Many methods of molecular biology, like PCR, cloning, and sequencing, require DNA extraction and purification. Proteinase K is a compound that is generally utilized in DNA extraction and decontamination systems.
Proteinase K is a serine protease catalyst that is disengaged from the organism Tritirachium collection. It has a 28.9 kDa molecular weight and is highly active and stable over a wide pH (4-12) and temperature range (up to 65°C). It is also resistant to numerous detergents, chelating agents, and denaturants.
In this blog post, we will talk about how Proteinase K helps to extract and purify DNA.
What Part Does Proteinase K Play In DNA Extraction?
The cell or tissue samples are first lysed to release the cellular components before DNA extraction can begin. To digest the proteins and remove them from the mixture, Proteinase K is added to the lysate. The lysate’s proteins have the potential to hinder subsequent processes like PCR, cloning, and sequencing. For this reason, high-quality DNA samples can only be obtained by eliminating proteins.
Proteinase K breaks down the structure of proteins by hydrolyzing the peptide bonds in the proteins. It breaks the peptide bond of aliphatic and aromatic amino acids like tryptophan, tyrosine, phenylalanine, and leucine close to the carboxyl group. The enzyme is active in the presence of SDS, which is a strong denaturant that disrupts the protein structure and exposes the cleavage sites.
Nucleases that have the potential to degrade DNA samples can also be effectively digested by Proteinase K. The DNA is broken up by nucleases, enzymes that break the phosphodiester bonds in the backbone of the DNA. They can be released during the lysis process and are found in cell or tissue samples. Proteinase K can process the nucleases and keep them from harming the DNA tests.
Function Of Proteinase K In DNA Purification
Following the removal of the proteins from the lysate, phenol-chloroform extraction, spin columns, and magnetic beads are used to purify the DNA samples. In addition, some of these purification procedures employ Proteinase K to eliminate any remaining proteins from DNA samples.
The DNA is extracted using phenol and chloroform, organic solvents that separate the DNA from the lysate’s other components, in the phenol-chloroform extraction method. To digest any remaining proteins in the aqueous phase, Proteinase K is added to the mixture. This makes it easier to get rid of the contaminants and get pure DNA samples.
Proteinase K is added to the binding buffer or lysis buffer during the spin column and magnetic bead purification procedures to digest any proteins in the sample. After that, the contaminants are washed away as the DNA samples are bound to the spin columns or magnetic beads. To digest any remaining proteins in the final DNA samples, Proteinase K is also added to the elution buffer.
DNA purification and extraction both rely heavily on Proteinase K. It is used to get rid of proteins and nucleases from the lysate, which can cause problems for applications that follow. After the purification process, it is also used to digest any proteins that remain in DNA samples. Proteinase K is an excellent enzyme for use in molecular biology because it is highly stable and active across a wide pH and temperature range. Obtaining high-quality DNA samples for a variety of molecular biology techniques can be made easier with an understanding of the function that Proteinase K plays in DNA extraction and purification. You can get in touch with MBP Inc. We have everything from Proteinase K 100 mg to a storage buffer set that can help in DNA extraction.