Silica Membrane Spin Columns The use of glass beads or silica gel particles has become a popular method for isolating DNA. The evolution of this principle has resulted in the introduction of silica membrane spin columns. The basic principle of silica gel solid support spin columns is fairly simple. DNA is bound to the silica membrane spin columns in the presence of high concentrations of chaotrophic salts, contaminants are washed away, and DNA is eluted from the silica membrane in water or low-salt buffer. The major advantage of silica membrane spin columns is the fact that the silica is bound to a solid support, which eliminates the problem of glass-bead contamination of DNA samples. This method of DNA purification, modified from principles originally described in Vogelstein and Gillespie (1979) provides a quick, convenient, nontoxic method and can produce high yields of pure DNA. However, the yields may somewhat lower, generally ranging from 50% to 75% of the starting material. The procedure seems to work best with DNA fragments larger than 500 bp, as some shorter fragments may bind tightly and irreversibly to silica membranes. An advantage of silica membrane spin columns relative to glass beads (Vogelstein and Gillespie, 1979) is that they help decrease shearing of DNA fragments that are larger than 3 to 10 kb.
Binding Mechanism The principle of silica matrices purification is based on high affinity of negatively charged DNA backbone towards the positively charged silica particles (Esser et al., 2006). DNA binds to silica through hydrogen-binding interacation with an underivatised hydrophilic matrix provided by silica, under concentrated chaotrophic salt conditions (usually sodium iodide, sodium perchlorate, guanidinium thiocyanate) (Berensmeier, 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). It was inferred from the isotherms that (i) shielded intermolecular electrostatic forces, (ii) dehydration of the DNA and silica surface and (iii) intermolecular hydrogen bond formation in the DNA silica contact layer are the major contributing driving force for adsorption. Under high salt concentrations, nucleic acids selectively bind to silica membranes while other contaminants, mainly proteins, pass through the membranes. By and large, guanidinium thiocyanate and guanidinium hydrochloride are used for binding nucleic acids to silica membranes. Guanidinium thiocyanate at a concentration of 4M ot 6M works best, while guanidinium hydrochloride is used at higher concentrations (in excess of 6M). In order to control pH of the binding reagent, sodium acetate and Tris-HCl buffers, ranging from pH 6.00 to 7.50 are used. Binding of smaller fragments and preferable binding of RNA over DNA appear to be a function of pH and membrane characteristics. The binding efficiency is significantly improved in the presence of ethanol. Sodium iodide and sodium perchlorate are also used to a lesser extent for nucleic acid binding. Guanidinium thiocyanate is an excellent protein denaturant and hence very effective in inactivating nucleases. However, detergents such as sodium dodecyl sulfate is not compatible with guandinium thiocynate, but works well with guanidinium hydrochloride.
Our Membranes Our membranes are made of binder-free microbifer glass with defined pore size and thickness, both of which are critical for efficient nucleic acid binding. We have validated these membranes for effective binding of both DNA and RNA, in the presence of chaotrophic and non-chaotrophic salts.
Thickness and pore size influence nucleic acid binding capacity of the membranes. Thickness of our membranes ranges from 0.3 mm to 2 mm, while pore size ranges from 0.7 microns to 1.3 microns. Smaller pores size is ideal for smaller nucleic acid fragments and RNA molecules. Surface area is not related to membrane thickness but quality of purification, when dealing with contaminants. Thinner membranes offer ease in isolation and membrane drying.
The next important property is tensile strength and our membranes have a higher tensile strength, in the order of 5 lb/in which offer durability during binding, repeated washing and elution.
The binding capacity of our mini columns is in excess of 30 µg total nucleic acid and the binding capacity of our midi columns is in excess o 200 µg total nucleic acid.
Applications Plasmid isolation Agarose Gel DNA Extraction PCR Product Purification Clean-up after Enzymatic Digestions Probe Label Clean-up Nucleic acid Purification and Concentration
Limitations A major disadvantage of silica membrane is its inability to purify nucleic acids in the presence of phenolic compounds and humic substances. These are compounds with a phenolic ring structure and are polyanionic in nature with net negative charge. They mimic DNA in their chemical structure and could oxidize to form covalent bonds with nucleic acids. These compounds, bind to silica membranes, in the presence of high salt concentrations and elute in low salt solutions. High salt washes have little effect in dislodging them from the membranes. Hence, it is necessary to remove these compounds from DNA solutions before DNA is bound to silica membranes.