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Nucleic acidseparation is an increasingly important technique in molecular biology.Various techniques have been developed in the past few decades. However, the traditional nucleic acid isolation methods have time- and work-consuming process based on several extraction and centrifugation steps. And they are often limited by small yields, low purities or low product concentrations. Therefore, when it comes to some precious samples with ultra-low concentration and volume, these methods may face the risk of no product collection or product over-dilution.
During the last few years, particle-based nucleic acid isolation technique has been developed, which allows a rapid and efficient nucleic acid purification with hardly any restrictions with respect to the sample concentrations and volumes. These particles are universally built with certain materials or characterized with delicate surface modifications that make them able to extract the total nucleic acid or a certain RNA and DNA directly from crude samples.
Among all the particles that have been developed for the separations, magnetic particles stand out by its unique magnetic property. Magnetic particles acquire a magnetic moment when placed in a magnetic field and therefore can be displaced. Thus, these particles can be removed readily by the application of a magnetic field, which is a quick, simple and efficient way to separate the particles from mixtures and a far less rigorous method than traditional techniques.
Fig 1. Schematic illustration of nucleic acids isolation using magnetic beads.
Table 1. Filters vs. Beads in nucleic acid isolation
|Filters (solid phase)||Beads|
|Matrix||Stationary, irregular density||Mobile dispersion, regular density|
|Exposure to nucleic acid||Rapid, during centrifuge||Thorough, mixing in solution|
|Isolation method||Centrifuge||Magnetic force|
|Elution||Requires large volume for efficient elution||Efficient elution in small volumes|
Types of Particle-based Nucleic Acid Extraction
Total DNA and RNA can be separated by particles through their interactions with these particles’ surface. Due to silica’s unique property to trap nucleic acids through their hydrogen-bindings with an underivatized hydrophilic matrix, silica particles and silica-coated particles have been widely used for the extraction of total nucleic acids.
For the purpose of removing RNA from DNA, the RNA is destroyed before the DNA separation step. Vice versa, RNA can be separated if the DNA is degraded with DNase.
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Absolute Mag™ Plain Silica Magnetic Particles
Due to particles’ modifiable surface, multiple ligands, such as DNA or RNA sequences and DNA-binding proteins, can be attached to particles in order to selectively capture the nucleic acids of interest.
Two complementary DNA/RNA strands can bind together through hybridization. Thus, by attaching the single-stranded DNA/RNA with a certain sequence to the particles, their complementary oligonucleotides can be isolated by the application of a centrifugation or a magnetic field.
The streptavidin-biotin binding is the strongest known non-covalent interaction between a protein and a ligand and has been exploited for use in many bioseparation processes. Through labeling biotin to the 3' End of DNA/RNA, the complementary oligonucleotides of interest can be easily trapped by streptavidin modified particles.
Polyadenylation occurs in the process of producing mature messenger RNA (mRNA). Thus, mature mRNA is characterized by a poly(A) tail that consists of multiple adenosine monophosphates. In order to isolate mRNA from mixtures, oligonucleotide dT sequences are bound covalently to the particle surface or indirectly by biotinylated oligonucleotides and the interaction of streptavidin-coated particles. These oligonucleotide dT sequences can attract mRNA through hybridizing with the stable poly(A) 3 termini of the mRNA. Thus, the trapped mRNA can be isolated by centrifugation or magnetic field force.
Fig 2. Schematic illustration of mRNA isolation using Absolute Mag™ Oligo(dT) magnetic particles
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Absolute Mag™ Oligo(dT) Magnetic Particles
The traditional isolation of nucleic acids in the fluid phase is one of the most commonly used techniques for nucleic acid extraction. This method is generally based on complex series of precipitation and washing steps. (Fig. 3) However, highly toxic chemicals, such as phenol and chloroform, are used in this isolation. Furthermore, due to its laborious and protracted process, samples are facing degradation, loss and cross-contamination during the whole process.
Fig. 3 Schematic illustration of traditional isolation of nucleic acids in the fluid phase.
Nucleic acid isolations in the solid phase were developed as the alternative choice. Silica-based columns and anion-exchange resin columns are the two most commonly used columns used in nucleic acid isolations. Silica-based columns trap nucleic acids through their hydrogen-bindings with an underivatized hydrophilic matrix and anion-exchange resin columns isolate nucleic acids through their bindings to DEAE groups. However, isolating nucleic acid using columns still face the risk of sample loss. Meanwhile, a certain quantity of buffer is needed for the target elution, which may result in a product over-dilution.