DNA isolation- What, Why and How ?

Here, we can have a brief discussion on the fundamentals of DNA isolation.

What is DNA isolation?

The process of isolating DNA from variable sources such as blood, tissue, cell culture, buccal swab etc. A source of DNA is needed for every experiment in molecular biology, forensic, genetic labs etc. This has become a routine procedure. 

Why the DNA isolation is important?

• To study about the cancer markers 
• To detect genetic diseases
• To develop new drugs
• To detect bacterial and viral diseases
• Paternity tests
• Criminal investigations

How the DNA is to be isolated?

The common features of all types of DNA isolation are,

Now we can see the details of the DNA extraction,

1. Cell lysis or disruption can be done either in physical/chemical method. 

• Physical method includes grinding/sonicating especially for plant cell (tough cell wall!). 
• Chemical method includes treating with sodium dodecyl sulphate (SDS) or sodium lauryl sulphate (SLS) which helps in solubilizing the cell membrane.
• Chemical method also includes the addition of enzymes which helps to digest DNA associated as well as other proteins. E.g., Proteinase K for animal cells, Lysozyme for bacterial cells etc.

2. Precipitation of DNA (separates the DNA from cell debris, detergents, proteins etc.)

• Ice cold absolute ethanol or isopropanol is used to precipitate the DNA. DNA is insoluble in alcohol.
• Monovalent salt such as sodium acetate is also added. The sodium ions (positively charged) react with phosphate ions in the DNA and make the DNA less hydrophilic means less soluble in water. This accelerates the precipitation process by alcohol.
• The salt and alcohol are removed by 70% alcohol.

3. Purification of DNA

• DNA can be re-suspended in TE buffer (Tris-EDTA buffer, pH 8).

What is Next? 

Now, we must quantify the DNA obtained. DNA quantification means checking for its purity and concentration.

• The agarose gel electrophoresis can be used to confirm the presence of DNA.
• UV spectrophotometer or Nanodrop can be used to determine concentration and purity of DNA. The ratio of absorbance at 260nm and 280nm (A260nm/A280nm) should be equal to 1.8 for pure DNA. If it is <1.8, protein contamination and if it is >1.8, RNA contamination.
• By using the fact that 1 absorbance is equal to 50 µg ml^-1, determine the DNA concentration using the following equation.  

50*A260nm = Concentration of DNA in µg ml^-1

References

• P. Arora, M. (2005). Genetic Engineering (1st ed.). Himalaya Publishing House
• W. (2010). Principles and Techniques of Biochemistry and Molecular Biology (7^th ed.). Cambridge India 

• DNA extraction. (2009, June 18). Science Learning Hub. https://www.sciencelearn.org.nz/resources/2036-dna-extraction

Have a great day!

Thank you!