Silica Membrane Spin Columns
DNA isolation from biological samples is a foundational step in molecular biology. Isolating quantitatively functional DNA is vital for the success of a wide array of molecular assays that are critical in understanding and solving our day-to-day scientific problems. Vogelstein and Gillespie (1979) pioneered the use of glass powder in isolating DNA from agarose slabs. They demonstrated binding DNA to glass powder after dissolving the agarose embedded with DNA in sodium iodide. This procedure was found to be a satisfactory means of removing DNA from agarose and the binding capacity of glass powder exceeded 1 mg of DNA bound per gram of glass. The other important aspect of this protocol is DNA recovery in any low ionic salt buffer that will have minimal impact on assays such as RT-PCR and sequencing applications. This method is rapid, convenient, and yields DNA of high purity suitable for downstream applications.
Binding Salts – Vogelstein and Gillespie (1979) used sodium iodide as the salt to bind DNA to glass particles while Marko et al. (1982) used sodium perchlorate to isolate plasmid DNA using crushed Whatman GF/A, 10 cm diameter glass-fiber filters. Boom et al. (1990) purified nucleic acid from clinical samples using glass particles in the presence of guanidinium thiocyanate (GuSCN) or guanidinium hydrochloride GuHCl). Guanidinium salts have been shown to be a critical agent in the purification and isolation of both DNA and RNA because of its potential to lyse cells, combined with its ability to inactivate nucleases by acting as a powerful denaturant (MacDonald et al. 1987, Chirgwin et al. 1979, Chomczynski and Sacchi, 1987). Inactivation of nucleases parallels the kinetic efficiency of protein denaturation, with guanidinium thiocyanate performing better than guanidinium hydrochloride, followed by urea (MacDonald et al. 1987). Both, Von Hippel and Wang (1964) and Castellino and Barker (1968) found that guanidinium thiocyanate was about 2.5-fold more effective on a molar basis than guanidinium hydrochloride as an equilibrium denaturant. In the former salt both cation and anion are strong chaotropes, while in the latter, only the guanidinium cation is chaotropic and hence active in denaturation (Jencks, 1969). The most important advantage of glass particles binding to nucleic acid is the option to directly elute DNA without the need for ethanol precipitation.
Silica Membrane Spin Columns - The evolution of this solid-phase extraction which is based on the ability of nucleic acids to adsorb on to silicon dioxide surface (glass particles) has resulted in the introduction of silica membrane spin columns. This nucleic acid binding technique in the presence of high ionic strength solutions was developed in 1990 (Bogdanov et al., 2013). Now it is used in many commercial kits and automated nucleic aid isolation systems (Tan and Yiap (2009), Parida et al. (2006), and Poeckh et al. (2008). The basic principle of silica gel solid support spin columns is simple. DNA is bound to silica membrane spin columns in the presence of high concentrations of chaotriphic salts, while contaminants are washed away, and DNA is eluted from the silica membranes with water or low ionic salt buffers. The two major advantages of silica membrane spin columns over glass beads or silica gel particles are quicker drying time after ethanol wash, and avoiding glass particle contamination of DNA samples. In glass particle method, after ethanol wash, the glass particles must be dried at room temperature to prevent ethanol contamination. In silica membrane spin columns, a quick spin for a minute will remove residual ethanol from the membranes. Likewise, DNA elution is also simple, where the elution buffer is added on the membranes and a simple 1-minute centrifugation will release the bound DNA into the elution buffer. In the case of glass particles, while eluting DNA, it is necessary to avoid disturbing the pelleted glass particles. Rothe and Nagy (2016) reported higher DNA yield with silica membrane spin columns over silica beads but also reported a higher risk of co-extracting impurities with membrane-based methods over glass particle-based ones.
Binding Mechanism – Understanding the adsorption mechanism of DNA molecules on silica (SiOx) surfaces is crucial for optimizing this technology in DNA isolation and purification from a variety of biological samples. The three main effects, driving adsorption of DNA to silica particles are (i) shielded intermolecular electrostatic forces, (ii) dehydration of the DNA and silica surfaces, and (iii) intermolecular hydrogen bond formation in the DNA-silica contact layer (Melzak et al. (1996). Sorption processes typically occur based on hydrogen-binding interaction with an underivatized hydrophilic matrix such as silica, under chaotropic conditions (Berensmeir, 2006). Sodium plays a role as a cation bridge that attracts the negatively charged oxygen ions in silica under high salt conditions (pH ≤ 7.00). DNA is tightly bound, and extensive washing removes all contaminants. The adsorption of plasmid and chromosomal DNA on microcrystalline silica surface and the effect of ionic strength, temperature, pH, DNA size and conformation on the adsorption phenomenon were reported by Melzak et al. (1996). DNA superstructure can have a dramatic influence on adsorption characteristics. Supercoiled DNA, for instance, may be limited in its ability to form long stretches of favorable contact points with the flat silica because of the topology of the supercoil (Melzak et al. 1996). Linearized pUC18 binding is nearly twice compared to supercoiled pUC18, indicating that linearization increases the binding affinity, which agrees with the argument that linearization increases the number of favorable energetic contact points between the furanose-phosphate exterior of the double helix and the silica surface. Under high salt concentrations, nucleic acids selectively bind to silica membranes while other contaminants, mainly proteins, pass through the membranes.
Limitations – In general, there is a percentage of DNA loss in sample processing which is very much controlled by the biological nature of samples. Several publications regarding DNA extraction efficiency focus on comparing the methods rather than assessing where potential DNA loss occurs (Lee et al. 2010, Kemp et al. 2014 Barta et al. 2014, and Jakubowska et al. 2012). Biological samples based on their chemical composition is the main source of DNA loss.
In the case of cotton swabs, it has been suggested that DNA is being trapped in the substrate itself and that the swab material, such as tightly wound cotton fibers, can affect the ability to efficiently retrieve DNA useable for downstream STR analysis (Brownlow et al., 2012, Daley et al. 2006, van Oorschot et al. 2010, Dentinger et al. 2010, and Adamowicz et al. 2014).
Environmental samples such as soils, plant materials and fecal samples are intrinsically problematic when it comes to nucleic acid extractions. Clay components of soil samples have long known for their affinity for naked DNA and humic acids can mimic DNA in their chemical structure with a net negative charge, and co-elute along DNA. DNA released in the presence of soil is often adsorbed by the soil solid phase and clay minerals generally account for more than 90% of the soil solid phase (Agnelli et al., 2004). Previous studies have reported the adsorption capacities of several raw clay minerals (Saeki et al., 2010; Pedreira-Segade et al., 2016; Gardner and Gunsch, 2017). Humic acids could compete for binding sites on silica membranes along with DNA and co-elute with DNA, thereby reducing DNA yield and quality. This fact must be considered while designing appropriate lysis buffer and other purification methods for nucleic acid isolation from soil samples.
Plant species are biochemically heterogenous, and several plants are recalcitrant to extraction of good quality DNA (Krishnan et al., 2024). Young tissues, usually seedling leaves, are the prime choice for DNA extraction in most cases, as mature tissues were reported to have high polysaccharides and polyphenolic compounds (Porebski et al. 1997). Practically, it is not possible to have control on the type of plant tissues to choose for nucleic acid extractions. The main challenge comes from phenolic and polysaccharide compounds present in plants. During sample homogenization, disruption of cells releases these phenolic acids, simple phenols and polyphenols from the vacuoles, and they freely undergo oxidative reactions (Pratyusha, 2022). This oxidative reaction results in the irreversible binding to the phosphate backbone of DNA resulting in the browning of the tissues and making the extraction of good quality DNA difficult for downstream molecular biology applications (Tibbits et al. 2006, Sahu et al. 2012, Healey et al. 2014, Carey et al. 2023). The other component in plant tissues, polysaccharides, also complicate nucleic acid isolation. Polysaccharides co-precipitate with DNA, making it gelatinous and thus reduces the quality of isolated DNA (Tibbits et al. 2006 and Haely et al. 2014).
Fecal samples have their own set of inhibitory substances that influence template DNA quality in PCR. They include bile salts, bilirubin, urobilinogens, polysaccharides, and large amount of background DNA (Brain et al. 1992 and Stacy-Phipps et al. 1995).
Alternatively, DNA loss could also occur in the silica column itself when performing solid-phase extraction methods; however, there is little information on which step of the extraction process is most responsible, or if it is caused by excessive tube transfers and manipulation (Schiffner et al. 2005). It is possible that not all the DNA is properly binding to the column when the sample lysate is filtered through, or that some DNA is prematurely released from the column during subsequent washes. Lastly, a portion of the DNA may not be released from the column during the final elution step. One study comparing extraction kits reported that DNA yields increased as the amount of silica in the column increased implying that inefficient initial binding of DNA to the silica (or possibly an overloading of the silica column in the case of high-yield DNA samples) could be a primary source of DNA loss (Lee et al. 2010). Elution efficiency can be improved by increasing the temperature of the elution solution. A higher temperature can break the hydrogen bond between the nucleic acid and the surface of silicon dioxide, so that the nucleic acid can be detached and washed down more easily (Hu et al. 2020).
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