Magnetic DNA Purification: History and recent developments

Magnetic DNA purification is a simple and reliable way to isolate DNA.

Nucleic acid (DNA and RNA) isolation and amplification is an important tool for molecular biology and 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) prompting the wide applications of nucleic acid detection for the on-site immunological technologies and rapid 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, hybridisation, cDNA synthesis, etc. cannot be carried out with the crude sample material, as the presence of large amounts of cellular or other contaminating materials (e.g. proteins or carbohydrates) in such complex mixtures often impedes many of the subsequent reactions and techniques.

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 methods that are used today and rely on the detection of DNA or RNA (e.g. diagnosis of microbial infections, forensic science, tissue and blood typing, detection of genetic variations, etc).

Before the advent of modern technologies, nucleic acid isolation and purification was a laborious and time-consuming procedure with numerous extraction and centrifugation steps, often limited by small yields and low purities of the products, and not suited for automation and up-scaling. During the last few years however specifically functionalized magnetic particles were developed which together with an appropriate buffer system allow for the quick and efficient purification directly after their extraction from crude cell extracts.

How is DNA isolation procedure done?

Compared with immunological methods, nucleic acid detection is a direct method with higher sensitivity and takes lesser time to prepare the kits. There are numerous “amplification-free” and “amplification-based” methods for nucleic acid detection, however, the sensitivity of nucleic acid detection increases 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 and extraction/purification of nucleic acid.

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^[³^].

DNA purification methods

Traditional non-magnetic methods of DNA purification are divided into two types of fluid and solid phase.

Fluid phase

A range of methods are known for the isolation of nucleic acids in the fluid phase, but they are generally based on complex series of precipitation and washing steps and are not only time-consuming, cumbersome and laborious to perform but 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 comparatively high.

Solid phase

Alternative separation techniques have been developed. Sorption processes based on (a) hydrogen-binding interaction with an underivatised 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 were used for DNA purification. Solid-phase systems which adsorb DNA — silica-based particles, glass fibres, and anion-exchange carriers – are used in chromatographic separation columns.

These carriers are applied for 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.

DNA 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. DNA isolation is relatively easy, quick, and standardized. However, the process still requires many buffers, solvents, expensive centrifuges, laboratory space, and time to complete.

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. When exposed to a magnetic field these beads have a magnetic “moment” by which they can be directed to the side of their vessel after nucleic acid binding and elution step to pour away the remaining solvent and collect a pure DNA sample ready for quantification and analysis.

Magnetic carriers with immobilized affinity ligands or prepared from a biopolymer exhibiting affinity to the target nucleic acid are used for the isolation process. Such materials are magnetic particles produced from different synthetic polymers, biopolymers, porous glass, or magnetic particles based on inorganic magnetic materials such as surface-modified iron oxide. Different magnetic carriers have been marketed.

How does magnetic bead DNA extraction work?

The early magnetic separation techniques 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.

Especially suited 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. When magnetic aggregation and clumping of the particles are prevented during the reaction, easy suspension of the particles and uniform nucleic acid extraction are ensured.

Materials with a large surface area are preferred for binding the nucleic acids. The nucleic-acid-binding process may be assisted by the nucleic acid ‘wrapping around’ the support. Such supports generally have an irregular surface and may be porous for example. Particulate materials, e.g. beads and in particular polymer beads, are generally preferred due to their larger binding capacity. Conveniently, a particulate solid support used will comprise spherical beads. Monodisperse particles (particles of mainly uniform size) have the advantage of providing for a very uniform reproducibility of magnetic separation.

How are functionalized magnetic beads used for nucleotide 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 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 [4].

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 isolation

There 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.

Advantages of magnetic beads for DNA purification

Magnetic beads offer a quick, simple and efficient way to separate DNA than traditional techniques generating shear forces leading to the degradation of the nucleic acids and the risk of cross-contamination. It is also possible to isolate components of the cell lysate, which inhibit for example the DNA polymerase of a following PCR reaction pile polysaccharides, phenolic compounds or humic substances.

DNA can be isolated directly from crude sample materials such as blood, tissue homogenates, cultivation media, water, etc. The particles are used in batch processes where there are hardly any restrictions with respect to the sample volumes. Due to the possibility of adjusting the magnetic properties of the solid materials, they can be removed relatively easily and selectively even from viscous sample suspensions and small-volume samples.

Magnetic separation is the only 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 purifications.

These techniques also serve as a basis of various automated low-to high-throughput procedures that allow saving time and money without requiring columns. Various types of magnetic particles are commercially available for nucleic acid purification offering the manual and automated mode.

References:

J.L. Zhong, X.H. Zhao, Isothermal amplification technologies for the detection of foodborne pathogens, Food Anal. Methods 11 (2018) 1543-1560.
J.D. Fox, Nucleic acid amplification tests for detection of respiratory viruses, J. Clin. 581 Virol. 40 (2007) S15-S23.
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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