Magnetic DNA purification is a simple and reliable way to isolate DNA.
This method for purifying DNA has a wide range of applications. In fact, genomic sequencing and molecular analysis have become so integral to biological research that they are now all but required for work to be published in high profile journals.
Outside of scientific research, magnetic DNA purification is also fundamental to forensic analysis in the criminal justice system. This requires the ability to rapidly extract and purify high-quality DNA and RNA from a variety of tissues. As a result of this demand, improvements to current techniques are needed.
History of DNA purification
Our understanding of genetic material has increased substantially since Friederich Miescher performed the first DNA extraction in 1869. That first extraction led to the simple discovery that a material exists within cells that precipitates out of acidic solution and dissolves into alkaline solution. After that, it took until 1953 for the full structure of DNA to be uncovered. Shortly after this, standard laboratory procedures for isolating nucleic acids emerged. But what are the modern methods used for DNA purification?
How does magnetic DNA purification using nucleic acid isolation work?
Nucleic acid isolation and purification consists of three basic steps:
- Disruption (or lysis) of cells
- Removal of protein and contaminants
- Recovery of DNA
The disruption of the cells is a mechanical process: tissue is typically frozen in liquid nitrogen and crushed to break the macroscopic structure and to rupture cell walls. This slurry of tissue is then mixed with solvents and salts to wash out unwanted proteins, disable DNAses, and collect the DNA into a usable solution. Finally, a phenol-chloroform organic solvent is used to concentrate DNA in a hydrophilic phase. This hydrophilic solution can then be collected, centrifuged, and washed until pure DNA is recovered.
Earlier methods involved the DNA collecting in the pellet at the bottom of the tube, which would then be dissolved in water and collected. To increase yield and streamline protocols between laboratories, solid phase support systems were developed. Currently, commercial kits using a combination of solvents and solid-phase support columns are widely used to isolate and purify DNA and RNA.
What is column-based DNA isolation?
This method uses proprietary columns, sold commercially in isolation kits, that are made of silica matrices, glass particles, diatomaceous earth, or anion-exchange resins. The DNA or RNA binds to the column while proteins and contaminants are washed out by ethanol using a centrifuge. This process is repeated, and finally the remaining nucleic acids are collected with water.
Due to the widespread availability and affordability of these commercial kits, DNA isolation is relatively easy, quick, and standardized. However, the process still requires many buffers, solvents, expensive centrifuges, laboratory space, and time to complete.
The need to increase the efficiency of this process led to the development of magnetic DNA purification methods. The advances in nanotechnology over the past 20 years have made magnetic bead DNA extraction a viable and increasingly attractive method of nucleic acid purification. Magnetic DNA purification doesn’t require columns or centrifugation, and enables the process to be automated.
How does magnetic bead DNA extraction work?
Magnetic beads are a collection of uniform particles, 500 nanometers by 500 micrometers in diameter, which are made with magnetic materials. These magnetic materials are typically composites containing nanoparticles of magnetite. The particles become magnetized in the presence of an external magnet, making them easy to manipulate.
The United States patent entitled ‘DNA purification and isolation using magnetic particles’, was published in 1998. This early magnetic separation technique used particles consisting of an iron-oxide core coated with silane. The surface of the particles was then bound to molecules containing a free carboxylic acid, which in turn bound to DNA or RNA. The salt concentration determined the strength of the bonds between functional groups and the nucleic acids, which allowed for controlled reversible binding. At the correct salt concentration, the nucleic acids would bind to the magnetic particles. Next, a magnet was placed outside of the tube containing the solution to create a strong external magnetic field. The magnetic particles bound with nucleic acids were attracted to the field and “stuck” to the outer edges of the tube.
An alternative technique used a magnet at the bottom of the tube to draw particles downward. In both cases, the magnetic field is maintained while the unwanted solution is exchanged and contaminating proteins are washed away. After the washing steps, the DNA or RNA is released from the magnetic particles with an elution buffer, leaving behind a pure sample ready for quantification and analysis.
How are functionalized magnetic beads used for nucleotide extraction?
In recent years, modifications to the patented magnetic DNA purification method have been developed. The magnetic particles can now be made from synthetic polymers embedded with iron oxide ‘pigments’, or metallic cores of iron-oxide coated with a polymer surface to enable functionalization. These uniform particles can be coated with functional moieties, or can be left uncoated.
Some functional coatings work via electrostatic interactions (positively charged amine or imidazole moieties), while others work via salt- or pH-mediated attractions (silica and carboxyl groups). More specific DNA (or RNA) isolation is enabled by the introduction of target oligonucleotides to the magnetic bead surface. These target oligonucleotides are useful for the extraction of specific sequences of ssDNA or RNA.
Magnetic DNA purification is a clear improvement on centrifuge-dependent isolation techniques when using semi-automatic or fully automatic systems. These systems are used when rapid purification of many samples is necessary.
In the absence of magnetic DNA isolation, the need to move the samples into and out of a centrifuge is inefficient, and can increase analysis time. In contrast, the use of magnetic particles allows the tubes to remain in one machine throughout the entire process.
What are some examples of DNA isolation by magnetic beads?
Magnetic beads are used to purify DNA in a wide range of different situations. These include:
Plasmid DNA isolation
Magnetic beads are used to isolate plasmid DNA from crude extract. This is possible through carefully optimized solutions that separate plasmid DNA from genomic DNA and proteins, before allowing the plasmid DNA to bind the magnetic beads and introducing them to a magnetic field.
Genomic DNA isolation
Magnetic beads are also used to separate genomic DNA from proteins and RNA from crude extract. The optimization of salt, pH and charge in solution means only the genomic DNA can bind to the beads, which can then be placed in a magnetic field for separation.
DNA fragment isolationThere are kits available for many types of DNA isolation, including for DNA fragments. DNA fragments may need to be isolated for next generation sequencing protocols, where the fragments can be separated using beads that can select for the size of a molecule.
Magnetic beads makes DNA purification simple
The ease-of-use and scalability of magnetic DNA purification techniques will be increasingly useful as scientists dig ever deeper to untangle the complicated molecular bases of observable phenotypes. Genetic analysis and manipulation is the way of the future, and using magnets to isolate pure DNA and RNA will help to accelerate the pace of discovery.
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