Genomic sequencing and molecular analysis have become so standard to biological research that they are now all but required for work to be published in high profile journals. Outside the scientific realm, magnetic DNA purification is also fundamental to forensic analysis in the criminal justice system. Therefore, a method to rapidly extract and purify high-quality DNA and RNA from a variety of tissues is indispensable, and improvements to the technique are desired.
Our understanding of genetic material has substantially increased since Friederich Miescher performed the first DNA extraction in 1869. That first extraction was the simple discovery that a material exists within cells that precipitates out of acidic solution and dissolves into alkaline solution. It took until 1953 for the structure of DNA to be elucidated. Shortly thereafter, standard laboratory procedures to isolate nucleic acids emerged.
Magnetic DNA Purification: Nucleic Acid Isolation
Nucleic acid isolation and purification consists of three basic steps: disruption/lysis, removal of protein and contaminants, and 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 in order to wash out unwanted proteins, disable DNAses, and to collect the DNA into a workable solution. 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. In the early systems the DNA would collect in the pellet at the bottom of the tube and would then be dissolved and collected in water. With the goal to increase yield and to 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 .
Columm-based dna isolation
Proprietary columns sold commercial in isolation kits 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 with the aid of a centrifuge. After a series of repeated washing and centrifuging steps the nucleic acid is collected with water. Due to the widespread availability and affordability of the 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. An interest in slimming and streamlining the process led to the development of magnetic DNA purification methods. The advancement of nanotechnology over the past 20 years has introduced magnetic bead dna extraction as a viable method of nucleic acid purification. This method eliminates the need for columns and centrifugation, and enables process automation.
Magnetic bead DNA extraction
Magnetic beads are a collection of uniform particles 500 nanometers to 500 micrometers in diameter which are made with magnetic materials, typically composites containing nanoparticles of magnetite. The particles become magnetized in the presence of an external magnet, making them easy to control.
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. That surface was bound with molecules containing a free carboxylic acid, which in turn bound to DNA or RNA. Salt concentration determined the strength of the bonds between functional groups and nucleic acid, which allowed for controlled reversible binding. In correct salt concentrations the nucleic acids bind to the magnetic particles. Then a magnet is placed outside of the tube to create a strong external magnetic field. The magnetic particles bound with nucleic acid are attracted to the field and “stick” to the outer edges of the tube. An alternative technique uses a magnet at the bottom 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, which results in a pure sample ready for quantification and analysis.
Functionalized magnetic beads for nucelotide extraction
Modifications on the patented method have been developed over recent years. Magnetic particles used for magnetic DNA purification can be made from synthetic polymers embedded with iron oxide ‘pigments’, or metallic cores of iron-oxide coated with a polymer surface to enable functionalization. The uniform particles can be coated with functional moities or can be left uncoated. Some functional coatings work via electrostatic interactions (positively charged amine or imidazole moities), while others work via salt- or pH-mediated attractions (silica and carboxyl groups). The ability for more specific isolation is introduced by the attachment 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 upon centrifuge-dependent isolation techniques when semi-automatic or fully automatic systems are considered. These systems are used when rapid purification of many samples is necessary. The need to move the samples into and out of a centrifuge is inefficient in these situations and can increase analysis time. The use of magnetic particles allows for the tubes to remain in one machine throughout the entire process.
Examples of DNA isolation by magnetic beads
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 protein before allowing the plasmid DNA to bind the magnetic beads and introducing them to a magnetic field.
Genomic DNA isolation: magnetic beads are used to separate genomic DNA from proteins and RNA from crude extract. The optimization of salt, pH and charge in solution permits just the genomic DNA to bind beads which can then be placed in a magnet for separation.
DNA fragment isolation: there are kits available for many types of DNA isolation, including for DNA fragments. DNA fragments may need to be isolated for a next generation sequencing protocol, and the fragments can be separated using beads that can select for the size of a molecule.
The ease-of-use and scalability of magnetic DNA purification techniques will surely be 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 maintain the rapid pace of discovery.
DNA purification and isolation using magnetic particles. A Hawkins, T. http://www.google.com/patents/US5705628. 1998. Google Patents.
Tan SC, Yiap BC. DNA, RNA, and Protein Extraction: The Past and The Present. Journal of Biomedicine and Biotechnology 2009;2009:574398. doi:10.1155/2009/574398.
Z.M. Saiyed, C. Bochiwal, H. Gorasia, S.D. Telang, C.N. Ramchand, Application of magnetic particles (Fe3O4) for isolation of genomic DNA from mammalian cells, Analytical Biochemistry, Volume 356, Issue 2, 15 September 2006, Pages 306-308.
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