Methods of nucleic acid extraction and purification

Sample preparation of nucleic acid involves a wide range of methods to obtain a sample that fits analytical technique requirements from a sample that cannot be analyzed directly. Nucleic acid samples can be used for PCR or reverse transcription (RT). Since nucleic acid quality can affect downstream reactions’ performance, in experimental workflow sample purification and quality assessment are two important steps. 

RNA Isolation 
The first step of nucleic acid purification process when working with tissues or cells as the starting material is membrane permeabilization or cell lysis. The cell membranes are broken open in this process and a cell lysate is created by disrupting the cellular structure. The nucleic acid of interest can to be accessed through this process. Cell lysis can be achieved either chemically with a lysis buffer or via mechanical techniques such as sonicating, douncing, and grinding. 

Lysis buffers are designed to rupture cells by osmosis and their compositions can be tailored for specific applications. In order to disrupt membrane lipids, lysis buffers typically contain Tripton or NP-40 detergent. The lysis buffer can also contain chelating agents like EGTA and EDTA in order to inhibit nuclease activity. Cell lysis can be facilitated by proteases such as proteinase K. In order to isolate the nucleic acid, the resulting cell lysate may go through a chemical separation such as phenol / chloroform extraction, a solid phase-based separation, or a physical separation such as centrifugation. 

DNA Isolation 
DNA can be purified from yeast, bacteria, mammalian cell cultures as well as animal and plant tissue by using a DNA filtration column. DNA of interest can be separated from unwanted contaminants or cellular debris by adjusting the salt and pH of the solution. The DNA is binding to a silica membrane located at a filter column’s bottom. In plasmid and genomic DNA purification, this is a popular method. 

Centrifugation and vacuum protocols are available. The DNA is subjected to a low and high stringency wash being bound to the membrane, in order to remove contaminants such as lipids proteins, and RNA. An elution buffer is used for extracting the purified DNA from the membrane into a collection tube. Depending on the final concentration of DNA or plasmid desired, the volume of the elution buffer used can vary. The DNA recovered can be used with Southern blot and PCR analysis. 
Cesium chloride gradients have been traditionally used for purifying plasmid DNA from genomic DNA. Highly pure DNA is provided by this method. However, this requires removal from the recovered plasmid of cesium chloride and ethidium bromide, being time consuming. A more efficient and faster means of purification is provided by plasmid purification kits. Protein RNA and other cellular components are removed, while DNA is adsorbed onto the silica-based membrane. 

Automated extraction systems 
An automated nucleic acid extraction system can also be used. Automatic extraction systems are using robotic liquid handling technology in order to streamline the tasks involved in nucleic acid sample extracting, such as cherry picking and serial dilution. These automated systems also include typically functions such as temperature control, shaking, and PCR protocols. 

Nucleic acid extraction can be used in various types of biological research including pathology, forensics, molecular biology, drug discovery, and environmental research. When choosing an automated nucleic acid extraction system is recommended to look for features such as full start-to-finish automation, integration with laboratory information management systems, safeguards gains contamination, and error control.