The key steps of DNA extraction is give below.
- Sample disruption
- Clearing cell debris
- Purification
- Concentration DNA
Sample disruption.
rapid and complete disruption of cells in sample releasing DNA into lysate. there are several methods to disruption of the cell wall.
- Physical methods – grinding with mortar and pestle under liquid nitrogen.
- Chemical methods – Detergents are used (SDS,CTAB) it inhibit the DNases activity and denature the proteins.
- Enzymatic disruption – used disrupt the cell walls. (Lysozyme, proteinase K)
Main components of the lysis buffer.
- Tris buffer – DNA is pH sensitive, buffer maintains maintains the pH solution.
- EDTA – DNase inhibitor
- NaCl – maintains the ionic strength of the lysis solution
- Detergent – inhibit the DNases activity and denature the proteins,aiding the removal of proteins from the solution
- Other salts
Clearing cell debris
Cell debris are removed in order to prevent the clogging during the purification. Done by centrifugation and filtration.
Purification
Removal of RNA – Carry out by RNase A which degraded the RNA molecules.
Deproteinization –
Organic solvent-based purification – Nucleic acids hydrophilic molecules therefore easily soluble in water, protein are mainly hydrophobic residues making them partially soluble in organic solvents. main organic solvents are phenol and chloroform. limitations of this method – time consuming, phenol is highly toxic, require the mixing of phenol phase with water phase it cause to damage of the DNA molecules.
Inorganic solvent based purification – Proteinase K method – breakdown the proteins and inactivate the nucleases. Salting out method – at high salt concentration protein will precipitate. but this method is not very suitable for downstream analysis.
Solid-phase extraction (spin-column based)- It depends on the ionic strength and the pH of the surrounding medium. In presence of a high concentration of salts DNA will bind to silica surface, protein will not binds. then nucleic acids are eluted under the low or non – salt conditions.
Concentration DNA
Two alcohols are used ethanol and isopropanol. usually carried out 70% ethanol in the sodium or ammonium salts.
Significant of the CTAB based extraction methods
At high NaCl concentration nucleic acids do not form complexes with CTAB however form complexes with the proteins and polysaccharides.
