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 different 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.
Lysis is the breaking down of the membrane of a cell, often by viral, enzymic, or osmotic mechanisms that compromise its integrity. A fluid containing the contents of lysed cells is called a lysate. In molecular biology, biochemistry, and cell biology laboratories, cell cultures may be subjected to lysis in the process of purifying their components, as in protein purification, DNA extraction, RNA extraction, or in purifying organelles.
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. Ideally, to study a protein of interest, it must be separated from other components of the cell so that contaminants will not interfere in the examination of the protein of interest's structure and function. 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. DNA extraction is the process of isolating DNA from the cells of an organism isolated from a sample, typically a biological sample such as blood, saliva, or tissue. It involves breaking open the cells, removing proteins and other contaminants, and purifying the DNA so that it is free of other cellular components. The purified DNA can then be used for downstream applications such as PCR, sequencing, or cloning. Currently, it is a routine procedure in molecular biology or forensic analyses.
Affinity chromatography is a method of separating a biomolecule from a mixture, based on a highly specific macromolecular binding interaction between the biomolecule and another substance. The specific type of binding interaction depends on the biomolecule of interest; antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid binding interactions are frequently exploited for isolation of various biomolecules. Affinity chromatography is useful for its high selectivity and resolution of separation, compared to other chromatographic methods.
Ion chromatography is a form of chromatography that separates ions and ionizable polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecule—including small inorganic anions, large proteins, small nucleotides, and amino acids. However, ion chromatography must be done in conditions that are one pH 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.
In molecular biology, gel extraction or gel isolation is a technique used to isolate a desired fragment of intact DNA from an agarose gel following agarose gel electrophoresis. After extraction, fragments of interest can be mixed, precipitated, and enzymatically ligated together in several simple steps. This process, usually performed on plasmids, is the basis for rudimentary genetic engineering.
Protein methods are the techniques used to study proteins. There are experimental methods for studying proteins. Computational methods typically use computer programs to analyze proteins. However, many experimental methods require computational analysis of the raw data.
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.
A plasmid preparation is a method of DNA extraction and purification for plasmid DNA. It is an important step in many molecular biology experiments and is essential for the successful use of plasmids in research and biotechnology. Many methods have been developed to purify plasmid DNA from bacteria. During the purification procedure, the plasmid DNA is often separated from contaminating proteins and genomic DNA.
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 salt and ethanol as an antisolvent.
Acid guanidinium thiocyanate-phenol-chloroform extraction is a liquid–liquid extraction technique in biochemistry and molecular biology. It is widely used 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. Furthermore, an RNA column is typically unsuitable for purification of short RNA species, such as siRNA, miRNA 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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