DNA Isolation

Introduction

DNA, also known as Deoxyribonucleic acid, is the primary genetic material of a human living cell. The DNA was a double helix structure when we talked about the microstructure of this genetic material. 

The double helix structure of DNA comprises nucleotides. The DNA molecule consists of two different strands, which are more about wind around each other to form a double helix. The double helix is made up of strands made up of sugar. These sugar bases have phosphoric acid as their acidic content. You will also get to see a special diester bond in the strand. Different nucleotides are attached to the sugar bases; these nucleotides are the basic makeup of the DNA.

The DNA is what gets transferred from a parent to a child and is responsible for giving the younger ones specific genetic traits. All the sugars are attached to different bases, basically four in numbers: adenine, cytosine, thiamine and guanine. 

DNA Isolation Techniques

Different techniques can make DNA isolation. Some of these are relatively new and can help in numerous functions. The technique is all about extracting the DNA from the given sample and purifying and  getting the nucleic acid to get the double helix’s accurate structure.

In the world of advanced technologies and DNA analysis, real-time PCR is something significant in the entire process of DNA isolation. PCR, also known as a polymerase chain reaction, is one of the reactions where the DNA is multiplied numerous times to make the extraction very easy.

PCR is of very high importance and hence one must know about the different techniques and the basics of PCR. The polymerase chain reaction is where sole or multiple pieces of DNA are inserted in special machines to let it replicate according to the given data. 

DNA Purification

There are 5 basic steps to purify a DNA

• Distribution of every cellular structure to create a lysate

• The soluble DNA must be separated from the cell and other impurities

• The DNA must be inserted correctly and should be inserted into the purification mix

• The proteins are washed, and all the contaminants are thrown away from the matrix

• Solution for extraction of DNA

The Creation of Lysate

The first process in any DNA purification would be to purify the nucleic acid. Before one purifier is the nucleic acid, it must be extracted so as to release the DNA or RNA into the fluid medium. The entire goal of creating a lysate would be to completely destroy the cell in a sample and just release the nucleic acid. One must know that there are four different methods for lysate formation-

Physical method

The physical method of the creation of a lysate is something very unique. Basically, in this method, we are trying to grind or crush the entire sample so as to break it physically. One of the best methods out there for physical description is to freeze the entire sample. Once you freeze the entire sample, you can use a motor and a pastel with some liquid nitrogen to grind it thoroughly.

The chemical method

The chemical method is not as complicated or as physical as the method mentioned above. One can use some harsh chemicals so as to break the cell and form the lysate. Disturbing the cell via different regions can quickly help to denature the protein, but one must remember the fact that using specific chemicals is very necessary. If the chemical used is very harsh that it can denature the actual content of the particular cell, then one can expect numerous problems to occur while using this method. Some of the chemicals which are commonly used are SDS or some alkaline salt solutions.

Enzymatic methods

Enzymes are particular proteins that can help in numerous functions. If you are not aware of the fact that enzymes also exist in the human body, then be ready to be amazed as there are thousands of enzymes in a running human body that are basically responsible for maintaining the static metabolism.

The selection for enzymes must be dependent on the type of cell wall and the different materials one can get inside the cell to be denatured or to be destroyed—enzymes such as lysozyme, proteinase K, lipases are suitable. The enzyme activities out there are dependent on numerous factors such as temperature, PH and more. 

Clearing the Lysate

You must notice that due to the usage of harsh methods such as physical and chemical methods, the entire matrix is covered with debris. One must remember that nucleic acid purification is essential to isolate the DNA. Depending upon the material, the dust particles from the cells like proteins, lipids and polysaccharides must be removed. 

One of the most significant ways of clearing the entire lysate would be by centrifugation. Filtering can be swift and rapid, but with vast amounts of debris, the filter becomes clogged. Once the lysate is cleared, the DNA could be purified and could be used with different chemicals so as to store and precipitate.

Binding to the Purification Matrix

Once the nucleic acid is purified, it needs to be bound to a specific purification Matrix such as silica or cellulose, which can give a firm base on the nucleic acid. For instance, if you don’t bind It to the matrix, there is a high chance that even after the excellent isolation of the DNA and purification of the left DNA, you won’t be able to precipitate the DNA.

Depending upon the chemistries and the selective binding of RNA and DNA, there are basically two different preparation Matrix that is used these days-

• The first one is solution-based chemistry- this one is a unique chemistry that does not release the binding Matrix but is basically dependent on alcohol preparation and precipitation. DNA could be precipitated in isopropanol

• The other one would be silica-based chemistry, in which the binding of DNA to the silica under huge salt concentration takes place

Isolation of DNA from Plant

The Isolation of DNA from plants is basically dependent on the same procedure as mentioned above.

The cell walls are first broken, and as the main component of cell walls in most of the plant Kingdom is cellulose, you must need liquid nitrogen and enzymes that can digest cellulose.

After the cell membrane is disrupted, the DNA is released into a particular extraction buffer.

In the next step, DNA is also protected from the restriction enzymes by using special chelating agents.

DNA could be broken by turbulence or by extreme vibrations. The primary genetic material must be released by the shock; things can go wrong for the initial DNA isolation. 

Some of the special types of plant isolation methods include genomic extraction, crude method, and ionic detergent CTAB (cetyltrimethylammonium bromide). 

Conclusion

From the above article, we will like to mention that DNA isolation is a complicated process, and there are a lot of things to be taken care of before extracting and isolating the DNA. The above article mentions everything you need to consider regarding DNA isolation.