The present invention relates to a chromatography column and a method for isolating nucleic acid. It is particularly suitable for co-isolating RNA and DNA, for isolating nucleic acid contained in a particle, e.g., virus, and for isolating diluted nucleic acid in a large volume, e.g., plasma.
Nucleic Acid Isolation
Nucleic acid isolation is an important step for many biochemical and diagnostic uses, e.g., cloning, transformation, restriction digestion, in vitro transcription, amplification, sequencing. However, it can not be easily carried out because of the presence of large amounts of cellular and other contaminants, e.g., proteins, carbohydrates, and small metabolites in crude samples. Thus, simple, fast, efficient, reliable, and cost-effective methods are needed.
Silica-Based Isolation
The silica- or silicon dioxide-based method has become a popular method for isolating nucleic acid that use controlled pore glass, filters embedded with silica particles, silica gel particles, resins comprising silica in the form of diatomaceous earth, and glass fibers1 2 3 4 5 6 7 8 9 
The basic procedure is simple. DNA or RNA is bound to the surface of silica resin or membrane in the presence of a high concentration of chaotrophic salts, contaminants are washed away, and DNA is eluted in water or low-salt buffer.
The principle of silica-based isolation is based on the high affinity of the negatively charged DNA backbone towards the positively charged silica surface10 under concentrated chaotrophic salt conditions, e.g., sodium iodide (NaI), sodium perchlorate (NaCIO4), guanidinium hydrochloride (GuHCl), and guanidinium thiocyanate (GuTC).
The effects of ionic strength, temperature, pH, DNA size and conformation on the binding of nucleic acid to the silica surface were investigated11. For example, the binding capacity of the silica surface is linearly related to the chaotrophic salt concentration. Additionally, at a given chaotrophic salt concentration, the binding capacity to the silica surface of any type of DNA is higher at a lower pH value.
GuTC and GuHCl are commonly used for binding nucleic acid to the silica surface. NaI and NaCIO4 are also used to a lesser extent. GuTC at a concentration of 4 M to 6 M works best, while GuHCl is used at a higher concentration of 6 M. The binding efficiency is significantly improved in the presence of ethanol or propanol. In order to control the pH value of the binding reagent, sodium acetate and Tris-HCl buffers, ranging from pH 6 to 7.5 are often used. Moreover, guanidinium salts, e.g., GuTC, are known to efficiently lyse cells and denature proteins4, eliminating the need for adding denaturing enzymes, e.g., proteinase.
Spin and vacuum chromatography columns are commonly used within which silica resin or membrane is packed as the stationary phase, e.g., those of PureYield plasmid midiprep system and WizardPlus minipreps DNA purification system from Promega, QIAprep miniprep kit from Qiagen, and EZgene plasmid purification miniprep kit from Bioland.
The silica-based method provides a quick, convenient, and efficient method. The purified DNA is qualified and ready to use for a wide variety of downstream applications. However, the starting volume of samples, e.g., plasma, is limited because up to 3 volumes of chaotrophic salt solution should be added to one volume of the DNA or RNA samples to reach the effective chaotropic salt concentration required by silica binding.
Anion Exchange-Based Isolation
The method of anion exchange resin and membrane is also commonly used for isolating nucleic acid. In this procedure, DNA is bound to the anion exchanger in the presence of a low salt concentration at a low pH. DNA is eluted in the presence of a high salt concentration at a high pH value12 13 14 15 16 17 18 19 20 9.
The binding principle is based on the interaction between the negatively charged phosphates of the DNA backbone and the positively charged group, e.g., DEAE, on the surface. The salt concentration and pH value of a solution used determine whether DNA is bound or eluted.
Anion exchange bead-based columns, often gravity flow driven, are common, e.g., QIAGEN genomic-tips kit. The QIAGEN resin contains porous silica bead as support coated with diethylaminoethanol (DEAE) functional group. Others include Pall AcroSep chromatography columns with DEAE weak anion exchange ceramic HyperD F bead and with Q strong anion exchange ceramic HyperD F bead.
In contrast to the bead-based columns, anion exchange membrane columns, often spin or vacuum flow driven, are available. They have the advantages of high flow rate, low cost, and high throughput, e.g., Vivapure weak anion exchange mini D membrane spin column and Vivapure strong anion exchange mini Q membrane spin column21.
Anion exchange columns provide an easy, safe, and reliable method for the isolation of nucleic acid from various types of samples. The prepared nucleic acid is of superior purity equivalent to two rounds of purification on CsCl gradient centrifuge. However, the eluted nucleic acid is not ready to use because of the high salt concentration in the elution buffer. An extra step of ethanol or isopropanol precipitation is typically needed to remove the salt.