🧬 DNA and RNA EXTRACTION | Nucleic Acid Isolation | Adwoa Biotech

The goal in DNA extraction is to break open cells: this involves breaking open not only the cell membrane but also the nuclear membrane.
Friedrich Miescher in 1869 did DNA isolation for the first time
(Nalina Gupta, J Cytol. 2019 Apr-Jun; 36(2): 116-117).
When you're extracting DNA, the aim is to use a technique that gives you a decent quantity and quality of DNA. And by quality we're referring to pure DNA that is devoid of contaminants, such as RNA and proteins.
Or if you're extracting RNA, then you want pure RNA without DNA or proteins - which are components of a cell.
Nucleic acid extraction is achieved with reagents such as detergents; these are added to a buffer to help make holes in the cell membrane (permeabilises the membrane). This permeabilisation allows the contents of the cell to spill out. The next steps are to remove the protein fractions of the cell. The process includes addition of Protease (proteases are enzymes that degrade proteins), as well as heating the lysate so that the proteins denature and become single threads of amino acid. In this process, a reagent such as chloroform may be added for what is termed a liquid phase extraction of the nucleic acid (DNA/RNA). THe purpose of the Chloroform is to emulsify the mixture so that we can separate it into an organic and aqueous layer. The proteins move into the organic layer, while the nucleic acids move into the aqueous layer on top. In between the aqueous and organic layer is a very thin interface layer which contains lipids and other insoluble matter.
The aqueous layer is removed to a separate tube and ethanol is added to precipitate the DNA out of solution and concentrate it. The purpose of the ethanol (or isopropanol) is to reduce the solubility of the nucleic acid, allowing it to pellet out of solution when centrifuged.
Several wash steps (with ethanol or isopropanol) is further performed to remove salts that are soluble in alcohol.
Finally, the DNA is resolubilised in a small volume of MiliQ water or salt buffer such as Tris EDTA (TE), in order to have concentrated nucleic acids for use in other applications.
An alternative and safer method, is to use what is termed a solid phase extraction. Here the DNA after lysing the cell, is bound to s solid support such as silica or magnetic beads. DNA adsorbs to silica beads/particles at a specific pH in the presence of specific salts while the use of magnetic beads encourages DNA to bind reversibly to magnetic beads coated with DNA-binding antibodies. then the ethanol washes to remove salts are performed. Magnetic beads uses a coating that can bind nucleic acids reversibly by just adjusting buffer conditions. This approach removes the need for vacuum or centrifugation, which minimizes stress or shearing forces on the target molecules, requires fewer steps and reagents.
Silica-based nucleic acid purification methods use a bind-wash-elute process. Nucleic acids bind to the silica membrane in the presence of chaotropic salts. Polysaccharides and proteins do not bind well to the column and residual traces are removed during the alcohol-based wash steps, along with the salts.
There are yet other methods that can be used. So keep in mind that DNA extraction techniques are numerous and may involve either organic extraction such as the phenol-chloroform method presented), or a nonorganic method (involving salting out and proteinase K treatment), or adsorption method (silica-gel membrane or magnetic beads).
How much can we recover?
Each diploid cell in a typical mammalian cell contains ~6 picograms (pg) of DNA.
If you're looking at haploid germ cells like sperms and eggs, they will have ½ of that (3 pg).
So an average white blood count in a ml of adult blood (where they range from 5 - 10 x10^6 cells), will yield 30-60 ng/ml
RNA on the other hand is sightly different. Depending on the state of the cell (whether it's quiescent, replicating, metabolically active), it will have different amounts of RNA in the cell. This can range from 10-30 pg of total RNA per cell.
Thus, to calculate the theoretical yield of DNA of a sample, we need to know how many cells are in the sample.
Additional notes;
The ethanol precipitation is performed with 95% (or higher) ethanol. This is followed by 70% ethanol to gently break the pellet loose and wash it. This removes some of the salts present in the leftover supernatant and bound to DNA pellet making the final DNA cleaner.
Finally, the pellet is air-dried before resuspending the DNA in sterile water or other desired buffer. It is important not to over-dry the pellet as it may lead to denaturation of DNA and make it harder to resuspend.
Isopropanol can also be used instead of ethanol; the precipitation efficiency of the isopropanol is higher making one volume enough for precipitation. However, isopropanol is less volatile than ethanol and needs more time to air-dry in the final step. The pellet might also adhere less tightly to the tube when using isopropanol.

[@adwoabiotech]

Which DNA extraction method does your lab use?

[@Dinka-EnglishTV5]

I did DNA extraction by method of Maxi prep for the first time. I didn’t even understand the others steps. I’m willing to learn more methods and other steps.

[@adwoabiotech]

It takes time to digest all the information but repeat it several times and it'll be locked in. The Maxiprep allows isolation of larger amounts of DNA. What was your sample source for the extraction?

[@Dinka-EnglishTV5]

@@adwoabiotech we were extracting Plasmid DNA of bacteria E.coli

[@adwoabiotech]

Journal Reference: www.ncbi.nlm.nih.gov/pmc/articles/PMC6425773/

[@jacquimurphy4394]

I struggled to listen to it due to the music which is a shame as sounds like it may have been useful 😢

[@adwoabiotech]

The music in this video is barely audible. You may have had another browser opened, that was playing music?

[@albertofabbri1543]

@@adwoabiotech Sorry but I do not agree with you. The music is not as low as you say and it makes harder to understand what is being said.

[@adwoabiotech]

@@albertofabbri1543 Ok, thanks. I appreciate you letting me know. I will keep it in mind for future videos.