Spin column-based nucleic acid purification is a solid phase extraction method to quickly purify nucleic acids. This method relies on the fact that nucleic acid will bind to the solid phase of silica under certain conditions.
Spin column-based nucleic acid purification is a solid phase extraction method to quickly purify nucleic acids. This method relies on the fact that nucleic acid will bind to the solid phase of silica under certain conditions.
The stages of the method are lyse, bind, wash, and elute. [1] [2] More specifically, this entails the lysis of target cells to release nucleic acids, selective binding of nucleic acid to a silica membrane, washing away particulates and inhibitors that are not bound to the silica membrane, and elution of the nucleic acid, with the end result being purified nucleic acid in an aqueous solution.
For lysis, the cells (blood, tissue, etc.) of the sample must undergo a treatment to break the cell membrane and free the nucleic acid. Depending on the target material, this can include the use of detergent or other buffers, proteinases or other enzymes, heating to various times/temperatures, or mechanical disruption such as cutting with a knife or homogenizer, using a mortar and pestle, or bead-beating with a bead mill.
For binding, a buffer solution is then added to the lysed sample along with ethanol or isopropanol. The sample in binding solution is then transferred to a spin column, and the column is put either in a centrifuge or attached to a vacuum. The centrifuge/vacuum forces the solution through a silica membrane that is inside the spin column, where under the right ionic conditions, nucleic acids will bind to the silica membrane, as the rest of the solution passes through. With the target material bound, the flow-through can be removed.
To wash, a new buffer is added onto the column, then centrifuged/vacuumed through the membrane. This buffer is intended to maintain binding conditions, while removing the binding salts and other remaining contaminants. Generally it takes several washes, often with increasing percentages of ethanol/isopropanol, until the nucleic acid on the silica membrane is free of contaminants. The last 'wash' is often a dry step to allow the alcohol to evaporate, leaving only purified nucleic acids bound to the column.
Finally, elution is the process of adding an aqueous solution to the column, allowing the hydrophilic nucleic acid to leave the column and return to solution. This step may be improved with salt, pH, time, or heat. Finally, to capture the eluate/eluent, the column is transferred into a clean microtube prior to a last centrifugation step.
Even prior to the nucleic acid methods employed today, it was known that in the presence of chaotropic agents, such as sodium iodide or sodium perchlorate, DNA binds to silica, glass particles or to unicellular algae called diatoms which shield their cell walls with silica. This property was used to purify nucleic acid using glass powder or silica beads under alkaline conditions. [3] This was later improved using guanidinium thiocyanate or guanidinium hydrochloride as the chaotropic agent. [4] For ease of handling, the use of glass beads was later changed to silica columns. And to enable use of automated extraction instruments, there was development of silica-coated paramagnetic beads, more commonly referred to as "magnetic bead" extraction.
Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for the specification of the function, structure and interactions of the protein of interest. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps usually exploit differences in protein size, physico-chemical properties, binding affinity and biological activity. The pure result may be termed protein isolate.
A chaotropic agent is a molecule in water solution that can disrupt the hydrogen bonding network between water molecules. This has an effect on the stability of the native state of other molecules in the solution, mainly macromolecules by weakening the hydrophobic effect. For example, a chaotropic agent reduces the amount of order in the structure of a protein formed by water molecules, both in the bulk and the hydration shells around hydrophobic amino acids, and may cause its denaturation.
The first isolation of deoxyribonucleic acid (DNA) was done in 1869 by Friedrich Miescher. Currently it is a routine procedure in molecular biology or forensic analyses. For the chemical method, there are many different kits used for extraction, and selecting the correct one will save time on kit optimization and extraction procedures. PCR sensitivity detection is considered to show the variation between the commercial kits.
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Column chromatography in chemistry is a chromatography method used to isolate a single chemical compound from a mixture. Chromatography is able to separate substances based on differential adsorption of compounds to the adsorbent; compounds move through the column at different rates, allowing them to be separated into fractions. The technique is widely applicable, as many different adsorbents can be used with a wide range of solvents. The technique can be used on scales from micrograms up to kilograms. The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process. The latter prevents cross-contamination and stationary phase degradation due to recycling. Column chromatography can be done using gravity to move the solvent, or using compressed gas to push the solvent through the column.
Ion chromatography separates ions and polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecule—including large proteins, small nucleotides, and amino acids. However, ion chromatography must be done in conditions that are one unit away from the isoelectric point of a protein.
Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins. Immunoprecipitation requires that the antibody be coupled to a solid substrate at some point in the procedure.
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Guanidinium thiocyanate(GTC) or guanidinium isothiocyanate (GITC) is a chemical compound used as a general protein denaturant, being a chaotropic agent, although it is most commonly used as a nucleic acid protector in the extraction of DNA and RNA from cells.
Phenol extraction is a processing technology used to prepare phenols as raw materials, compounds, or additives for industrial wood processing and for chemical industries. Phenol extraction is also a laboratory process to purify DNA samples.
A plasmid preparation is a method of DNA extraction and purification for plasmid DNA. Many methods have been developed to purify plasmid DNA from bacteria. These methods invariably involve three steps:
Ethanol precipitation is a method used to purify and/or concentrate RNA, DNA, and polysaccharides such as pectin and xyloglucan from aqueous solutions by adding ethanol as an antisolvent.
Acid guanidinium thiocyanate-phenol-chloroform extraction is a liquid–liquid extraction technique in biochemistry. It is widely used in molecular biology for isolating RNA. This method may take longer than a column-based system such as the silica-based purification, but has higher purity and the advantage of high recovery of RNA: an RNA column is typically unsuitable for purification of short RNA species, such as siRNA, miRNA, gRNA and tRNA.
DNA separation by silica adsorption is a method of DNA separation that is based on DNA molecules binding to silica surfaces in the presence of certain salts and under certain pH conditions.
Chromatography is a physical method of separation that distributes the components you want to separate between two phases, one stationary, the other moving in a definite direction. Cold ethanol precipitation, developed by Cohn in 1946, manipulates pH, ionic strength, ethanol concentration and temperature to precipitate different protein fractions from plasma. Chromatographic techniques utilise ion exchange, gel filtration and affinity resins to separate proteins. Since the 1980s it has emerged as an effective method of purifying blood components for therapeutic use.
TRIzol is a widely used chemical solution used in the extraction of DNA, RNA, and proteins from cells. The solution was initially used and published by Piotr Chomczyński and Nicoletta Sacchi in 1987.
RNA extraction is the purification of RNA from biological samples. This procedure is complicated by the ubiquitous presence of ribonuclease enzymes in cells and tissues, which can rapidly degrade RNA. Several methods are used in molecular biology to isolate RNA from samples, the most common of these is guanidinium thiocyanate-phenol-chloroform extraction. The filter paper based lysis and elution method features high throughput capacity.
Boom method is a solid phase extraction method for isolating nucleic acid from a biological sample. This method is characterized by "absorbing the nucleic acids (NA) to the silica beads".
Phenol–chloroform extraction is a liquid-liquid extraction technique in molecular biology used to separate nucleic acids from proteins and lipids.
Solid-phase reversible immobilization, or SPRI, is a method of purifying nucleic acids from solution. It uses silica- or carboxyl-coated paramagnetic beads, which reversibly bind to nucleic acids in the presence of polyethylene glycol and a salt. A common application of SPRI technology is purifying samples of DNA amplified by PCR for sequencing reactions:.
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