Nucleic Acid Isolation or Purification Reagent
Nucleic acid isolation
Our understanding of genetic material has increased substantially since Friederich Miescher first extracted DNA in 1869. He discovered that there is the material inside cells that precipitates from an acidic solution and dissolves in an alkaline solution. He called it nuclein because it seemed to be located within the nucleus.
It was not until 1953 that the structure of DNA was elucidated. It was during this time that procedures for isolating DNA began to emerge. Later, during the 1960s and 1970s, scientists were furiously untangling the cellular environment, and the discovery of RNA with its various forms and functions further refined DNA purification procedures.
There are three general steps for the isolation of nucleic acids.
- cell lysis
- Elimination of proteins, nucleic acids and contaminating salts and deactivation of DNases or RNases.
- DNA or RNA recovery
Step 1: cell lysis
The first step in nucleic acid isolation or purification reagent is to break the cell wall and/or membrane to release the genetic material. This is accomplished with the use of a lysis buffer, rotor homogenizer, bead mill, freeze-thaw cycles, or sonication. Lysis buffer contains a detergent to help break down cell membranes and an enzyme such as protease K to digest protein components. The homogenizer and bead mill provide harsh mechanical shear to break up tissue and cells.
Cell lysis produces a solution in which the cell contents are no longer clearly compartmentalized. Thus, DNases and RNases threaten to enzymatically destroy genetic material. EDTA can be used to deactivate DNases by chelating divalent ions that are necessary for enzyme activity, and RNases are permanently destroyed with beta-mercaptoethanol. Additionally, to decrease the possibility of contamination with environmental RNases or DNases, it is important to use buffers and DEPC-treated water, wear gloves, and maintain a clean workspace.
Steps 2 and 3: removal of contaminants and recovery of DNA and RNA
Guanidinium thiocyanate-phenol-chloroform extraction
This early purification technique takes advantage of the variable solubility of cellular components in organic or aqueous solvents and sensitivity to salt concentration. The addition of phenol and chloroform to the cell lysate causes the solution to fall into a hydrophobic phase and a hydrophilic phase. Nucleic acids will remain in the hydrophilic phase while proteins will be in the hydrophobic phase.
Guanidinium thiocyanate is a chaotropic agent that disrupts hydrogen bonds. When guanidinium thiocyanate is used in a phenol-chloroform extraction, it helps to separate the RNA and DNA into two different aqueous layers. After centrifugation, the RNA will dissolve in the top aqueous layer, with the DNA below, and the proteins will be in the organic hydrophobic layer at the bottom. Guanidinium thiocyanate also denatures proteins, including RNases.
Solid Phase Column Support Systems and Ethanol Precipitation
Nucleic acids can also be collected by ethanol precipitation methods. This technique requires solid phase support systems. The first support system developed for this purpose was a column. The column is static and the solution is poured through it. Under appropriate salt concentrations and pH, nucleic acids bind to the column while other contaminants flow through it. This procedure relies on multiple washing and centrifugation steps to remove contaminants before finally eluting and capturing the DNA or RNA.
Magnetic purification of nucleic acids
More recently, superparamagnetic particles and magnetic separation have been used to capture nucleic acids. This method allows free mobility of the particles within the solution, which improves nucleic acid adsorption and capture efficiency. The superparamagnetic nature of the particles allows them to be manipulated by an external magnet and held in place while contaminating proteins and salts are removed. These systems may be based on salt concentration and ethanol precipitation or may use more sophisticated chemistry for the reversible binding of DNA or RNA in a specific or non-specific manner.