🧬DNA Isolation 🧬

 You probably heard of the Blueprint of life, these term refer to DNA. DNA is a very long molecule made up of chain of nucleotides. The DNA isolation mostly use in Molecular biology and Forensic 

History of DNA Isolation

The very first DNA isolation was done by a Swiss physician, Friedrich Miescher in 1869.

To separate DNA from the proteins Miescher developed new protocol to separate the cells nuclei from cytoplasm and then isolated DNA. However his first protocol failed to yield enough material to continue further analysis. He had develop second protocol to obtain larger quantities of purified nuclei, which had been named as 'nucleic acid' later by his student, Richard Altman.

When the Miescher managed to obtain DNA from cell, many other have followed suit which lead to further advancement in the DNA isolation and purification protocol. 

The initial routine laboratory procedures for DNA extraction were developed from density gradient centrifugation strategies. Meselson and stah used this method in 1958.

Later procedures made use of the differences in solubility of large chromosomal DNA, plasmids and proteins in alkaline buffer. 

Steps of DNA Isolation 

There is a three basic steps of DNA Isolation as follows 

1. Preparation of cell extract or lysis of cell
2. Purification of DNA from extract or Precipitation 
3. Concentrating DNA sample or Purification 

1. Lysis of cell :

There is two ways of lysis 

a. Mechanical disruption, it also refers as physical method. This can be done by using homogenizer (like a small blender), with a mortar and pestle, or by cutting into small pieces. This method particularly important when using plant cells because they have tough cell wall. 

b. Lysis by using chemical like detergent or enzyme. (eg. EDTA, SDS, Proteases) 

2. Precipitation : 

Most common used procedures are 

• Ethanol Precipitation 
• Phenol-chloroform extraction 
• Minicolumn purification 

i. Ethanol Precipitation :

This method done by using a ice - cold ethanol or isopropanol. DNA is insoluble in this alcohol, so it will aggregate together and giving a pellet upon centrifugation. 

ii. Phenol-chloroform extraction  :

In this type of extraction phenol denatures proteins in the samples. After centrifugation of sample, the denatured proteins stay in the organic phase while with the chloroform that remove phenol residues from solution. 

iii. Minicolumn Purification :

It relies on the fact that the nucleic acid may bind (adsorption) to the solid phase (silica or other) depending on the pH and salt concentration of buffer. 

3. Purification : 

Most common method is use of absolute ethanol. Centrifuge with it then air dry the pellet contain the purified DNA threads mix it with TE buffer, slight alkaline buffer or ultra pure water.

   Fig. DNA isolation 

🔶 Analyse the isolated DNA by PCR or gel electrophoresis. 

🔶 To check the DNA purity measures it's intensity at 260 nm and 280 nm wavelengths.