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Nucleic acid (DNA and RNA) purification and amplification is an important tool for molecular biology and an important step before many biochemical and diagnostic processes. These techniques have made great progress recently[1] [2] due to the increasing number of sudden and public health-threatening infectious diseases (e.g. Ebola virus, Zika virus and more recently SARS-Covid). For quickly and reliably diagnosing these diseases, nucleic acid detection are key tools used in on-site immunological technologies and rapid test kits (for magnetic mRNA purification refer to “Oligo dT-coated magnetic beads: the benefits of their application for mRNA purification”).

Many downstream applications such as detection, cloning, sequencing, amplification, hybridization, and cDNA synthesis cannot be carried out with crude sample material. This is because the presence of large amounts of cellular debris or other contaminating materials, like proteins or carbohydrates, in such complex mixtures often impedes many of the subsequent reactions and disease detection techniques.

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Furthermore, DNA may contaminate RNA preparations and vice versa. Thus, methods for the efficient, reliable, and reproducible isolation of nucleic acids from complex mixtures are needed for many nucleic acid purification methods that rely on the detection of specific DNA or RNA sequences. Utilization of these technologies is wide ranging, and can be used in the diagnosis of microbial infections, forensic science, tissue and blood typing, and even for the detection of genetic variations.

Before the advent of modern technologies, nucleic acid isolation and purification was a laborious and time-consuming procedure with numerous extraction and centrifugation steps. The process was often limited by small yields, low purities of the products, and not suited for automation and up-scaling. During the last few years, however, specifically functionalized magnetic particles (also termed magnetic beads) have been developed. Coupled with an appropriate buffer system, magnetic particles allow for the quick and efficient purification of nucleic acids directly after their extraction from crude cell extracts. Thuse, the advent of magnetic beads DNA purification has provided a simple and reliable way to isolate DNA.

How is DNA isolation Performed?

Compared with immunological methods, nucleic acid detection is more direct, provides higher sensitivity, and takes less time in preparation. There are numerous “amplification-free” and “amplification-based” methods for nucleic acid detection, however, the sensitivity of nucleic acid detection will always increase after amplification. The  main  steps  in  all  nucleic  acid  extractions  include cell lysis (or cell disruption), inactivation  of  cellular  nucleases, removal of protein and contaminants, extraction, and purification  of  the target nucleic  acid.

The initial steps in DNA purification starts with the disruption of the cells through a mechanical process. Starting tissues are typically frozen in liquid nitrogen and then crushed to break macroscopic structures and to rupture the cell walls. This slurry of tissue is then mixed with solvents and salts to wash out unwanted proteins, disable DNAses, and allows the DNA to be collected 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 required that DNA be pelleted at the bottom of the tube through centrifugation, 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[³]. More recent methods utilize magnetic beads and/or microfluidic systems as the solid-phase support systems, which has helped greatly improve the efficiency of the procedure, quality and quantity of the resulting products.

Traditional Non-Magnetic Methods of DNA Purification

Liquid-Liquid Extraction (LLE)

A range of methods can be used for the isolation of nucleic acids in the fluid phase (also termed, liquid phase), but they are generally based on complex series of precipitation and washing steps and are time-consuming, cumbersome, and laborious to perform. The relatively large number of steps required increases the risk of degradation, sample loss, or cross-contamination of samples, especially when several samples are processed simultaneously. In the case of RNA isolation, the risk of DNA contamination is also comparatively high, as opposed to other methods.

Solid Phase Extraction (SPE)

Due to the inherent downsides of fluid phase extraction, alternative separation techniques have been developed. Sorption processes based on (a) hydrogen-binding interaction with an underivatized hydrophilic matrix, typically silica, under chaotropic conditions, (b) ionic exchange under aqueous conditions by means of an anion exchanger, (c) affinity and (d) size exclusion mechanisms have all greatly enhanced DNA purification. Solid-phase systems which adsorb DNA, including those that utilize silica-based particles, glass fibers, and anion-exchange carriers, are employed in chromatographic separation columns.

These carriers are used in DNA isolation or purification together with highly concentrated chaotropic salt solutions (e.g. sodium iodide, sodium perchlorate, guanidinium thiocyanate). Other approaches are based on detergence together with a nucleic acid-binding material or on the usage of a solid carrier with DNA-binding functional groups combined with polyethylene glycol and salts at high concentrations.

In the process, DNA binds to the solid-support column while proteins and contaminants are washed out by ethanol using a centrifuge. This process is repeated, and finally the remaining target nucleic acids are collected. DNA isolation using solid phase extraction is relatively easy, quick, and standardized. However, the process still requires many buffers, solvents, expensive centrifuges, laboratory space, and time to complete.

DNA Purification Using Magnetic Separation

Magnetic beads are a collection of uniform particles (500 nm to 500 mm in diameter) which are typically made with composites containing nanoparticles of magnetite. After binding to target nucleic acids, these beads have a magnetic “moment” when exposed to a magnetic field. At this step, the magnetic beads are directed to the side of their vessel, and then wash and elution steps can be performed to remove the remaining solvent. The collected, pure, DNA sample is then ready for quantification and analysis.

In magnetic beads DNA purification, magnetic carriers contain immobilized affinity ligands or are prepared from a biopolymer exhibiting affinity to the target nucleic acid. Materials used to make magnetic particles have been produced from different synthetic polymers, biopolymers, and porous glass based on inorganic magnetic materials such as surface-modified iron oxide. Different magnetic carriers are commercially available for DNA isolation and purification, expanding the utilization and ability of magnetic beads technologies.

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How does Magnetic Beads DNA Purification Work?

Early magnetic separation techniques used particles consisting of an iron-oxide core coated with silane. The surface of the magnetic particles binds to molecules containing a free carboxylic acid, which, in turn, binds to DNA or RNA. The salt concentration in the binding buffer determines the strength of the bonds between functional groups on the magnetic beads and the nucleic acids, to allow for controlled, reversible, binding. At the correct salt concentration, generally near the physiological pH, nucleic acids will bind tightly to the magnetic particles.

Especially suited for nucleic acid purification are superparamagnetic particles, which do not interact among each other in the absence of a magnetic field. These particles will magnetize under a strong magnetic field, but retain no permanent magnetism once the field is removed. This phenomena prevents magnetic aggregation and clumping of the particles during the reaction, ensuring easy suspension of the magnetic particles and uniform nucleic acid extraction.

How are Functionalized Magnetic Beads Used For Nucleic Acid Extraction?

The magnetic particles are made from synthetic polymers embedded with iron oxide ‘pigments’, or metallic cores of iron-oxide coated with a polymer surface. These magnetic particles can be left uncoated, or can be coated with functional moieties to enable functionalization. Such functionalization provides new or enhanced binding properties and features to the magnetic particles by changing the surface chemistry of the beads.

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 introducing target oligonucleotides to the magnetic bead surface. These target oligonucleotides are useful for the extraction of specific sequences within the  ssDNA or RNA [4] .

What are Some Uses for Magnetic Beads DNA Purification?

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 extracts. 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. The isolation of plasmid DNA from a crude sample is an essential step prior to DNA sequencing or cloning efforts, transfection experiments, or gene therapies.

Genomic DNA isolation

Magnetic beads are also used to separate genomic DNA from proteins and RNA from crude extracts. 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. Genomic DNA isolation has found particular importance in studying the genetic causes of diseases, for expanding diagnostics, and in drug development.

DNA fragment isolation

Magnetic bead-based kits are 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 magnetic beads that can target DNA fragments depending on the size of a molecule.

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Advantages of Magnetic Beads DNA Purification

Magnetic beads offer a quick, simple, and efficient way to separate DNA over traditional techniques that may generate shear forces and lead to the degradation of the nucleic acids or present the risk of cross-contamination. With magnetic beads DNA purification, it is also possible to isolate components of the cell lysate like polysaccharides, phenolic compounds, or humic substances. These components may inhibit downstream experiments, and for example, can prevent the success of DNA polymerase in a following PCR amplification.

When using magnetic beads DNA purification, DNA can be isolated directly from crude sample materials such as blood, tissue homogenates, cultivation media, and water. Magnetic particles can be used in batch processes with little restrictions with respect to experimental sample volumes. Due to the possibility of adjusting the magnetic properties of the solid materials, magnetic particles can be removed relatively easily and selectively from the reaction, even from viscous sample suspensions and small-volume samples.

Magnetic beads DNA purification can easily be applied to a wide range of applications, from extremely small to large scale. Magnetic separation is the most feasible method for the recovery of small particles (diameter approx. 0.05 – 1 micrometer) in the presence of biological debris and other fouling material of similar size. The efficiency of magnetic separation is especially suited for large-scale purification processes. These techniques can also be applied in various automated low- to high-throughput procedures that allow saving time and money without requiring the use of columns. Various types of magnetic particles are commercially available for nucleic acid purification for a range of  manual and automated operations.

 

References:

  1. J.L. Zhong, X.H. Zhao, Isothermal amplification technologies for the detection of foodborne pathogens, Food Anal. Methods 11 (2018) 1543-1560.
  2. J.D. Fox, Nucleic acid amplification tests for detection of respiratory viruses, J. Clin. 581 Virol. 40 (2007) S15-S23.
  3. 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.
  4. 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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Published on March 11, 2015 and updated on February 22, 2024.

 

Lluis M. Martínez | SEPMAG Chief Scientific Officer

Founder of SEPMAG, Lluis holds a PhD in Magnetic Materials by the UAB. He has conducted research at German and Spanish academic institutions. Having worked in companies in Ireland, USA and Spain, he has more than 20 years of experience applying magnetic materials and sensors to industrial products and processes. He has filed several international patents on the field and co-authored more than 20 scientific papers, most of them on the subject of magnetic particle movement.

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