Introduction
What is Nucleic Acid Extraction?
In the very simplest of terms, nucleic acid extraction is the removal of the RNA and/or DNA from a sample and all the excess that is not necessary. The process of extraction isolates the nucleic acids from a sample and yields them in the form of a concentrated eluate, free from diluents and contaminants that could affect any downstream applications.
Applications of Nucleic Acid Extraction
Purified nucleic acids are used in a plethora of different applications, ranging across multiple different industries. Healthcare is perhaps the area where it is used most, with purified RNA and DNA required for a host of different of different testing purposes.
Applications of nucleic acid extraction in healthcare include :
- Next Generation Sequencing (NGS)
- Amplification-based SNP Genotyping
- Array-based Genotyping
- Restriction Enzyme Digestion
- Analyses using Modifying Enzymes (e.g. Ligation and Cloning)
There are also other fields beyond healthcare where nucleic acid extraction is used, including but not limited to paternity testing, forensics and genomics.
A Brief History of Nucleic Acid Extraction
DNA extraction dates back a long way, with the first known isolation having been performed by a Swiss physician named Friedrich Miescher in 1869. Miescher was hoping to solve the fundamental principles of life by determining the chemical composition of cells. After failing with lymphocytes, he was able to obtain a crude precipitate of DNA from leucocytes found in pus on discarded bandages. He did this by adding acid and then alkali to the cell to leave the cell’s cytoplasm, and then developed a protocol to separate the DNA from the other proteins.
Following Miescher’s ground-breaking research, many other scientists have gone on to advance and develop techniques to isolate and purify DNA. Edwin Joseph Cohn, a protein scientist developed many techniques for protein purification during WW2. He was responsible for isolating the serum albumin fraction of blood plasma, which is important in maintaining the osmotic pressure in the blood vessels. This was crucial for keeping soldiers alive.
In 1953 Francis Crick, along with Rosalind Franklin and James Watson, determined the structure of DNA, showing that it was made up of two strands of long chains of nucleic acid nucleotides. This breakthrough discovery paved the way for Meselson and Stahl, who were able to develop a density gradient centrifugation protocol to isolate DNA from E. Coli bacteria as they demonstrated the semi-conservative replication of DNA during their 1958 experiment.
Techniques of Nucleic Acid Extraction
What are the 4 stages of DNA extraction?
All extraction methods boil down to the same fundamental steps.
Cell Disruption. This stage, also known as cell lysis, involves breaking down the cell wall and/or the cell membrane, in order release the intra-cellular fluids containing the nucleic acids of interest.
Removal of Unwanted Debris. This includes membrane lipids, proteins and other unwanted nucleic acids which can interfere with downstream applications.
Isolation. There are a number of different ways to isolate the nucleic acids of interest from the cleared lysate you created, which fall between two main categories: solution based or solid state (see next section).
Concentration. After the nucleic acids have been isolated from all other contaminants and diluents, they are presented in a highly-concentrated eluate.
The Two Types of Extraction
There are two types of nucleic acid extraction – solution based methods and solid state methods. The solution based method is also known as the chemical extraction method, as it involves using chemicals to break down the cell and access the nucleic material. This can be using either organic compounds such as phenol and chloroform, or the less harmful and therefore more recommended inorganic compounds such as Proteinase K or silica gel.
Examples of different chemical extraction methods to break down a cell include:
- Osmotic rupturing of membrane
- Enzymatic digestion of cell wall
- Solubilisation of membrane
- With detergents
- With alkali treatment
Solid state techniques, also known as mechanical methods, involves exploiting how DNA interacts with a solid substrate. By selecting a bead or molecule which the DNA will bind onto but the analyte will not, it is possible to separate the two. Examples of solid-phase extraction techniques including using silica and magnetic beads.
Magnetic Bead Extraction Explained
The Magnetic Bead Extraction Method
The potential for extraction using magnetic beads was first recognised in a US patent filed by Trevor Hawkins, for the Whitehead Institute research institution. This patent acknowledged that it was possible to extract genetic material by binding them to a solid support carrier, which could be a magnetic bead. The principle is that you use a highly functionalised magnetic bead onto which the genetic material will bind onto, which can then be separated from the supernatant by applying a magnetic force to the outside of the vessel holding the sample.
Why Use Magnetic Bead Extraction?
Magnetic bead extraction technology is becoming increasingly prevalent, due to the potential it holds for rapid and efficient extraction procedures. In recent times there have been developments of highly functionalised magnetic beads with suitable buffer systems, which have made possible automation of nucleic acid extraction and a workflow which is very resource light and cost-efficient. Also, magnetic bead extraction methods do not involve the centrifugation steps that can cause shear forces which break up longer pieces of DNA. This means that longer strands of DNA remain intact, which is important in genomics testing.
Post time: Nov-25-2022