Nucleic Acid Isolation & Purification
Overview
The isolation and purification of nucleic acids from different sample types typically includes these steps:
Sample Lysis and Homogenization
Nucleic acid extraction and isolation begin with breaking down cellular structures or homogenizing samples that contain genetic material—such as bacterial and mammalian cells, plant tissue, fungal tissue, mammalian tissue, blood, plasma, serum, viruses, buccal and nasal swabs, gel matrices, PCRs, and other enzymatic reactions. This process is typically carried out using compatible detergents, mechanical disruption, and/or heat.
Nucleic acid purification methods
Several methods for nucleic acid purification are available, and each method is based on a different biochemical principle.
Extraction using organic solvents:
In this protocol the cell lysate is mixed with phenol, chloroform and isoamyl alcohol for the separation of nucleic acids from proteins. This method gives a high yield, but traces of organic solvents often contaminate the sample. Furthermore, the use of these substances toxic to health make it necessary to carry out the entire process is in a chemical fume hood.
Adsorption on a silica column:
In the presence of certain salts, nucleic acids are retained by adsorption on silica columns. The columns are then washed with salt solutions to remove unbound particles, and the nucleic acids are finally eluted with water or a low-salt solution.
Magnetic separation:
In this method the lysed samples are mixed with magnetic beads that can bind to nucleic acids. After a series of washes for maximum purification of the genetic material, the beads are removed from the solution by a magnetic separator.
The final application will determine the most suitable purification method. These applications include PCR, qPCR, restriction enzyme digestion, ligation, cloning, genotyping, gene expression analysis, next-generation sequencing (NGS), as well as Northern and Southern blotting.
