article, we will discuss the DNA extraction by proteinase K enzyme. The major topics of the article are:
Introduction to DNA extraction:
DNA extraction is an important step in any genomic technique ranging from the polymerase chain reaction to microarray and from real-time PCR to next-generation sequencing.
For extracting DNA or isolating DNA from the cell we have to break cell membrane (or cell wall in case of Plant cell) and nuclear membrane.
For breaking nuclear membrane as well as cell membrane different chemicals are utilised which are enlisted in the previous article: Different types of DNA extraction methods.
The first DNA extraction attempt had performed by Friedrich Miescher in 1869.
We can break the cell membrane and nuclear membrane of the cell by different methods such as mechanical method, chemical method and enzymatic method.
In this article, we will explain the enzymatic method in combination with the chemical method for extracting pure DNA.
Notably, obtaining High quality DNA is the prime importance of any DNA extraction method. The DNA obtained from each DNA extraction method must be nearby ~1.80 260/280 absorbance ratio with more than 100μg of concentration.
The detailed explanation of DNA extraction is discussed in The article: Different types of DNA extraction methods. Read it first.
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Introduction of proteinase K
Proteinase K= Protein (long chain of amino acid) + ase (suffix for enzyme) + K (keratin)
Why keratin is associated with proteinase is a very interesting story. The keratin is a hair protein and the hardest form of protein. Proteinase is the first enzyme isolated from the fungus Tritirachium album which can digest the hardest form of protein, “Keratin”. Therefore the K is given as suffix for the enzyme.
Proteinase K is a member of the family subtilisin, typically a proteinase. The molecular mass of the proteinase K is ~28, 930kDa. The enzyme is typically a protein which is made up of the long chain of approximately 278 amino acids.
As we all know every enzyme required a cofactor for performing a reaction. For example, the Mg2+ ion is the cofactor for the Taq DNA polymerase enzyme similarly, calcium ion is the cofactor for the enzyme proteinase K.
The first proteinase K is isolated in the year 1974 and it is the most commonly used endopeptidase; proteinase in DNA extraction.
Role of proteinase K digestion in DNA extraction
Of course, it digests the protein, but how?
The major portion of the cell is made up of the protein. The nuclear membrane, enzymes, ligands, receptors, chaperons, antibodies and other receptor-specific legends are made up the only protein.
Also, the cell membrane and nuclear membrane is made up of the composite protein such as phospholipid, glycoprotein and sphingomyelin. These are the several molecules which are made up of the proteins along with some lipids, carbohydrates and other molecules.
The proteinase K digest the protein portion of these molecules. Further, it digests the protein associated with DNA. The protein is made up of the long chain of amino acids. Depending upon their nature of interaction with water, it is categorised into the hydrophilic (attract water ) and hydrophobic (water repel) amino acids.
The proteinase K digests mainly the hydrophobic amino acids ( both aromatic as well as aliphatic). The cofactor, calcium ion provides stability to the enzyme, however, it cannot increase the activity of the reaction.
Highest proteinase K activity is reported at the temperature 60ºC ranging from 37ºC to 70ºC. The temperature vs enzymatic activity graph is shown below.
Furthermore, the stability of the enzyme reported highest at the pH 8.0 (which is required in DNA extraction). However, the enzyme is active at pH 4.0 to up to 10.0. the graph of pH vs the activity of the enzyme is shown below,
The proteinase K gives the best results in the presence of several other chemicals. Chemicals such as Sodium dodecyl sulphate, EDTA, urea, CTAB and Nonidet P40 can be used for protein digestion. In combination with these chemicals, proteinase K increases the efficiency of the result by increasing the purity of the DNA.
Further, proteinase K works incredibly good along with several other enzymes such as trypsin, chymotrypsin and RNase. It depends on us, what types of tissue we are using for the DNA extraction. We can use a different combination of proteinase K, enzyme and chemicals.
Preparation of proteinase K solution
The activity of enzyme decreases over a period of time so we have to store the proteinase K enzyme below 4ºC temperature. At the Lower temperature, the enzyme remains inactive.
The commercially available proteinase K enzyme comes in a powder form. It is extracted from the fungus. We have to prepare a stock solution as well as working proteinase K solution depending upon our requirements.
The proteinase K powder lyophilised either into the water or into the buffer.
If it is lyophilised into the buffer, we do not need to add any other extra ingredients such as buffer or glycerol. Just add the desired amount of water to the vial.
If it is lyophilised only into the water we have to add all other ingredients which are required for the optimum activity of the enzyme. Other ingredients are 10mM Tris (pH 8.0), 1mM EDTA (pH 8.0), 3mM CaCl2 and 50% glycerol. We can use TE buffer directly (at pH 8.0).
For the DNA extraction, 200μg/ml of proteinase K working solution is sufficient. Generally, the proteinase K provided in 20mg/ml concentrations by the manufacturer.
Let’s do some calculations,
20mg = 20,000μg
1ml = 1000μL
20,000μg/ 1000μL is our stock solution (provided by the manufacturer)
The requirement for the working solution is 200μg/ml
C1= 20,000μg/ 1000μL
V1 is a volume given is unknown (?)
C2 is concentration required = 200μg
V2 is volume required= 1000μl
V1= 1000 * 200/ 20,000
When we add 10μL of proteinase K (from the 20mg/ml stock) to the 990μL water, the working solution becomes 200μg/ ml, we can use 1μL directly.
Proteinase K DNA extraction protocol
There are many variations in proteinase K DNA extraction protocol. However, the variation depends on the requirement of the researcher and the type of tissue.
I am describing the protocol for DNA extraction from the blood.
- Take 2 ml of the blood sample and add 10 to 20μL of TE buffer to the sample. Mix well.
- Centrifuge the sample at 2500 rpm for 20 minutes
- Discard the supernatant and add 10 to 15μL of TE buffer to the pallet and mix it gently.
- Centrifuge the sample at 2500 rpm for 15 minutes. Discard the supernatant and add 20μL of proteinase K solution (commercially available ready to use) and 2 ml of DNA extraction buffer to the falcon tube.
- Incubate the sample at 56℃ to 60℃ for 1 to 2 hrs or until the pallet dissolve properly.
- Now add 1 to 2 ml of chilled isopropanol and a pinch of NaCl to the falcon tube and invert the tube for some time to obtain the precipitate.
- Centrifuge the tube at 8000 to 10,000 rpm to settle the pellet at the bottom.
- Discard the supernatant and add 1ml ethanol and centrifuge it at 10,000rpm for 1 to 2 minutes.
- Discard the ethanol and dry the pellet in the dryer now add TE buffer (pH 8.0) to the pellet and put it in a water bath for 2 to 3 hrs.
This protocol is for 2ml of the sample, you can design your own protocol for 1 ml or even 500μL sample. calculate each chemical accordingly.
The DNA extraction buffer composition varies from protocol to protocol as well. For the broad use include Tris, EDTA, NaCl, SDS or Triton X 100 or any other salt into the DNA extraction buffer.
The concentration required for DNA extraction buffer is mentioned in the previous article: Different types of DNA extraction methods.
Use of Phenol and chloroform: by using the combination of Phenol chloroform and isoamyl alcohol in the Proteinase K DNA extraction method can increase the efficiency of the result.
After the TE wash, we can add PCI follow by proteinase K addition step.
Use of CTAB and RNase: in case of plant DNA extraction, a combination of CTAB along with RNase will give the best result.
Also, we can use triton X 100 or Nonidet P40 along with DNA extraction buffer.
Application of proteinase K DNA extraction method
The proteinase K DNA extraction method is one of the most successful DNA extraction methods reported to date. It is the first choice for genomic DNA extraction for microarray and sequencing.
The DNA obtained from the proteinase K method is highly pure and the quantity of DNA is also very good. Further, the method is simple and rapid.
It is also used in mRNA extraction. It is very effective for deactivation of DNase and RNase.
Limitation of proteinase K DNA extraction method
The only limitation of this method is the proteinase K itself. It is an enzyme, hence the shelf time of the proteinase K is very less. We have to preserve it at 4℃ temperature. Also, it can only be processed on ice otherwise the activity will decrease.
I had worked on this method during my college days However, I personally not recommend this method because of the instability of the enzyme proteinase. But recently I am quite interested in working with proteinase K DNA extraction method because of the Qiagen.
The Qiagen developed a unique type of chemistry for proteinase K which is even working at room temperature. We do not need to store it in a cool place.
I personally prefer to use Qiagen proteinase K in DNA extraction. It is the best product and the yield of DNA is also very good. If you are a genetics student you must have to learn proteinase K DNA extraction method.